Pseudomonas aeruginosa

Int J Biochem Cell Biol, 1999 Jun, 31(6), 645 - 51
Human beta-defensin-2; Schroder JM et al.; Human beta-defensin-2 (HBD-2) is a cysteine-rich cationic low molecular weight antimicrobial peptide recently discovered in psoriatic lesional skin . It is produced by a number of epithelial cells and exhibits potent antimicrobial activity against Gram-negative bacteria and Candida, but not Gram-positive Staphylococcus aureus . HBD-2 represents the first human defensin that is produced following stimulation of epithelial cells by contact with microorganisms such as Pseudomonas aeruginosa or cytokines such as TNF-alpha and IL-1 beta . The HBD-2 gene and protein are locally expressed in keratinocytes associated with inflammatory skin lesions such as psoriasis as well as in the infected lung epithelia of patients with cystic fibrosis . It is intriguing to speculate that HBD-2 is a dynamic component of the local epithelial defense system of the skin and respiratory tract having a role to protect surfaces from infection, and providing a possible reason why skin and lung infections with Gram-negative bacteria are rather rare.

N Engl J Med, 1999 Jul 15, 341(3), 156 - 62
Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism; Kerem E et al.; BACKGROUND: Active sodium absorption is the dominant mechanism of ion transport in airway epithelium, but its role in pulmonary physiology and airway host defense is unknown . To address this question, we studied the function of airway epithelial cells and determined the frequency of pulmonary symptoms in patients with systemic pseudohypoaldosteronism, a salt-losing disorder caused by loss-of-function mutations in the genes for the epithelial sodium channel . METHODS: In nine patients 1.5 to 22 years of age who had systemic pseudohypoaldosteronism, we tested for mutations in the genes for the epithelial sodium channel, estimated the rate of sodium transport in the airway, determined the volume and ion composition of airway surface liquid, reviewed clinical features, collected laboratory data pertinent to pulmonary function, and, in three adults, measured mucociliary clearance . RESULTS: The patients with systemic pseudohypoaldosteronism had loss-of-function mutations in the genes for the epithelial sodium-channel subunits, no sodium absorption from airway surfaces, and a volume of airway surface liquid that was more than twice the normal value . The mean (+/-SE) mucociliary transport rate was higher in the 3 adult patients than in 12 normal subjects (2.0+/-0.7 vs . 0.5+/-0.3 percent per minute, P=0.009) . Young patients (those five years of age or less) all had recurrent episodes of chest congestion, coughing, and wheezing, but no airway infections with Staphylococcus aureus or Pseudomonas aeruginosa . Older patients (those more than five years of age) had less frequent respiratory symptoms . CONCLUSIONS: Patients with systemic pseudohypoaldosteronism fail to absorb liquid from airway surfaces; the result is an increased volume of liquid in the airways . These results demonstrate that sodium transport has a role in regulating the volume of liquid on airway surfaces.

J Med Microbiol, 1999 Jul, 48(7), 701 - 3
Use of a low nutrient culture medium for the identification of bacteria causing severe ocular infection; Horgan SE et al.; A low nutrient culture medium was used to identify the pathogens in four cases of persisting ocular infection . Bacto R2A agar was used in addition to conventional liquid- and solid-phase media to culture pathogenic bacteria from one case of recurrent keratitis, one case of suture-related keratitis with endophthalmitis and two eyes (two patients) with post-operative endophthalmitis . In each case, a pathogen was identified solely with R2A agar after culture for 6 days . Species isolated were Pseudomonas aeruginosa (one), Propionibacterium acnes (two) and Staphylococcus aureus (one) . Antibiotic therapy was tailored to conform to the sensitivity of the cultured organism in each case . The use of Bacto R2A low nutrient agar should be considered in culture negative eyes not showing clinical improvement, or for chronic cases where bacteria may have become adapted to more stringent ocular environments.

J Bacteriol, 1999 Jul, 181(14), 4146 - 53
Overexpression of Methanococcus voltae flagellin subunits in Escherichia coli and Pseudomonas aeruginosa: a source of archaeal preflagellin; Bayley DP et al.; Methanococcus voltae is a flagellated member of the Archaea . Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated . While the flagellins of M . voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa . Here we describe the expression of a M . voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa . Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa . This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M . voltae . In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M . voltae FlaB2 mature protein fused to the P . aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P . aeruginosa and a prepilin peptidase (pilD) mutant of P . aeruginosa . Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells . However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product . When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P . aeruginosa pilin) and expressed in both wild-type and pilD mutant P . aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type . Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P . aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.

J Int Med Res, 1998 Dec, 26(6), 304 - 12
Oral ciprofloxacin in the treatment of pseudomonas exacerbations of paediatric cystic fibrosis: clinical efficacy and safety evaluation using magnetic resonance image scanning; Redmond A et al.; Ciprofloxacin is effective for treating pulmonary infection in adult cystic fibrosis patients, and demonstrates excellent efficacy against Pseudomonas aeruginosa, but its use in paediatric cystic fibrosis patients has been limited because quinolone-induced cartilage toxicity has been observed in juvenile animals and has been considered a potential risk for children . Children with cystic fibrosis (n = 26; aged 6-16 years), with proven P . aeruginosa colonization of their sputum, were enrolled into a 14-day, open, non-comparative study . Patients were assigned to twice-daily treatment with oral ciprofloxacin 250 mg, 500 mg or 750 mg, depending on their body weight . None of the patients exhibited any signs or symptoms of arthropathy, as assessed by magnetic resonance imaging of the right knee, during or immediately after treatment, or at the 3-month post-therapy assessment . Cough, sputum production and sputum purulence were improved in more than 70% of patients . Patients showed a mean weight increase of 0.4 kg (95% confidence interval 0.1 to 0.7 kg) over the study period . Only one patient required a repeat chest radiograph, which showed no resolution of the abnormal radiographic appearances . Three patients reported adverse events during the trial, none of which were considered to be related to the study treatment.

Arch Microbiol, 1999 Jul, 172(1), 59 - 62
Discontinuous expansion linked to sector formation in Pseudomonas aeruginosa colonies; Grondin A et al.; Colonies of Pseudomonas aeruginosa exhibit sectors that were shown to be located at specific intervals within the colony . Maxima in the distribution of sectors were observed every 5 mm as measured from the center of the colony . These maxima correlated with changes in the expansion rates of colonies . The absolute average number of sectors per colony was higher for colonies grown at higher temperatures . These results increase our understanding of colony pattern formation.

Arch Microbiol, 1999 Jul, 172(1), 31 - 9
Functional assembly of the lambda S holin requires periplasmic localization of its N-terminus; Graschopf A et al.; Bacteriophage-lambda-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase . At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan . The lambda S gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor . Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105) . The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107 . It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein . To study the membrane topology of the S proteins, we used the topology probe TEM beta-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein . We show that both S proteins have a type III (Nout/Cin) topology . The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent "hole" in the membrane.com/link/service/journals/00203/bibs/172n1p31.html

Chemotherapy, 1999 Jul-Aug, 45(4), 296 - 302
Ionic binding of 3H-gentamicin and short-time bactericidal activity of gentamicin against Pseudomonas aeruginosa isolates with different lipopolysaccharide structures; Saika T et al.; The clinical isolates of Pseudomonas aeruginosa can be roughly classified into long- and short-LPS strains and LPS-deficient strains . Ionic binding of 3H-gentamicin was the highest in the long-LPS strains followed in descending order by short-LPS and LPS-deficient strains . However, PAC605 strain, completely lacking in the repeated units of O-polysaccharide and also lacking in some of the neutral sugar residues of the core oligosaccharide region, highly bound to 3H-gentamicin and it is suggested that the negatively charged sites on the deep-core oligosaccharide region and/or on lipid A participated in the binding to 3H-gentamicin . This manner of binding may be also applied to P . aeruginosa No . 45 (LPS-deficient), a clinical isolate.

Chemotherapy, 1999 Jul-Aug, 45(4), 284 - 95
Pharmacodynamics of intermittent- and continuous-infusion cefepime alone and in combination with once-daily tobramycin against Pseudomonas aeruginosa in an in vitro infection model; Tessier PR et al.; Cefepime, a fourth-generation cephalosporin, is currently one of the primary agents used in combination with an aminoglycoside when treating Pseudomonas aeruginosa infections . The bactericidal activity of cefepime administered as intermittent doses (IT) or continuous infusion (CI) both alone and in combination with once-daily tobramycin (ODT) against two clinical strains of P . aeruginosa was compared using an in vitro infection model . Cefepime concentrations simulated human pharmacokinetics after a 1-gram Q12 regimen, or a 1-gram loading dose followed by a 2-gram Q24 CI regimen; the ODT regimen mimicked peak concentrations of >/=10 x MIC . All regimen simulations were run in duplicate over 48 h and a growth control (no antimicrobials added) was run concurrently . Strains tested, PSA5 and PSA10, had MICs of 2 and 8 microg/ml to cefepime, respectively; both MICs to tobramycin were 1.0 microg/ml . CI regimens resulted in concentrations approximately 6x and 2x the MIC for PSA5 and PSA10, respectively . The change in log10 colony-forming units (CFU) per milliliter over time for both P . aeruginosa isolates was compared to initial inocula for all treatment regimens . Initial bolus doses of both IT and CI regimens resulted in a similar decrease in the log10 CFU/ml of both organisms over the first 12 h of the study . After subsequent doses, however, both IT regimens showed greatly diminished bactericidal activity, while both CI regimens were persistently bactericidal without the observation of significant regrowth . As a result, a statistical difference in log10 CFU/ml between both IT and CI regimens with and without ODT was realized at 24, 36 and 48 h for each isolate . Unlike IT dosing, CI cefepime alone or in combination with ODT optimizes bactericidal activity by maximizing the percent of the dosing interval that concentrations remained above the MIC resulting in undiminished bacterial inhibition when compared to IT regimens . These data further suggest that CI is the most efficient method of administration of beta-lactam antibiotics.

Clin Diagn Lab Immunol, 1999 Jul, 6(4), 537 - 41
Effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis in mice; Matsumoto T et al.; We evaluated the effect of antiflagellar human monoclonal antibody on gut-derived Pseudomonas aeruginosa sepsis . Mice were given a suspension of P . aeruginosa SP10052 in their drinking water and were simultaneously treated with ampicillin (200 mg/kg of body weight) to disrupt the normal bacterial flora . Cyclophosphamide was then administered to induce leukopenia and translocation of the P . aeruginosa that had colonized the gastrointestinal tract, thereby producing gut-derived generalized sepsis . In this model, intraperitoneal injection of 100 microg of antiflagellar human monoclonal antibody (SC-1225) per mouse for 5 consecutive days significantly (P < 0.01) increased the survival rate compared with that for mice treated with bovine serum albumin (BSA) . Treatment with SC-1225 significantly reduced the average number of viable bacteria in portal blood, liver, and heart blood compared with the average number after treatment with BSA . Furthermore, the presence in serum of the inflammatory cytokines tumor necrosis factor alpha and interleukin 6 were evaluated as markers of severity of infection, and the results showed that the levels of these cytokines in mice treated with SC-1225 were significantly decreased in comparison with those in BSA-treated control mice . Although there was no significant difference in the number of bacteria that colonized the intestine, SC-1225 treatment significantly increased bacterial opsonophagocytosis by cultured peritoneal macrophages from mice with or without cyclophosphamide pretreatment . Our results indicate that antiflagellar human monoclonal antibody SC-1225 protects mice against gut-derived sepsis caused by P . aeruginosa and suggest that such an effect is due to its opsonophagocytic activity and the reduced motility of the translocated bacteria once the bacteria move from the intestine into the bloodstream.

Am J Respir Crit Care Med, 1999 Jul, 160(1), 186 - 91
Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients; Muhlebach MS et al.; Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF) . The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems . Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications . Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P . aeruginosa . The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen . Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF . We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P . aeruginosa) . These data may provide further rationale for anti-inflammatory therapy early in CF.

Antimicrob Agents Chemother, 1999 Jul, 43(7), 1609 - 15
Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa peritonitis in a murine model; Barekzi NA et al.; Infectious peritonitis results from bacterial contamination of the abdominal cavity . Conventional antibiotic treatment is complicated both by the emergence of antibiotic-resistant bacteria and by increased patient populations intrinsically at risk for nosocomial infections . To complement antibiotic therapies, the efficacy of direct, locally applied pooled human immunoglobulin G (IgG) was assessed in a murine model (strains CF-1, CD-1, and CFW) of peritonitis caused by intraperitoneal inoculations of 10(6) or 10(7) CFU of Pseudomonas aeruginosa (strains IFO-3455, M-2, and MSRI-7072) . Various doses of IgG (0.005 to 10 mg/mouse) administered intraperitoneally simultaneously with local bacterial challenge significantly increased survival in a dose-dependent manner . Local intraperitoneal application of 10 mg of IgG increased animal survival independent of either the P . aeruginosa or the murine strains used . A local dose of 10 mg of IgG administered up to 6 h prophylactically or at the time of bacterial challenge resulted in 100% survival . Therapeutic 10-mg IgG treatment given up to 12 h postinfection also significantly increased survival . Human IgG administered to the mouse peritoneal cavity was rapidly detected systemically in serum . Additionally, administered IgG in peritoneal lavage fluid samples actively opsonized and decreased the bacterial burden via phagocytosis at 2 and 4 h post-bacterial challenge . Tissue microbial quantification studies showed that 1.0 mg of locally applied IgG significantly reduced the bacterial burden in the liver, peritoneal cavity, and blood and correlated with reduced levels of interleukin-6 in serum.

Antimicrob Agents Chemother, 1999 Jul, 43(7), 1584 - 90
Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate; Lauretti L et al.; Production of a metallo-beta-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy) . The metallo-beta-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem . Sequencing of the cloned gene revealed that it encoded a new class B beta-lactamase that was named VIM-1 . At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the beta-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes . Among these, VIM-1 showed the highest degree of similarity to Bc-II . Similarly to blaIMP, blaVIM was also found to be carried on a gene cassette inserted into a class 1 integron . The blaVIM-containing integron was located on the chromosome of P . aeruginosa VR-143/97, and the metallo-beta-lactamase-encoding determinant was not transferable to E . coli by conjugation . Expression of the integron-borne blaVIM gene in E . coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

Zhonghua Yi Xue Za Zhi (Taipei), 1999 Jun, 62(6), 362 - 8
Malignant otitis externa; Yu LH et al.; BACKGROUND: Malignant otitis externa is an infrequent but severe infection of the external auditory canal, most often affecting elderly diabetic patients . Early diagnosis is necessary due to its high morbidity and mortality . METHODS: From 1990 to 1997, all patients with malignant otitis externa at the Veterans General Hospital-Taipei were reviewed retrospectively . The clinical features and the strategy of diagnosis and treatment are discussed . RESULTS: Twelve patients with an average age of 65.3 years were included . Eleven of these patients were diabetic . All had the presenting symptoms of otalgia and otorrhea at diagnosis . Bacterial cultures grew Pseudomonas aeruginosa in eight patients and methicillin-resistant Staphylococcus aureus in four patients . The mean duration of admission was 82 days . Appropriate antibiotics were given according to the results of bacterial culture and sensitivity test . 99Technetium scans and 67gallium scans were performed to evaluate the extent of involvement and monitor the effects of treatment . Eventually, four patients died due to renal failure, meningitis, pneumonia and upper gastrointestinal bleeding, respectively . CONCLUSIONS: Malignant otitis externa is a life-threatening infection arising from the external auditory canal . A high degree of suspicion for malignant otitis externa is mandatory . Vigorous local and systemic antimicrobial treatment should be initiated early in the course of the disease to achieve a satisfactory outcome . 99Technetium and 67gallium scans are important for the diagnosis and evaluation of the treatment results.

Zhonghua Yi Xue Za Zhi (Taipei), 1999 May, 62(5), 300 - 7
Pseudomonas aeruginosa central nervous system infections: analysis of clinical features of 16 adult patients; Chuang YC et al.; BACKGROUND: The purpose of this study was to analyze the clinical features and therapeutic outcome of 16 adult patients with Pseudomonas aeruginosa central nervous system (CNS) infection . We also attempted to identify the factors that significantly influence the prognosis of this potentially fatal CNS infection . METHODS: Sixteen adult patients with P aeruginosa CNS infection, nine men and seven women, aged from 18 to 86 years, were included in this retrospective study . The clinical features and the laboratory data of these patients were analyzed . Potential prognostic factors were compared by means of Fisher's exact test and the relative risks were estimated by odds ratio . RESULTS: Of the 16 patients, 13 had meningitis and three had focal suppuration (two with brain abscess and one with spinal epidural abscess) . The 13 meningitis patients with nosocomial or community-acquired infections were classified into two forms: the spontaneous form and the neurosurgical form . The overall mortality rate was 37.5% (6/16) . In the meningitis group, the patients with the neurosurgical form had a lower mortality rate (11.1%; 1/9) than the patients with the spontaneous form (100%; 4/4), and those with community-acquired meningitis had a higher mortality rate (80%; 4/5) than those with nosocomial infections (12.5%; 1/8) . All the meningitis patients who did not receive appropriate antibiotic treatment expired . The statistically significant prognostic factors included the acquisition of infection, form of infection, bacteremia, initial level of consciousness and the use of appropriate antibiotics . CONCLUSIONS: Vigilance for P aeruginosa is particularly important in patients with predisposing factors such as head injury, neurosurgical procedures and long-term debilitating diseases . Early appropriate antibiotic therapy and neurosurgical intervention for patients with suppurative infections can bring about improved therapeutic results.

J Appl Microbiol, 1999 Jun, 86(6), 1039 - 46
Ortho-phthalaldehyde: a possible alternative to glutaraldehyde for high level disinfection; Walsh SE et al.; Ortho-phthalaldehyde (OPA) was tested against a range of organisms including glutaraldehyde-resistant mycobacteria, Bacillus subtilis spores and coat-defective spores . Glutaraldehyde (GTA) and peracetic acid (PAA) were tested for comparative purposes . Both suspension and carrier tests were performed using a range of concentrations and exposure times . All three biocides were very effective (> or = 5 log reduction) against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in suspension tests . OPA and GTA (PAA was not tested) were also very effective against Staph . aureus and Ps . aeruginosa in carrier tests . OPA showed good activity against the mycobacteria tested including the two GTA-resistant strains, but 0.5% w/v OPA was found not to be sporicidal . However, limited activity was found with higher concentrations and pH values . Coat-defective spores were more susceptible to OPA, suggesting that the coat may be responsible for this resistance . The findings of this study suggest that OPA is effective against GTA-resistant mycobacteria and that it is a viable alternative to GTA for high level disinfection.

J Appl Microbiol, 1999 Jun, 86(6), 944 - 54
Effect of nutrient limitation on adhesion characteristics of Pseudomonas aeruginosa; Cowell BA et al.; Pseudomonas aeruginosa causes a variety of diseases in humans including lung and ocular infections . Infections of the cornea are usually associated with wearing contact lenses and can result in loss of vision . This study aimed to determine the effect of carbon or nitrogen limitation on the adhesion to contact lenses of a strain of Ps . aeruginosa isolated from contact lens-related corneal inflammation . Cells were grown in a continuous culture apparatus in varying levels of glucose or ammonia to effect nutrient limitation . Adhesion to contact lenses was measured as total counts and viable counts . The cell surface hydrophobicity and charge were measured using adhesion to surface-modified Sepharose . Changes in lipopolysaccharide were determined using 1D SDS-PAGE and changes in cell-surface proteins were measured using 2D gel electrophoresis . The more the cultures were nitrogen limited, the greater the increase in adhesion to unworn hydrogel contact lenses 0.3 x 10(3) - 2.2 x 10(3) cells/mm2 on Etafilon A lenses . Cells that were carbon limited showed a greater increase in adhesion to contact lenses when the lenses had been coated in artificial tears . It appeared that lipopolysaccharide may have been involved in the constitutive adhesion to unworn lenses that occurred during C-limitation, whereas changes in the outer membrane proteins contributed to the increased adhesion under nitrogen limitation, or the change in adhesion that occurred to carbon-limited cells using contact lenses coated in artificial tears . Nine cell-surface proteins appeared during nitrogen limitation with kDa/pI of 75/4.8, 4.9, 5.0; 62/5.6; 89/6.5; 38/6.4; 28/1.5; 18/6.4; 12/4.5 . Any or all of these may have been involved in the increased adhesion and further experiments are underway to examine this possibility.

Int J Pharm, 1999 Jul 20, 184(2), 179 - 87
New cytokine dressings . II . Stimulation of oxidative burst in leucocytes in vitro and reduction of viable bacteria within an infected wound; Grzybowski J et al.; Recently, we have developed the new dressings containing rhG-CSF or rhGM-CSF . In the present study we investigated either in vitro or in vivo biological activity of the dressings . Human whole blood samples were incubated with extracts from the collagen- or polyurethane-based dressings containing rhG-CSF or/and rhGM-CSF and phagocytic and oxidative metabolic activities were quantitated using Phagotest or Bursttest kits . The results indicate that both the number of phagocyting cells and the intensity of phagocytosis per cell, as well as the level of the oxidative burst in particular, were stimulated by one or both of the cytokines extracted . Next, the experimental skin wounds in mice were infected with 107 CFU of Pseudomonas aeruginosa strain ATCC 27853 and treated locally with the rhG-CSF-containing dressing . The analysis of the biopsies taken from the wounds indicated that in mice treated with the cytokine-containing dressing on the third day the log of CFU per biopsy was 5.0 vs . 6.2 in the control (P<0.001), and on the 8th day was lower than 4 vs . 5.4 in control (P<0.0001) . Our findings clearly suggest that the newly designed dressings containing the incorporated CSFs can be used as effective topical cytokine-delivery system in the treatment of bacterial infections in wounds . Copyright

Curr Microbiol, 1999 Jul, 39(1), 1 - 8
Kinetic properties of purified Pseudomonas aeruginosa phosphorylcholine phosphatase indicated that this enzyme may be utilized by the bacteria to colonize in different environments; Salvano MA et al.; Pseudomonas aeruginosa phosphorylcholine phosphatase from periplasmic extracts of bacteria grown on choline as the sole carbon and nitrogen source was purified to homogeneity . The enzyme represented nearly 1% of the total protein found in the periplasmic space and is a monomer of approximately 53 kDa with an isoelectric point of 7.5 . The optimum pH with phosphorylcholine was in the range of 5-8; with phosphorylethanolamine there was a peak at pH 6, and with p-nitrophenyl-phosphate (p-NPP) the optimum was at pH 5 . Studies carried out at pH 5 indicated: i) For the three substrates above, Mg2+, Zn2+, or Cu2+ was necessary for maximal activity . ii) With p-NPP, these cations bound to the free enzyme in an ordered bireactant system . iii) With phosphorylethanolamine, Mg2+, Zn2+, or Cu2+ bound to the free enzyme in an at random bireactant system . iv) With phosphorylcholine, maximal activity was obtained with cation concentrations as low as 100 nM . v) Al3+ ions were inhibitors of the enzyme activity . The n (Hill coefficient) values for the inhibition by Al3+ with phosphorylcholine or p-NPP were 1 or 4, respectively . vi) The enzyme exhibited two affinity sites for phosphorylcholine . With Mg2+, a site with a Km value of 0.5 mM was detected; the corresponding Vmax was 25 micromol min-1 (mg protein)-1; without Mg2+, the enzyme displayed Km and Vmax values of 0.09 mM and 4.2 micromol min-1 (mg protein)-1, respectively . Studies carried out at pH 7.4 indicated: i) The enzyme could not catalyze the hydrolysis of p-NPP, and phosphorylethanolamine was a poor substrate in either the presence or absence of divalent cations . ii) The enzyme activity measured with phosphorylcholine was independent of divalent cations or was not inhibited by Al3+ ions . iii) With or without Mg2+, the enzyme exhibited two affinity sites for phosphorylcholine; the Km values were 0.05 mM and 0.5 mM with their corresponding Vmax of 5.6 and 25 micromol min-1 (mg protein)-1, respectively.

Orthopedics, 1999 Jun, 22(6), 601 - 4
Sterilization of contaminated bone-tendon autografts using 10% povidone-iodine solution; Stanford R et al.; This study evaluates methods of sterilizing contaminated bone-tendon autografts using 10% povidone-iodine solution . Sterile grafts were prepared from human cadavers . Grafts were immersed in a suspension of either Staphylococcus aureus or Pseudomonas aeruginosa, and three sets of sterilization experiments were performed in 10% povidone-iodine for 30 minutes: one each with S . aureus and P . aeruginosa by static soaking and a third with S . aureus by serial washing with agitation . Of grafts inoculated with S . aureus, six of six grew the test organism after soaking at room temperature, as did five of six after soaking at 36 degrees C and also eight of nine after washing with agitation . Of grafts inoculated with P . aeruginosa, five of six grew the test strain after soaking at room temperature, as did six of six after soaking at 36 degrees C . Thirty minutes of exposure to aqueous 10% povidone-iodine does not adequately sterilize an inoculated graft.

FEBS Lett, 1999 Jun 11, 452(3), 309 - 13
Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens; Simpson AJ et al.; Elafin (elastase-specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung . Using synthetic peptides corresponding to full-length elafin (H2N-1AVT.....95Q-OH), the NH2-terminal domain (H2N-1AVT.....50K-OH) and the COOH-terminal domain (H2N-51PGS.....95Q-OH), we demonstrate that elafin's anti-elastase activity resides exclusively in the COOH-terminus . Several characteristics of elafin suggest potential anti-microbial activity . The anti-microbial activity of elafin, and of its two structural domains, was tested against the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus . Elafin killed both bacteria efficiently, with 93% killing of P . aeruginosa by 2.5 microM elafin and 48% killing of S . aureus by 25 microM elafin . For both organisms, full-length elafin was required to optimise bacterial killing . These findings represent the first demonstration of co-existent anti-proteolytic and anti-microbial functions for elafin.

Farmaco, 1999 Apr 30, 54(4), 232 - 6
Gold(I) complexes as antimicrobial agents; Novelli F et al.; Seven gold complexes were prepared and investigated for biocidal activity against Gram-positive and -negative bacteria, fungi and protozoa . All of them were active against the tested microorganisms with the exception of Pseudomonas aeruginosa . In many, cases minimum inhibitory concentrations (MIC) were well below 1 microgram/ml . The activity is not simply related to the gold content, but also to the nature of both the phosphine and the aminothiol to which the metal is bound.

J Bacteriol, 1999 Jul, 181(13), 4118 - 24
The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS; Stintzi A et al.; A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced . Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore . pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.

J Bacteriol, 1999 Jul, 181(13), 4012 - 9
Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa; Bleves S et al.; The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope . After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane . This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein . XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium . Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane . This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP . An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P . aeruginosa secretion machinery . Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein . Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.

J Bacteriol, 1999 Jul, 181(13), 3974 - 80
Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes; Jordan A et al.; Three separate classes of ribonucleotide reductases exist in nature . They differ widely in protein structure . Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria . Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes . Surprisingly, the genomic sequence of Pseudomonas aeruginosa contains sequences for each of the three classes . Here, we show by DNA hybridization that other species of Pseudomonas also contain the genes for three classes . Extracts from P . aeruginosa and P . stutzeri grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity . Unexpectedly, class I activity increased greatly during microaerobic conditions . The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form . The catalytic properties of all enzymes are similar to those of other bacterial reductases . However, the Pseudomonas class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding Escherichia coli enzyme but similar to the mouse enzyme . In similarity searches, the amino acid sequence of the class I enzyme of P . aeruginosa was more related to that of eukaryotes than to that of E . coli or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene . The results raise many questions concerning the physiological function and evolution of the three classes in Pseudomonas species.

J Bacteriol, 1999 Jul, 181(13), 3890 - 7
Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa; Suh SJ et al.; The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria . To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P . aeruginosa, including PAO1 . The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent . The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source . In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2 . In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain . We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P . aeruginosa . The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain . The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1 . The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine . This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model . In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected . Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium . On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain . Thus, our data indicate that although some of the functions of RpoS in P . aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

DNA Res, 1999 Apr 30, 6(2), 103 - 8
The gene for an exopolyphosphatase of Pseudomonas aeruginosa; Miyake T et al.; In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase {exopoly(P)ase; EC 3.6.1.11} of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk . Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon . The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX . The gene product of ppx (paPPX) was overproduced in E . coli, and its activity was evaluated . Orthophosphate (Pi) is released from polyphosphate {poly(P)}, the average chain lengths of which are 79 and 750, respectively . The amount of Pi released matched the amount of poly(P) lost . Thus ppx encodes an enzyme that has exopoly(P)ase activity.

J Antimicrob Chemother, 1999 May, 43(5), 651 - 7
Investigation of the synergic effects of aminoglycoside-fluoroquinolone and third-generation cephalosporin combinations against clinical isolates of Pseudomonas spp; Mayer I et al.; Antimicrobial synergy resulting from antibiotic combination therapy is often important in the treatment of serious bacterial infections . Previous studies have demonstrated synergy between an aminoglycoside and beta-lactam antibiotics in the treatment of Pseudomonas aeruginosa infections . The present paper investigates the synergic effects of aminoglycosides (amikacin and netilmicin) and fluoroquinolones (ciprofloxacin, ofloxacin and pefloxacin) in combination with third-generation cephalosporins (cefoperazone, ceftriaxone and ceftazidime) against 18 clinical isolates of Pseudomonas spp . The effects of these drugs were examined by three methods (disc diffusion, 'chequerboard' titration and the time-killing method), to evaluate the activities of the antibiotics alone and in combination against selected isolates . Fractional inhibitory concentration indices were calculated for all isolates with all combinations . Use of the disc diffusion method revealed that amikacin and netilmicin in combination with the three cephalosporins exhibited synergy against 7-12 isolates, whereas the combinations of quinolones and ceftazidime displayed synergic effects only in the case of 3-5 isolates . On 'chequerboard' titration, amikacin and ceftriaxone exerted synergy against seven of the isolates . The other combinations showed synergy against fewer isolates, but every combination demonstrated synergic effect against some of the isolates . The tested combinations had different effects against various Pseudomonas spp . With the time-killing method, the 1/2 x MIC of amikacin or ciprofloxacin in combination with the 1/2 x MIC of third-generation cephalosporins proved to be most effective . No antagonism was found with these combinations . Discrepancies in the detection of synergy were observed for the different methods.

J Antimicrob Chemother, 1999 Jan, 43(1), 61 - 70
Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli; Piddock LJ et al.; The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c . 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E . coli was investigated . The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E . coli was also determined . Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E . coli, despite a reduction in fluorescence at alkaline pH . For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH . The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E . coli and S . aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively . Above these temperatures the cell viability decreased . With an increase in drug concentration there was, for intact E . coli and 12/15 agents, and for S . aureus and 10/15 agents, a linear increase in the concentration of drug accumulated . However, for P . aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway . Assuming 100% accumulation into intact cells of E . coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts . CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species . The variation in the effect of CCCP upon accumulation of the different quinolones into E . coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone . Overall, these data suggest that accumulation of most quinolones into E . coli and S . aureus proceeds by simple diffusion, but that P . aeruginosa behaves differently.

Biosci Biotechnol Biochem, 1999 May, 63(5), 946 - 7
Production of rhamnolipid biosurfactant by fed-batch culture of Pseudomonas aeruginosa using glucose as a sole carbon source; Lee Y et al.; The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant . With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.

APMIS, 1999 Jun, 107(6), 585 - 92
Development of resistance and cross-resistance in Pseudomonas aeruginosa exposed to subinhibitory antibiotic concentrations; Wu YL et al.; The purpose of this study was to compare resistance and cross-resistance development in Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients to commonly used antipseudomonal antibiotics . Isolates were repeatedly exposed to subinhibitory concentrations of either azlocillin, tobramycin, ceftazidime or ciprofloxacin . On 10 consecutive occasions, samples were removed from the half-MIC well of a microtitre plate and regrown in drug-free medium to provide the next inoculum for MIC determination . The increase in MIC at the end of the treatment period was significant (p<0.05) for all selecting antibiotics . Cross-resistance to unrelated antibiotics was not observed, but was significant (p<0.05) in all beta-lactams (ticarcillin, piperacillin, ceftazidime and cefsulodin) studied where azlocillin was the selecting antibiotic . The addition of clavulanic acid to ticarcillin and of tazobactam to piperacillin had no effect on cross-resistance . The development of resistance to azlocillin was associated with increased beta-lactamase activity and a change in isoelectric point of the beta-lactamases . The result of this study supports a rotational policy for antipseudomonal antibiotics in CF patients.

Infection, 1999, 27 Suppl 1, S69 - 73
Reducing catheter-associated infections with silver-impregnated catheters in long-term therapy of children; Carbon RT et al.; Central venous long-term catheters offer reliable, large-lumen vascular access with high flow rates for delivery of nutrition or for cell-containing infusions and perfusions . Catheter-associated infections (CAI) pose the greatest threat to such vascular access, despite existing preventive measures . In this article one prospective and one retrospective study of CAI in pediatric therapy are presented . Study I: A retrospective investigation from 1990 through 1995 of 60 conventional long-term catheters in 50 patients . The total number of days in which the catheters were in place was 11,818 . The calculated CAI incidence was 1 per 1,000 days of catheter insertion . Bacteriologically demonstrated CAI (identical isolate on the catheter tip and in a blood culture) occurred in three instances (5%) . Five cases (8.3%) were diagnosed with a therapy-resistant, septic clinical picture . Study II: A prospective, randomized comparison of long-term silver-impregnated (Erlanger silver catheters) and control catheters (Quinton Instrument Co.) was made with 41 patients (20 with a silver catheter, 21 with a Quinton catheter) . To date, the silver catheters have been distinguished by sterile bacteriological findings, whereas three cases of CAI have been demonstrated with the comparative catheters . One patient recently underwent intensive care after becoming unstable with signs of septic shock and demonstrable Pseudomonas aeruginosa, and two other patients manifested coagulase-negative staphylococci on the catheter tips . In three of nine control catheters an incidence of 1.18 per 1,000 days of indwelling catheters was found, whereas no CAI has occurred with the eight microbiologically tested silver catheters.

Clin Orthop, 1999 Jun, (363), 232 - 9
Prolonged leaching time of peptide antibiotics from acrylic bone cement; Yaniv M et al.; The leaching time of antibiotics in growth inhibitory concentrations from polyacrylic bone cements was determined using Escherichia coli, methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and Pseudomonas aeruginosa as target bacteria . The leaching time of the peptide antibiotics vancomycin and polymyxin B nonapeptide was considerably longer than that of gentamicin, novobiocin, and erythromycin . Among the nonpeptide antibiotics, the leaching time of novobiocin lasted longer than did that of gentamicin . The acylated parent polymyxin B antibiotic leached for a considerably shorter period than did the deacylated polymyxin B nonapeptide derivative, suggesting that the lipids impede the leaching process from the cement . Cement beads impregnated with polymyxin B nonapeptide and introduced into the tibia of three rabbits receiving oral novobiocin protected bone infection against a challenge of Pseudomonas aeruginosa that otherwise caused infection in tibias containing gentamicin impregnated cement beads . The peptide antibiotics alone or in combination with other antibiotics (polymyxin B nonapeptide and novobiocin) impregnated in cements for orthopaedic procedures may provide longer periods of protection against a wide range of bacterial pathogens.

Infect Immun, 1999 Jul, 67(7), 3625 - 30
Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia; Comolli JC et al.; Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection . They are also important bacterial adhesins, and nonpiliated mutants of P . aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models . This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P . aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both . This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo . Mutants of pilT and pilU retain surface pili but have lost twitching motility . In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants . The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant . In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality . These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

Infect Immun, 1999 Jul, 67(7), 3542 - 7
Mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs; Bals R et al.; One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins . We describe in this report the isolation and characterization of a murine homologue of human beta-defensin 2 (hBD-2) called mouse beta-defensin 3 (mBD-3) . The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues . Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron . The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse alpha- and beta-defensins are found . Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung . After instillation of Pseudomonas aeruginosa PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver . Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against P . aeruginosa PAO1 (MIC of 8 micrograms/ml) and Escherichia coli D31 (MIC of 16 micrograms/ml) in a salt-dependent manner . This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces . These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.

Infect Immun, 1999 Jul, 67(7), 3494 - 503
Differential sensitivity of human epithelial cells to Pseudomonas aeruginosa exoenzyme S; McGuffie EM et al.; Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa . Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence . To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines . Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS . Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS . Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing . Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells . In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras . Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.

Infect Immun, 1999 Jul, 67(7), 3207 - 14
Pili binding to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial internalization by Pseudomonas aeruginosa; Comolli JC et al.; The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated . To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1 . Compared to an untreated monolayer, P . aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1 . Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU . Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold . These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1 . Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells . These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P . aeruginosa exoproducts solely determine the steps subsequent to adhesion.

J Mol Biol, 1999 Jun 25, 289(5), 1459 - 67
Folding mechanism of Pseudomonas aeruginosa cytochrome c551: role of electrostatic interactions on the hydrophobic collapse and transition state properties; Travaglini-Allocatelli C et al.; We report on the folding kinetics of the small 82 residue cytochrome c551from Pseudomonas aeruginosa . The presence of two Trp residues (Trp56 and Trp77) allows the monitoring of fluorescence quenching on refolding in two different regions of the protein . A single His residue (the iron-coordinating His16) permits the study of refolding in the absence of miscoordination events . After identification of the kinetic traps (Pro isomerization and aggregation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which we show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compact, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transition state(s) possibly involving the formation of a specific salt bridge between Lys10 and Glu70, at the contact between the N and C-terminal helices . Comparison with the widely studied horse cytochrome c allows the discussion of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %) .

Ned Tijdschr Geneeskd, 1999 May 29, 143(22), 1140 - 3
{Use and adverse reactions of local antibiotics and disinfectants on the skin}; Smeenk G et al.; The local antibiotics are an important addition to the treatment of moderately severe skin infections . Development of contact allergy and bacterial resistance, however, are some of the adverse reactions that may occur . Fusidic acid and tetracycline are the most suitable products for the treatment of superficial primary infections of the skin caused by Gram-positive cocci and secondary infected dermatoses . In case of insufficient effect mupirocin may be tried . Mupirocin is an effective antibiotic in the treatment of nasal carriage of Staphylococcus aureus in cases of increased risk of infection (haemodialysis, continuous peritoneal dialysis, after thoracic surgery) . Besides it is an important local antibiotic in the treatment of meticillin-resistant staphylococci in the nose . In order to prevent development of bacterial resistance its further use has to be restricted . Silver sulfadiazine is a good option for the local treatment of infections by Pseudomonas aeruginosa and other Gram-negative bacteria . Erythromycin and clindamycin are useful local antibiotics for the treatment of mild acne vulgaris . Disinfectants are mainly suitable for the use on the intact skin.

J Mol Biol, 1999 Jun 18, 289(4), 1017 - 28
MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association; Brown K et al.; The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR) . It shows as high levels of activity and affinity for the P . nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa) . Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions . Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely.The three-dimensional structure of P . nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit . Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %) . Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area . Four tighly packed dimers form the eight molecules of the asymmetric unit.The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv) . The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates . The dimer observed in the crystal also exists in solution . It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker . In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion . The availability of the structure of the cytochrome c552-Pn and that of NiR from P . aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur .

Genetika, 1999 Mar, 35(2), 297 - 302
{x811--mutation of the Pseudomonas aeruginosa transposable phage D3112 with a pleiotropic effect}; Burkal'tseva MV et al.; Characteristics of Pseudomonas aeruginosa Transposable Phage D3112 carrying mutation x811 are described . x811 is a recessive mutation with pleiotropic effect . It determines a deteriorated lysis of infected or induced bacteria, a delayed replication, and a considerably decreased replication rate . In addition, the x811 mutation is expressed as the Kil phenotype, since high-temperature induction of prophage D3112 cts15 x811 does not cause an immediate decrease in the ability of bacteria to form colonies at 42 degrees C . Restriction analysis of DNA of D3112 cts15 x811 and its segregants has not revealed extended insertions or deletions . The characteristics of segregants of the D3112 cts15 x811 phage agree with the suggestion that the x811 mutation has emerged in a regulatory element (a gene or a site) that controls both expression of the entire early operon, including the "pre-early" function Kil, and the regulation of the repressor synthesis.

Minerva Pediatr, 1999 Mar, 51(3), 47 - 51
{Anti-pseudomonas-specific precipitins as a marker of chronic infection in patients with cystic fibrosis}; Castello D et al.; AIMS: The authors underline the characteristics of Pseudomonas aeruginosa and its methods of action on bronchial mucosa in cystic fibrosis . They then discuss the two concepts of "colonisation" and "chronic infection" . METHODS: The level of "infection" was evaluated using an immunoelectrophoretic method involving the precipitation of bands of specific precipitins . A technical description of the method is included . The authors illustrate the use of the method in 78 cases of cystic fibrosis, comparing the positive results obtained using precipitin electrophoresis with the results of direct bacteriological findings . RESULTS: Using the bacteriological criteria, a total of 26.9% of patients were diagnosed as infected, whereas this percentage rose to 32.1% using precipitins . 87.2% of cases were concordant using both methods . CONCLUSIONS: As a practical solution, the authors recommend that the two methods are combined, thus obtaining a marked reduction in the number of false positives with obvious consequences in terms of therapeutic decisions.

J Mol Evol, 1999 Jul, 49(1), 108 - 21
A probable mixed-function supraoperon in Pseudomonas exhibits gene organization features of both intergenomic conservation and gene shuffling; Xie G et al.; Sequencing of an 8182-bp chromosomal region in Pseudomonas stutzeri revealed the major portion of an apparent mixed-function supraoperon (defined as a nested organization of transcriptional units encoding gene products which function in more than one biochemical pathway) . A nearly identical supraoperon organization was apparent in the unpublished Pseudomonas aeruginosa genome database, where the complete Pseudomonas supraoperon was deduced . The serC(pdxF)-aroQp . pheA-hisHb-tyrAc-aroF-cmk-rpsA supraoperon encodes 3-phosphoserine aminotransferase, a bidomain chorismate mutase/prephenate dehydratase, imidazole acetol-phosphate aminotransferase, cyclohexadienyl dehydrogenase, 5-enolpyruvylshikimate 3-phosphate synthase, cytidylate kinase, and ribosomal protein S1 . The member genes were identified by homology analysis, enzyme assay, and/or functional complementation . Although SerC(PdxF) and HisHb exercise their primary functions in serine, pyridoxine, and histidine biosynthesis, they also have critical catalytic roles in provision of the sidechain amino groups of tryptophan, phenylalanine, and tyrosine . The likelihood of supraoperon-wide translational coupling is suggested by the highly compressed intergenic spacing (including overlapping stop and start codons), as well as by possible hairpin structures in mRNA which may sequester some of the ribosome-binding sites and thus provide a mechanism for translational coupling . A comparison of the organization of the supraoperon genes in other organisms represented in the database revealed unmistakable conservation of the linkage of these genes across wide phylogenetic boundaries, albeit with considerable gene shuffling . At least remnants and shuffled portions of the entire supraoperon are distributed throughout the Gram-negative bacteria with the hisHb-tyrA-aroF gene block being conserved as distantly as the gram-positive bacteria . Such conservation of mixed-function genes may reflect the selective value of still-unknown global relationships of protein-protein interaction or regulation.

J Bacteriol, 1999 Jun, 181(12), 3849 - 51
Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa; Baysse C et al.; Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853) . Killing was specifically inhibited by addition of type II ferripyoverdine . All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein . We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.

J Bacteriol, 1999 Jun, 181(12), 3730 - 42
Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa; Ma JF et al.; We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes . These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557) . Our goal was to determine the contributions of KatA and BfrA to the resistance of P . aeruginosa to hydrogen peroxide (H2O2) . When provided on a multicopy plasmid, the P . aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli . The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM . Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity . The katA and katA bfrA mutants possessed no detectable catalase activity . Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation . Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant . RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2 . Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria . Our results suggest that P . aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.

Clin Ther, 1999 Apr, 21(4), 675 - 90
Sparfloxacin versus ciprofloxacin for the treatment of community-acquired, complicated skin and skin-structure infections; Lipsky BA et al.; Fluoroquinolones have been shown to be effective in the treatment of complicated skin and skin-structure infections, in part because of their broad-spectrum antibacterial activity against causative pathogens that are resistant to older antimicrobial agents . We enrolled 603 adult patients (>58% male, >85% white) in a double-masked, double-dummy, randomized, multicenter trial to compare the efficacy and tolerability of sparfloxacin (400-mg loading dose followed by 200 mg once daily) with those of ciprofloxacin (750 mg twice daily) for 10 days in the treatment of community-acquired, complicated skin and skin-structure infections . The primary efficacy variable was clinical response, based on assessment of signs and symptoms, in the clinically assessable population . Patients in the sparfloxacin and ciprofloxacin groups were comparable with respect to demographic characteristics, underlying diseases, medical history, and laboratory test results . Wound infection was the most common diagnosis, and Staphylococcus aureus was the most frequently isolated pathogen . For the 475 clinically assessable patients, the clinical success rate (percentage of patients cured or improved) was 90.1% (210/233) with sparfloxacin and 87.2% (211/242) with ciprofloxacin . In this analysis (95% confidence interval {CI}, -2.8 to 8.6) and the intent-to-treat analyses (95% CI, -4.2 to 6.2), results with sparfloxacin were statistically equivalent to those with ciprofloxacin (95% CI, -1 to 15.3) . For bacteriologically assessable patients, eradication rates were 87.0% (141/162) with sparfloxacin and 79.9% (123/154) with ciprofloxacin (95% CI, -1 to 15.3) . Eradication rates of S . aureus and coagulase-negative staphylococcal infections were 90.2% (101/112) with sparfloxacin and 77.9% (88/113) with ciprofloxacin . For patients with 2 or more pathogens at baseline (mixed infections), bacteriologic success was 87.6% for sparfloxacin and 77.9% for ciprofloxacin . Pseudomonas aeruginosa infections were eradicated or presumed eradicated in 71.4% (10/14) of sparfloxacin-treated patients and 87.5% (7/8) of ciprofloxacin-treated patients . Overall success rates in the bacteriologically assessable patients for sparfloxacin (84.6% {137/162}) and ciprofloxacin (78.6% {121/154}) were statistically equivalent (95% CI, -2.5 to 14.5) . Tolerability was assessed in all patients who received study medication . The overall frequency of treatment-related adverse events was comparable in the 2 treatment groups (26.5% sparfloxacin, 23.3% ciprofloxacin) . Drug-related adverse events involving the digestive system occurred in 7.1% of sparfloxacin-treated patients and 19.0% of ciprofloxacin-treated patients; photosensitivity reactions were reported in 11.1% of patients in the sparfloxacin group and 0.7% of patients in the ciprofloxacin group (P < 0.001) . The mean change in QTc interval from baseline to the maximum on-treatment value was greater in the sparfloxacin group (9 milliseconds) than in the ciprofloxacin group (3 milliseconds) (P = 0.005; 95% CI, 0.002 to 0.010) . The efficacy of sparfloxacin was comparable to that of ciprofloxacin in the treatment of community-acquired, complicated skin and skin-structure infections, including those caused by staphylococci, the most common pathogens . Sparfloxacin's once-daily regimen, high skin-tissue penetration, and improved activity against gram-positive cocci make it a therapeutic alternative to ciprofloxacin for patients who are not at risk for photosensitivity reactions or adverse events associated with prolongation of the QTc interval.

Conn Med, 1999 May, 63(5), 271 - 3
Community acquired Pseudomonas aeruginosa pneumonia; Vikram HR et al.; Pseudomonas aeruginosa is an uncommon cause of community acquired pneumonia in immunocompetent hosts . We report two cases that did well once appropriate and prolonged antimicrobial therapy was initiated . They had no evidence of immune deficiency . The initial consideration was pulmonary tuberculosis in both cases given the subacute presentation, significant weight loss, and findings on chest roentgenogram.

Am J Physiol, 1999 Jun, 276(6 Pt 1), L1037 - 45
Effects of a perfluorochemical emulsion on the fate of circulating Pseudomonas aeruginosa; Brain JD et al.; Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria . Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent . Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection . PFCE pretreatment decreased the rate and extent of vascular clearance of P . aeruginosa, with decreased uptake by the liver . Importantly, there were significant decreases in killing of P . aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals . PFCE did not alter the clearance of S . aureus from the circulation . However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake . Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization . These experiments demonstrate that the clearance kinetics and organ distribution of circulating P . aeruginosa and their subsequent killing are altered by PFCE . Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P . aeruginosa.

Am J Physiol, 1999 Jun, 276(6 Pt 1), L909 - 16
Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-kappaB expression; Leeper-Woodford SK et al.; Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury . Nuclear factor (NF)-kappaB is thought to regulate the expression of endotoxin {lipopolysaccharide (LPS)}-induced inflammatory cytokines such as TNF, and NF-kappaB may also be influenced by changes in O2 tension . It is thus proposed that acute changes in O2 tension surrounding AMs alter NF-kappaB activation and TNF secretion in these lung cells . AM-derived TNF secretion and NF-kappaB expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs . Adhered AMs (10(6)/ml) were incubated (37 degrees C at 5% CO2) for 2 h with LPS (Pseudomonas aeruginosa, 1 microgram/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2) . AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay . Electrophoretic mobility shift and supershift assays were used to determine NF-kappaB activation and to identify NF-kappaB isoforms in AM extracts . In addition, mRNAs for selected AM proteins were determined with RNase protection assays . LPS-exposed AMs in hypoxia had higher levels of TNF (P < 0.05) and enhanced expression of NF-kappaB (P < 0.05); the predominant isoforms were p65 and c-Rel . Increased mRNA bands for TNF-alpha, interleukin-1alpha, and interleukin-1beta were also observed in the hypoxic AMs . These results suggest that acute hypoxia in the lung may induce enhanced NF-kappaB activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.

FEMS Microbiol Lett, 1999 Jun 1, 175(1), 27 - 35
Characterisation of a chloramphenicol acetyltransferase determinant found in the chromosome of Pseudomonas aeruginosa; White PA et al.; The open reading frame (ORF) in the Pseudomonas aeruginosa chromosome, whose product resembles the chloramphenicol acetyltransferases (CAT) belonging to the CATB family, was cloned and shown to confer resistance to chloramphenicol (Cm) in Escherichia coli . The determinant was therefore named catB7 and the corresponding protein CATB7 . When the copy number and expression signals were identical, the catB7 gene conferred resistance to Cm at a level slightly lower than those of three other catB genes . CATB7 resembles other CATBs in that it acetylates Cm but not 1-acetoxy-Cm . For CATB7, the K(m) values for acetyl-CoA and Cm were 5.0-5.4-fold higher than the corresponding values for each of the three other CATB proteins (CATB1, CATB3 and CATB5) examined and the Vmax was 5-6 fold lower . Using PCR, the catB7 gene was found in all six P . aeruginosa strains examined but not in any other species of pseudomonad tested . Weak CAT activity was detected in crude cell extracts from five of the six P . aeruginosa strains . However, this activity did not correlate with the Cm susceptibility of the strains, indicating that catB7 is not likely to be the major determinant of intrinsic Cm resistance in P . aeruginosa.

Mol Microbiol, 1999 Jun, 32(5), 1054 - 64
ADP-ribosylation of oncogenic Ras proteins by pseudomonas aeruginosa exoenzyme S in vivo; Vincent TS et al.; The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388deltaexoS, were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells . Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria . Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively . ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras . The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts . Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing bacteria caused a decrease in the amount of Raf-1 associated with EGF-activated Ras and oncogenic Ras . The results from this study indicate that ExoS ADP-ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.

Biochemistry, 1999 Jun 8, 38(23), 7556 - 64
Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation; Wilson EK et al.; Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR) . We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively . We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1) . These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO . The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance . CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.

Anesthesiology, 1999 Jun, 90(6), 1650 - 62
The effects of two antiinflammatory pretreatments on bacterial-induced lung injury; Miyazaki H et al.; BACKGROUND: Two antiinflammatory therapies that have been effective in preventing acid-induced lung injury were evaluated . Specifically, their effects on a subsequent bacterial-airspace challenge were compared . Bacteria were instilled 24 h after acid-induced lung injury . Pseudomonas aeruginosa PAO-1 was used as the bacteria, because its effects in healthy lungs was documented previously . METHODS: New Zealand white rabbits were anesthetized and three pretreatments were administered: (1) pentoxifylline pretreatment (a 20-mg/kg bolus dose and then 6 mg x kg(-1) x h(-1) given intravenously), (2) 1 ml anti-tumor necrosis factor alpha antiserum given intravenously, or (3) normal saline given intravenously . The pretreatment doses were shown previously to prevent acid-induced lung injury . Then 1.2 ml/kg hydrochloric acid (HCl), pH 1.25, was instilled into the rabbits' right lungs . All the animals underwent mechanical ventilation for 8 h . Twenty-four hours after the acid instillation, the rabbits were anesthetized again and 2 ml/kg (10(9) colony forming units/ml) PAO-1 was instilled into their left lungs . The rabbits' breathing was aided by mechanical ventilation for another 8 h, and then they were killed and exsanguinated . RESULTS: Both pretreatments attenuated the acid-induced lung injury of the noninstilled left lungs . Arterial oxygen tension and the lung edema of pretreated, acid-exposed animals were significantly and almost equally improved (compared with no pretreatments) by either of the pretreatments . However, when the bacteria were instilled into the left lungs 24 h after the acid injury, the pentoxifylline pretreatment but not the anti-tumor necrosis factor alpha pretreatment prevented much of the bacteria-induced lung injury . Pentoxifylline pretreatment significantly improved the measurements of left lung edema and epithelial and endothelial permeability . There was also a trend for improved oxygenation in the pentoxifylline-pretreated and infected animals . In contrast, the anti-tumor necrosis factor alpha pretreatment did not prevent the bacteria-induced lung injury and increased some of the measurements of lung injury . CONCLUSIONS: Two antiinflammatory therapies that prevented acid-induced lung injury to the noninstilled left lungs had significantly different effects on a subsequent bacteria-induced lung injury to the left lungs . The therapies differed in their mechanism of tumor necrosis factor alpha blockade, and this may have affected the bacteria-induced injury to the lungs.

Biochem J, 1999 Jun 15, 340 ( Pt 3), 711 - 4
Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme; Farnaud S et al.; Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3 . Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding . Cys-166 is therefore implicated as the active-site nucleophile . Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue . Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32) . These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

J Biomater Sci Polym Ed, 1999, 10(5), 543 - 56
Polyelectroylte complex composed of chitosan and sodium alginate for wound dressing application; Kim HJ et al.; Drug-impregnated polyelectrolyte complex (PEC) sponge composed of chitosan and sodium alginate was prepared for wound dressing application . The morphological structure of this wound dressing was observed to be composed of a dense skin outer layer and a porous cross-section layer by scanning electron microscopy (SEM) . Equilibrium water content and release of silver sulfadiazine (AgSD) could be controlled by the number of repeated in situ PEC reactions between chitosan and sodium alginate . The release of AgSD from AgSD-impregnated PEC wound dressing in PBS buffer (PH = 7.4) was dependent on the number of repeated in situ complex formations for the wound dressing . The antibacterial capacity of AgSD-impregnated wound dressing was examined in agar plate against Pseudomonas aeruginosa and Staphylococcus aureus . From the behavior of antimicrobial release and the suppression of bacterial proliferation, it is thought that the PEC wound dressing containing antimicrobial agents could protect the wound surfaces from bacterial invasion and effectively suppress bacterial proliferation . In the cytotoxicity test, cellular damage was reduced by the controlled released of AgSD from the sponge matrix of AgSD-medicated wound dressing . In vivo tests showed that granulation tissue formation and wound contraction for the AgSD plus dihydroepiandrosterone (DHEA) impregnated PEC wound dressing were faster than any other groups.

Clin Rheumatol, 1999, 18(2), 167 - 9
Pseudomonas osteomyelitis of the symphysis pubis after inguinal hernia repair; Mader R et al.; Osteitis pubis (OP) is a term used to describe an entity characterised by severe pelvic pain, a wide-based gait and bony destruction of the margins of the pubic symphysis . It is usually assumed that OP is a non-infectious, self-limiting, relatively benign condition . Infectious osteomyelitis of the symphysis pubis (IOSP) is very unusual and the clinical presentation can resemble OP . IOSP following inguinal hernia repair is extremely rare . A case of IOSP caused by Pseudomonas aeruginosa is described . We reiterate the assumption that IOSP can be misdiagnosed as OP.

J Mol Biol, 1999 Jun 11, 289(3), 591 - 602
High resolution crystal structure of a Mg2+-dependent porphobilinogen synthase; Frankenberg N et al.; Common to the biosynthesis of all known tetrapyrroles is the condensation of two molecules of 5-aminolevulinic acid to the pyrrole porphobilinogen catalyzed by the enzyme porphobilinogen synthase (PBGS) . Two major classes of PBGS are known . Zn2+-dependent PBGSs are found in mammals, yeast and some bacteria including Escherichia coli, while Mg2+-dependent PBGSs are present mainly in plants and other bacteria . The crystal structure of the Mg2+-dependent PBGS from the human pathogen Pseudomonas aeruginosa in complex with the competitive inhibitor levulinic acid (LA) solved at 1.67 A resolution shows a homooctameric enzyme that consists of four asymmetric dimers . The monomers in each dimer differ from each other by having a "closed" and an "open" active site pocket . In the closed subunit, the active site is completely shielded from solvent by a well-defined lid that is partially disordered in the open subunit . A single molecule of LA binds to a mainly hydrophobic pocket in each monomer where it is covalently attached via a Schiff base to an active site lysine residue . Whereas no metal ions are found in the active site of both monomers, a single well-defined and highly hydrated Mg2+is present only in the closed form about 14 A away from the Schiff base forming nitrogen atom of the active site lysine . We conclude that the observed differences in the active sites of both monomers might be induced by Mg2+-binding to this remote site and propose a structure-based mechanism for this allosteric Mg2+in rate enhancement .

J Pediatr, 1999 Jun, 134(6), 734 - 9
Estimating effectiveness in an observational study: a case study of dornase alfa in cystic fibrosis . The Investigators and Coordinators of the Epidemiologic Study of Cystic Fibrosis; Johnson CA et al.; Patients with cystic fibrosis (CF) receiving dornase-alfa had improved pulmonary function relative to a control group in a large randomized phase III controlled study . We reviewed data from a large observational phase IV study to estimate the observed drug effect in patients receiving dornase alfa as part of their routine care . Patients 6 years or older and with a baseline forced expiratory volume in 1 second (FEV1) of at least 40% predicted who had been enrolled for at least 18 months were included (n = 283) . The control group consisted of 2382 patients who had never received dornase alfa . Patients in the study had a baseline spirometry and a second spirometry recorded 12 months later; a baseline observation period of 6 months preceded the initial spirometry, and dornase alfa had to have been started after the baseline spirometry (within 3 months) and to have continued through the 12-month follow-up spirometry . Patients treated with dornase alfa had lower pulmonary functions, more bacterial colonization, and more exacerbations at baseline (FEV1 : 76.0% vs 87.6%, Pseudomonas aeruginosa : 64.1% vs 46.7%, pulmonary exacerbations during the previous 6 months: 56.4% vs 22 . 2%) . Mean values of FEV1 for patients treated with dornase alfa improved by 3.9% of predicted compared with a decline of 1.6% in the untreated cohort . Covariate adjustment provided an estimated benefit of dornase alfa of 4.3% predicted FEV1 (SE = 0.9, P <.0001) . This analysis provides evidence for the effectiveness of dornase alfa therapy in clinical practice.

Undersea Hyperb Med, 1999 Spring, 26(1), 21 - 5
Effect of hyperbaric oxygen therapy in experimental subcutaneous and pulmonary infections due to Pseudomonas aeruginosa; Luongo C et al.; About 80% of nosocomial infections are caused by aerobic bacteria . Pseudomonas aeruginosa is a Gram-negative bacterium belonging to the Pseudomonadaceae family; P . aeruginosa is responsible for 6-22% of all hospital infections . The aim of this study was to evaluate the efficacy of hyperbaric oxygen (HBO2) therapy (2 atm abs x 55 min.day-1) alone for 8 days and combined with antibiotic chemotherapy (amikacin 15 mg.kg-1.day-1 for 8 days by intraperitoneal route) in rats infected subcutaneously and via the pulmonary route . In the rats infected by P . aeruginosa, HBO2 induced a reduction in mortality and morbidity with bacteria eradication in blood culture, bronchial aspirate, and skin biopsies when compared to control . These effects were increased by the use of amikacin, an antibiotic used for the treatment of sensitive Gram-negative bacteria.

J Antimicrob Chemother, 1999 Apr, 43(4), 517 - 22
Quality of antimicrobial susceptibility testing in the UK: a Pseudomonas aeruginosa survey revisited; Livermore DM et al.; As part of a programme to assess the usefulness of routine antimicrobial susceptibility data as a surveillance tool, we reviewed the results of a national survey of resistance in Pseudomonas aeruginosa, undertaken in 1993 . Twenty-four UK laboratories contributed isolates for centralized MIC testing, indicating also their own susceptibility test data . As reported previously (Chen et al . (1995) Journal of Antimicrobial Chemotherapy 35, 521-34), the rate of false resistance (isolates reported susceptible, but found resistant on MIC testing/all isolates reported susceptible) was 0.6-8%, according to the antimicrobial and breakpoint . Review showed that this favourable position reflected the fact that >88% of isolates were susceptible to any given antimicrobial and--in most cases--were correctly reported as such . Reporting was more erratic for resistant isolates: for beta-lactams and amikacin, isolates resistant at the highest MIC breakpoints were equally likely to be reported as 'susceptible' or 'resistant'; such misreporting was less common with ciprofloxacin and gentamicin but still occurred in 9-20% of cases . Conversely, up to 73% of the isolates reported as resistant proved to be susceptible at high breakpoints, and up to 44% were susceptible at low breakpoints . Miscategorizations did not reflect failure to detect particular mechanisms but, rather, the fact that MIC and zone breakpoints for P . aeruginosa serve to cut 'tails' of resistant organisms from continuous distributions, not to distinguish discrete populations . In this situation, some disagreement between routine tests and MICs is inevitable, but the frequency at which highly resistant isolates were reported as sensitive is disturbing . For surveillance, we conclude that resistance rates based on routine tests are unreliable for P . aeruginosa . This situation may improve with greater standardization of routine testing, but the continuous susceptibility distributions without discrete resistant and susceptible populations militate against perfect agreement . Despite these deficiencies, routine data should allow trend analysis.

J Antimicrob Chemother, 1999 Apr, 43(4), 483 - 90
Prediction of the antimicrobial effects of trovafloxacin and ciprofloxacin on staphylococci using an in-vitro dynamic model; Firsov AA et al.; To compare the pharmacodynamics of trovafloxacin and ciprofloxacin, three clinical isolates of Staphylococcus aureus with different MICs (0.03, 0.15, 0.6 and 0.1, 0.25, 1.25 mg/L, respectively) were exposed to decreasing concentrations of the quinolones according to their half-lives of 9.25 and 4 h, respectively . With each organism, single doses of trovafloxacin and twice-daily doses of ciprofloxacin were designed to provide 8-fold ranges of the ratio of area under the concentration-time curve (AUC) to the MIC, 58-466 and 116-932 (mg x h/L)/(mg/L), respectively . The antimicrobial effect was expressed by its intensity: the area between the control growth in the absence of antibiotics and the antibiotic-induced time-kill/regrowth curves (I(E)) . Linear relationships established between I(E) and log AUC/MIC were bacterial strain-independent but specific for the quinolones (r2 = 0.99 in both cases) . At a given AUC/MIC ratio, the I(E)s of trovafloxacin were greater than those of ciprofloxacin, suggesting that the antimicrobial effect of trovafloxacin compared with ciprofloxacin against staphylococci may be even greater than might be expected from the difference in their MICs . These data were combined with previous results obtained with three Gram-negative bacteria . Again, I(E) correlated well with the log AUC/MIC of trovafloxacin and ciprofloxacin in a strain- and species-independent fashion (r2 = 0.94 and 0.96, respectively) . On this basis, a value of the AUC/MIC of trovafloxacin which might be equivalent to Schentag's AUC/MIC = 125 (mg x h/L)/(mg/L) reported as the breakpoint value for ciprofloxacin was estimated at 71 (mg x h/L)/(mg/L) with the respective MIC breakpoint of 0.27 mg/L . Based on the I(E)-log AUC/MIC relationships, the I(E)s were plotted against the logarithm of trovafloxacin and ciprofloxacin dose (D) for hypothetical representatives of S . aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa with MICs corresponding to the MIC50s . These I(E)-log D relationships allow prediction of the effect of a given quinolone on a representative strain of the bacterial species.

Can J Microbiol, 1999 Jan, 45(1), 18 - 22
Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance; Li XZ et al.; Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants . Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P . aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system . Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants . These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.

Drug Dev Ind Pharm, 1999 Jun, 25(6), 781 - 8
Feasibility of an in vitro microbiological model as an alternative to the rabbit eye model; Devarajan PV et al.; Ocular inserts of gentamicin sulfate with polyvinyl alcohol (PVA) 1.5%, 2.0%, and 2.5% and a combination of methyl cellulose 2% and Eudragit NE 30D 30%, 35%, and 40% w/w of methyl cellulose were fabricated by a casting technique . The inserts were sterilized by gamma radiation at 25 kGy and tested for sterility . The microbiological efficacy of the ocular inserts against Staphylococcus aureus ATCC 6538P and Pseudomonas aeruginosa NCIM 2200 was evaluated by developing an in vitro microbiological model and an in vivo noninvasive rabbit eye model . Parameters of the in vitro microbiological model were varied, and the results correlated with a noninvasive rabbit eye model . The in vitro model proved to be a viable alternative to the rabbit eye model in evaluating the microbiological efficacy of gentamicin sulfate ocular inserts.

J Bacteriol, 1999 Jun, 181(11), 3478 - 85
Regulation of alginate biosynthesis in Pseudomonas syringae pv . syringae; Fakhr MK et al.; Both Pseudomonas aeruginosa and the phytopathogen P . syringae produce the exopolysaccharide alginate . However, the environmental signals that trigger alginate gene expression in P . syringae are different from those in P . aeruginosa with copper being a major signal in P . syringae . In P . aeruginosa, the alternate sigma factor encoded by algT (sigma22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway . In the present study, we cloned and characterized the gene encoding AlgR1 from P . syringae . The deduced amino acid sequence of AlgR1 from P . syringae showed 86% identity to its P . aeruginosa counterpart . Sequence analysis of the region flanking algR1 in P . syringae revealed the presence of argH, algZ, and hemC in an arrangement virtually identical to that reported in P . aeruginosa . An algR1 mutant, P . syringae FF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans . The algD promoter region in P . syringae (PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P . aeruginosa . Unlike P . aeruginosa, algR1 was not required for the transcription of algD in P . syringae, and PsalgD lacked the consensus sequence recognized by AlgR1 . However, both the algD and algR1 upstream regions in P . syringae contained the consensus sequence recognized by sigma22, suggesting that algT is required for transcription of both genes.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1379 - 82
Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents; Carmeli Y et al.; Pseudomonas aeruginosa is a leading cause of nosocomial infections . The risk of emergence of antibiotic resistance may vary with different antibiotic treatments . To compare the risks of emergence of resistance associated with four antipseudomonal agents, ciprofloxacin, ceftazidime, imipenem, and piperacillin, we conducted a cohort study, assessing relative risks for emergence of resistant P . aeruginosa in patients treated with any of these drugs . A total of 271 patients (followed for 3,810 days) with infections due to P . aeruginosa were treated with the study agents . Resistance emerged in 28 patients (10.2%) . Adjusted hazard ratios for the emergence of resistance were as follows: ceftazidime, 0.7 (P = 0.4); ciprofloxacin, 0.8 (P = 0.6); imipenem, 2.8 (P = 0.02); and piperacillin, 1.7 (P = 0.3) . Hazard ratios for emergence of resistance to each individual agent associated with treatment with the same agent were as follows: ceftazidime, 0.8 (P = 0.7); ciprofloxacin, 9.2 (P = 0.04); imipenem, 44 (P = 0.001); and piperacillin, 5.2 (P = 0.01) . We concluded that there were evident differences among antibiotics in the likelihood that their use would allow emergence of resistance in P . aeruginosa . Ceftazidime was associated with the lowest risk, and imipenem had the highest risk.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1362 - 6
OXA-17, a further extended-spectrum variant of OXA-10 beta-lactamase, isolated from Pseudomonas aeruginosa; Danel F et al.; Pseudomonas aeruginosa isolates 871 and 873 were isolated at Hacettepe University Hospital in Ankara and were highly resistant to ceftazidime (MIC, 128 microg/ml) . Each produced three beta-lactamases, with pIs of 5.3, 6.1, and 7.9 . The beta-lactamase with a pI of 5.3 was previously shown to be PER-1 enzyme . The antibiograms of the isolates were not entirely explained by production of PER-1 enzyme, insofar as ceftazidime resistance was incompletely reversed by clavulanate . The enzymes with pIs of 6.1 and 7.9 were therefore investigated . The enzyme with a pI of 6.1 proved to be a novel mutant of OXA-10, which we designated OXA-17, and had asparagine changed to serine at position 73 of the protein . When cloned into Escherichia coli XL1-blue, OXA-17 enzyme conferred greater resistance to cefotaxime, latamoxef, and cefepime than did OXA-10, but it had only a marginal (two- to fourfold) effect on the MIC of ceftazidime . This behavior contrasted with that of previous OXA-10 mutants, specifically OXA-11, -14, and -16, which predominately compromise ceftazidime . Extracted OXA-17 enzyme had relatively greater activity than OXA-10 against oxacillin, cloxacillin, and cefotaxime but, in terms of kcat/Km, it had lower catalytic efficiency against most beta-lactams . The enzyme with a pI of 7.9 was shown by gene sequencing to be OXA-2.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1340 - 6
Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa; Lomovskaya O et al.; Drug efflux pumps in Pseudomonas aeruginosa were evaluated as potential targets for antibacterial therapy . The potential effects of pump inhibition on susceptibility to fluoroquinolone antibiotics were studied with isogenic strains that overexpress or lack individual efflux pumps and that have various combinations of efflux- and target-mediated mutations . Deletions in three efflux pump operons were constructed . As expected, deletion of the MexAB-OprM efflux pump decreased resistance to fluoroquinolones in the wild-type P . aeruginosa (16-fold reduction for levofloxacin {LVX}) or in the strain that overexpressed mexAB-oprM operon (64-fold reduction for LVX) . In addition to that, resistance to LVX was significantly reduced even for the strains carrying target mutations (64-fold for strains for which LVX MICs were >4 microg/ml) . We also studied the frequencies of emergence of LVX-resistant variants from different deletion mutants and the wild-type strain . Deletion of individual pumps or pairs of the pumps did not significantly affect the frequency of emergence of resistant variants (at 4x the MIC for the wild-type strain) compared to that for the wild type (10(-6) to 10(-7)) . In the case of the strain with a triple deletion, the frequency of spontaneous mutants was undetectable (<10(-11)) . In summary, inhibition of drug efflux pumps would (i) significantly decrease the level of intrinsic resistance, (ii) reverse acquired resistance, and (iii) result in a decreased frequency of emergence of P . aeruginosa strains highly resistant to fluoroquinolones in clinical settings.

J Appl Microbiol, 1999 May, 86(5), 859 - 66
Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells; Mechin L et al.; The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of didecyldimethyl ammonium bromide (DDAB) was investigated . In this study, the strain was sub-cultured in a medium with increasing DDAB concentrations . After adaptation, Ps . aeruginosa was able to grow until the DDAB concentration in the medium was about five times greater than the Minimal Inhibitory Concentration . Resistance of cells to the bactericidal activity of DDAB also increased gradually during adaptation . This resistance was dependent on the presence of the biocide, as it quickly decreased when the cells were transferred to medium without biocide . Adapted cells showed changes in membrane fatty acid composition . The modifications mainly affected lauric, beta-hydroxylauric and palmitic acids, and they underlined the implication of the membranes in the cell response to the presence of the biocide . Simple linear regression analysis showed that the membrane fatty acid composition of Ps . aeruginosa played an important part in the resistance mechanisms of cells to the bactericidal activity of DDAB.

J Appl Microbiol, 1999 May, 86(5), 827 - 33
Disinfection of hospital waste sludge using hypochlorite and chlorine dioxide; Tsai CT et al.; Hypochlorite and chlorine dioxide were used to disinfect hospital waste-water sludge . Their abilities to inactivate pathogenic micro-organisms were compared . Reductions in indigenous coliform organisms and Pseudomonas aeruginosa were estimated . The results indicate that hypochlorite is a better disinfectant than chlorine dioxide for coliforms . Higher disinfection efficiency was obtained by treating a lower concentration of sludge . In addition, a higher agitation speed gave a higher disinfection efficiency with hypochlorite . The disinfection efficiencies of both disinfectants were higher against settled sludge than against thickened sludge . Therefore, it is recommended that disinfection should be performed on settled sludge rather than in a thickening tank.

Appl Environ Microbiol, 1999 Jun, 65(6), 2723 - 9
Molecular analysis of Pseudomonas aeruginosa: epidemiological investigation of mastitis outbreaks in Irish dairy herds; Daly M et al.; Pseudomonas aeruginosa is a pathogen in both humans and animals . This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds . Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism . Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P . aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak . Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital . With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes . Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains . However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns . Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array . The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates . Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods . These data support the view that the same P . aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.

Appl Environ Microbiol, 1999 Jun, 65(6), 2606 - 13
Characterization of Pseudomonas aeruginosa bacteriophage UNL-1, a bacterial virus with a novel UV-A-inducible DNA damage reactivation phenotype; Shaffer JJ et al.; UNL-1, a lytic virus of Pseudomonas aeruginosa, was observed to express a novel inducible DNA damage reactivation activity in UV-A-irradiated P . aeruginosa host cells . The expression of bacteriophage reactivation was quantified in hosts exposed to either UV-C or UV-A radiation . While reactivation of UV-C-damaged UNL-1 was not inducible in UV-C-irradiated host cells, an approximately 13-fold induction was observed in UV-A-irradiated host cells . When host cells were exposed to sunlight, reactivation of damaged UNL-1 virus increased eightfold . The UV-A induction of UNL-1 DNA damage reactivation was supported in hosts lacking recA gene function . This report is the first description of a recA-independent, UV-inducible virus DNA damage repair system . Our findings suggest that a combination of both host and virus DNA repair processes contribute to the persistence and sustained replication of some bacterial viruses in aquatic environments.

CLAO J, 1999 Apr, 25(2), 80 - 100
The relationship between contact lens oxygen permeability and binding of Pseudomonas aeruginosa to human corneal epithelial cells after overnight and extended wear; Ren DH et al.; PURPOSE: We designed a 3-year, prospective, randomized, masked clinical trial to evaluate the relationship of contact lens oxygen transmissibility and bacterial adherence to exfoliated surface epithelial cells in human overnight and extended lens wearers in a single center; corneal cell desquamation rate, surface epithelial cell size, and tear lactate dehydrogenase (LDH) levels were also determined concurrently . METHODS: One hundred nine human volunteers were successfully fit with test lenses prospectively and completed this study . Seven soft and three rigid gas permeable (RGP) lenses with stratified oxygen transmissibility were evaluated . After one week adaptation to daily wear, patients continually wore test lenses bilaterally for three months on a six nights wear, one night off basis . Before and after 24 hour, 1 month, and three months extended contact lens wear, exfoliated surface epithelial cells were collected using a modified corneal irrigation chamber . Bacterial binding was determined by measuring Pseudomonas aeruginosa (PA) adherence to exfoliated corneal epithelial cells . The number of exfoliated cells with adherent bacteria were counted using fluorescence microscopy . The effects of contact lens wear on the corneal surface were further assessed by alterations in tear LDH, and by surface epithelial cell size and epithelial thickness using in vivo tandem scanning confocal microscopy (TSCM) . Baseline values of outcome measures served as controls for individual patients; a concurrent group of controls were also followed to monitor seasonal or possible individual fluctuations . RESULTS: Quantitative evidence demonstrated that lens physical oxygen transmissibility properties and not lens type significantly correlated inversely with binding of PA to human exfoliated corneal epithelial cells after overnight and extended wear (R=0.258, P=0.0084); there was a significant decrease in surface epithelial cell desquamation and a significant increase in surface cell size following wear for all test lenses (P<0.05) . Epithelial thinning was also observed following lens wear (P<0.05) . CONCLUSIONS: These results establish for the first time a significant correlation between contact lens-induced increases in epithelial PA binding and lens oxygen transmissibility in humans . New ultra-oxygen permeable test lenses did not appear to increase bacterial binding over individual control levels; all test lenses suppressed surface epithelial cell shedding . Taken together, these findings suggest that a new generation of contact lenses constructed from ultra-transmissible oxygen materials may offer a significant potential advance in safety for extended wear.

J Antibiot (Tokyo), 1999 Feb, 52(2), 142 - 9
Structure-activity relationships of carbapenems to the antagonism of the antipseudomonal activity of other beta-lactam agents and to the beta-lactamase inducibility in Pseudomonas aeruginosa: effects of 1beta-methyl group and C-2 side chain; Kanazawa K et al.; The antagonism of the antipseudomonal activity of ceftazidime by meropenem (1a) was much less than those by imipenem (2a) and panipenem (2b) . To reveal the major structural features of carbapenem compounds responsible for the antagonism, we investigated the structure-activity relationships of carbapenems to their antagonism of the antipseudomonal activity of ceftazidime and to their beta-lactamase-inducibility in P . aeruginosa . The antagonistic effect of 1a was less than that of desmethyl-meropenem (1b) . Two other meropenem-analogues (3, 4), with the highly basic C-2 side chain, showed greater antagonistic effects than that of 1a, which has a weakly basic C-2 side chain . The beta-lactamase-inducibility of 1a in P . aeruginosa was lower than those of 2a, 1b and 4 . These results indicated that the antagonism of the antipseudomonal activity of ceftazidime by carbapenems was due to the induction of beta-lactamase in P . aeruginosa . As a result of the study on the structure-activity relationships, we clarified that the introduction of a 1beta-methyl group and/or the reduction of the basicity (cationic character) of the C-2 side chain in carbapenem skeleton decreased the antagonistic effect of carbapenems on the antipseudomonal activity of ceftazidime resulted mainly from the decreasing the beta-lactamase inducibility.

CLAO J, 1999 Apr, 25(2), 73 - 9
Short-term hypoxia downregulates epithelial cell desquamation in vivo, but does not increase Pseudomonas aeruginosa adherence to exfoliated human corneal epithelial cells; Ren DH et al.; PURPOSE: This study evaluates the effect of hypoxic and hypercapnic stress on bacterial adherence to surface corneal epithelial cells, as well as tear LDH levels, surface cell desquamation, and corneal swelling in normal human subjects . METHODS: Sixteen eyes of eight human volunteers were successively exposed to three gas mixtures (air, 100% N2, 95% N2-5% CO2) through tightly fitted goggles for six hours at two-week intervals . Exfoliated epithelial cells were collected and counted using a modified corneal irrigation chamber . Bacterial binding was determined by measuring Pseudomonas aeruginosa (PA) adherence to exfoliated corneal epithelial cells . The effects of hypoxic or hypercapnic stress on the corneal surface were also assessed by tear LDH measurement, and quantification of surface epithelial cell size and epithelial and stromal thickness were determined by in vivo confocal microscopy . RESULTS: Short-term precorneal hypoxia significantly decreased corneal epithelial cell desquamation . Both short-term hypoxia alone and combined with hypercapnia induced significant corneal stromal swelling (7 to 8%) but did not significantly enhance PA adherence to exfoliated human corneal epithelial cells . CONCLUSIONS: This study demonstrates, for the first time, that short-term precorneal hypoxia downregulates corneal epithelial cell desquamation in humans . These results also demonstrate that short-term hypoxia alone or combined with hypercapnia does not significantly increase PA adherence to exfoliated epithelial cells from the human cornea . The results reveal that either longer hypoxic exposure or other interactive factor(s), including but not limited to the mechanical effect of the contact lens itself, may be required for promotion of increased epithelial cell-PA binding following lens wear in humans.

Thorax, 1999 Jan, 54(1), 44 - 50
Specific IgG subclass antibody pattern to Aspergillus fumigatus in patients with cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA); Skov M et al.; BACKGROUND: IgG and IgG subclass antibodies to Aspergillus fumigatus (A fumigatus) were measured in a large population of patients with cystic fibrosis to elucidate a putative antibody pattern specific for allergic bronchopulmonary aspergillosis (ABPA) . METHODS: An ELISA technique using water soluble somatic hyphal (WSSH) A fumigatus antigens and subclass specific monoclonal antibodies was used for cross sectional quantification of IgG and IgG1-4 subclass antibody levels in the serum of 238 patients with cystic fibrosis and 107 healthy controls . RESULTS: In patients with cystic fibrosis persistently colonised with A fumigatus the subclass antibody levels were significantly increased compared with patients with cystic fibrosis never or rarely colonised (p < 0.001) . The group of patients persistently colonised with A fumigatus with ABPA (+Af+ABPA) had significantly increased levels of IgG antibodies to A fumigatus (Af-IgG) (median 69 ELISA units (EU) versus 31) and of subclasses Af-IgG1 (91 versus 27), Af-IgG2 (143 versus 56), and Af-IgG4 antibodies (72 versus 20), but not of IgG3 (17 versus 15), compared with the colonised patients without ABPA (+Af-ABPA) . Patients with cystic fibrosis with no or only rare isolates of A fumigatus without ABPA (-Af-ABPA) also had significantly increased subclass antibody levels (Af-IgG1 9 versus 3, Af-IgG2 28 versus 5, Af-IgG4 16 versus 4; p < 0.001) compared with healthy controls . Low, although detectable, levels of antibodies were demonstrated in healthy controls . ABPA seemed to occur independently of Pseudomonas aeruginosa infection . Using diagnostic cut off levels for ABPA, sensitivity and specificity were calculated . The highest specificity was found for IgG4 (88%); sensitivity was between 65% and 73% . The positive predictive values (PPV) were moderate, whereas the negative predictive values (NPV) were high (96% in all subclasses except IgG3 with 94%) . PPV increased to 50% if IgG1 as well as IgG2 and IgG4 were included . CONCLUSIONS: In a large number of unselected patients with cystic fibrosis significantly increased levels of Af-specific antibodies belonging to total IgG and all four subclasses were found in all groups of patients compared with healthy controls . In patients persistently colonised with A fumigatus these levels were significantly higher than in non-colonised patients, and the significantly highest levels (with the exception of IgG3) were found in patients with ABPA . Using a sensitive ELISA technique, measurements of IgG and IgG subclass antibodies to A fumigatus might be of importance in the management of ABPA, especially as a screening test to exclude the presence of ABPA; other tests are needed to confirm the diagnosis.

Appl Microbiol Biotechnol, 1999 Apr, 51(4), 498 - 503
Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane; Garnier PM et al.; A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane . P . aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source . The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane) . An inhibition model for P . aeruginosa grown on pentane was proposed . Pentane had an inhibitory effect on growth of P . aeruginosa, even at a concentration as low as 85 micrograms/l . This resulted in the calculation of the following kinetic parameters (mumax = 0.19 h-1, Ks = 2.9 micrograms/l, Ki = 3.5 mg/l) . Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane . This model depends only on two parameters: the concentrations of pentane and MTBE.

Am J Otol, 1999 May, 20(3), 369 - 72
Delayed intracranial abscess after acoustic neuroma surgery: a report of two cases; Staecker H et al.; OBJECTIVE: The use of antibiotics before and after surgery has made infectious complications of neurotologic surgery rare . The neurosurgical literature cites a rate of postoperative meningitis between 1% and 2% for "clean" cases and 1.5% to 2.5% for "clean contaminated" cases, such as cerebrospinal fluid contact with the middle ear or mastoid . Reports of infections after neurotologic procedures are rare in the otologic literature . In this report, two patients with brain abscess occurring in a delayed fashion after surgery are described . STUDY DESIGN: The study design was a retrospective chart review and case report . SETTING: The study was conducted at a tertiary referral center . RESULTS: Patient 1 underwent a suboccipital craniotomy for removal of an acoustic neuroma and had an uneventful postoperative recovery . Three months after surgery, he reported mild unsteadiness . Examination revealed mild ataxia, which led to repeat magnetic resonance imaging (MRI) and a diagnosis of cerebellar abscess . Patient 2 underwent translabyrinthine removal of an acoustic neuroma complicated by postoperative Pseudomonas aeruginosa meningitis, which responded promptly to intravenous antibiotics . Fifteen months after surgery, he visited a neurologist after having a seizure and was treated with anticonvulsants . After a second episode of seizure, imaging studies showed a temporal lobe abscess . CONCLUSIONS: The signs of intracranial abscess may be subtle and can occur weeks or months after surgery, requiring vigilance and a high index of suspicion for diagnosis . A change in postoperative symptoms after acoustic neuroma surgery should signal further investigation using MRI with gadolinium.

J Neuroimmunol, 1999 May 3, 96(2), 182 - 9
Dynamic norepinephrine alterations in bone marrow: evidence of functional innervation; Tang Y et al.; Efferent sympathetic nerve activity has been hypothesized to regulate the proliferation and maturation of leukocytes in the bone marrow . Although there is histological evidence for bone marrow innervation and documentation of measurable neurotransmitter, functional activation of these nerves to external stimulation has never been demonstrated . The present study was designed to assess the dynamics of norepinephrine (NE) release in bone marrow in response to well-established protocols known to elevate sympathetic activity . Toward this end, norepinephrine turnover was measured using isotopic and non-isotopic methods in mice in response to cold exposure and bacterial challenge . Cold exposure increased NE turnover rate in bone marrow by 36% from 0.33 to 0.45 ng g(-1) h(-1), while peritoneal Pseudomonas aeruginosa infection increased bone marrow NE turnover rate by 131% from 0.13 to 0.30 ng g(-1) h(-1) . These results demonstrate that the adrenergic innervation of the bone marrow is functionally dynamic and is responsive to generalized stress . Furthermore, these results lend credence to the premise that neural mechanisms participate in regulation of lympho- and myelopoietic cellular events.

Infect Immun, 1999 Jun, 67(6), 3151 - 4
Macrophages and epithelial cells respond differently to the Pseudomonas aeruginosa type III secretion system; Coburn J et al.; The multiple effects of Pseudomonas aeruginosa type III secretion have largely been attributed to variations in cytotoxin expression between strains . Here we show that the target cell type is also important . While lung epithelial cells showed significant changes in morphology but not viability when infected with P . aeruginosa, macrophages were efficiently killed by P . aeruginosa . Both responses were dependent on the type III secretion system.

Infect Immun, 1999 Jun, 67(6), 2847 - 54
Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S; Olson JC et al.; Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact . Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure . To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P . aeruginosa 388 and the non-ExoS-producing strain 388DeltaS . Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388DeltaS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388 . Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras . ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement . Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras . While microvillus effacement was detected in both 388- and 388DeltaS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388 . We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras . The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P . aeruginosa infectious process by rendering cells nonfunctional.

Microbiol Immunol, 1999, 43(3), 297 - 301
Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa; Okamoto K et al.; We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1 . The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602 . Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P . aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin . Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.

J Biol Chem, 1999 May 28, 274(22), 15646 - 54
A fluorescence investigation of the active site of Pseudomonas aeruginosa exotoxin A; Beattie BK et al.; Single tryptophan mutant proteins of a catalytically active domain III recombinant protein (PE24) from Pseudomonas aeruginosa exotoxin A were prepared by site-directed mutagenesis . The binding of the dinucleotide substrate, NAD+, to the PE24 active site was studied by exploiting intrinsic tryptophan fluorescence for the wild-type, single Trp, and tryptophan-deficient mutant proteins . Various approaches were used to study the substrate binding process, including dynamic quenching, CD spectroscopy, steady-state fluorescence emission analysis, NAD+-glycohydrolase activity, NAD+ binding analysis, protein denaturation experiments, fluorescence lifetime analysis, steady-state anisotropy measurement, stopped flow fluorescence spectroscopy, and quantum yield determination . It was found that the conservative replacement of tryptophan residues with phenylalanine had little or no effect on the folded stability and enzyme activity of the PE24 protein . Dynamic quenching experiments indicated that when bound to the active site of the enzyme, the NAD+ substrate protected Trp-558 from solvent to a large extent but had no effect on the degree of solvent exposure for tryptophans 417 and 466 . Also, upon substrate binding, the anisotropy of the Trp-417(W466F/W558F) protein showed the largest increase, followed by Trp-466(W417F/W558F), and there was no effect on Trp-558(W417F/W466F) . Furthermore, the intrinsic tryptophan fluorescence exhibited the highest degree of substrate-induced quenching for the wild-type protein, followed in decreasing order by Trp-417(W466F/W558F), Trp-558(W417F/W466F), and Trp-466(W417F/W558F) . These data provide evidence for a structural rearrangement in the enzyme domain near Trp-417 invoked by the binding of the NAD+ substrate.

Ann Otol Rhinol Laryngol, 1999 May, 108(5), 440 - 5
Antibiotics in chronic suppurative otitis media: a bacteriologic study; Indudharan R et al.; Conservative medical management of chronic suppurative otitis media (CSOM) is an important step in achieving a dry ear . Topical antibiotic ear drops and aural toilet form the mainstay of medical management of noncholesteatomatous CSOM . This study analyzes the causal organisms and their sensitivity to various antibiotics . Out of 382 swabs examined, the major organisms isolated were Pseudomonas aeruginosa (27.2%), followed by Staphylococcus aureus (23.6%) . The sensitivity of P . aeruginosa was 100% to ceftazidime, 98.9% to ciprofloxacin, 96.3% to gentamicin, and 95.4% to polymyxin B, whereas the sensitivity of S . aureus was 98.6% to ciprofloxacin, 97.4% to cloxacillin sodium, 96.5% to cotrimoxazole, and 90.7% to gentamicin . Pseudomonas aeruginosa was almost completely resistant to ampicillin (97.6%) and chloramphenicol (96.6%), whereas S . aureus was almost completely resistant to ampicillin (73.8%) and polymyxin B (98.3%) . Among the available topical antibiotic preparations for use in the ear, we found that ciprofloxacin and gentamicin are the best choices.

Arch Intern Med, 1999 May 24, 159(10), 1127 - 32
Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa; Carmeli Y et al.; BACKGROUND: Antimicrobial resistance is an increasing problem . OBJECTIVE: To examine the clinical and economic impact of antibiotic resistance in Pseudomonas aeruginosa . METHODS: In-hospital mortality, secondary bacteremia, length of stay, and hospital charges were examined in a cohort of 489 inpatients with positive clinical cultures for P aeruginosa . One hundred forty-four had a resistant baseline P aeruginosa isolate and 30 had resistance emerge during follow-up . Multivariable and survival analytic methods were used to adjust for confounding and effects of time . RESULTS: The overall in-hospital mortality rate was 7.6%, 7.7% in patients with a resistant isolate at baseline (relative risk {RR}, 1.3; 95% confidence interval {CI}, 0.6-2.8) and 27% in patients in whom resistance emerged (RR, 3.0; 95% CI, 1.2-7.8) . Secondary bacteremia developed in 1.4% of patients in whom resistance did not emerge and in 14% of those in whom resistance emerged (RR, 9.0; 95% CI, 2.7-30) . The median duration of hospital stay following the initial P aeruginosa isolate was 7 days . Emergence of resistance, but not baseline resistance, was significantly associated with a longer hospital stay (P<.001 and P=.71, respectively) . The average daily hospital charge was $2059 . Neither baseline resistance nor emergence of resistance had a significant effect on the daily hospital charge . In a matched cohort analysis, a trend was seen toward increased total charges in patients demonstrating emergence of resistance (difference, $7340; P=.14) . CONCLUSIONS: Emergence of antibiotic resistance in P aeruginosa results in severe adverse outcomes . Efforts should be directed toward early detection and prevention of emergence of antibiotic resistance.

J Med Microbiol, 1999 Mar, 48(3), 309 - 15
Relationship between morphological changes and endotoxin release induced by carbapenems in Pseudomonas aeruginosa; Horii T et al.; The relationship between morphological changes and endotoxin release induced in vitro by carbapenems in a clinical isolate of Pseudomonas aeruginosa was examined . The time-course and magnitude of endotoxin release induced varied among imipenem, panipenem, meropenem and biapenem and related to the morphological changes caused by these agents which variously affected cell shape, cell-wall disintegration and cell lysis . The amount of endotoxin released by carbapenem-treated cells correlated with both the cell-wall morphology and bacterial shape immediately before lysis . Meropenem and biapenem caused markedly increased endotoxin release during cell lysis and cell-wall disintegration, whereas imipenem and panipenem caused much less release of endotoxin.

Pharmacotherapy, 1999 May, 19(5), 620 - 6
A pilot study of the efficacy of constant-infusion ceftazidime in the treatment of endobronchial infections in adults with cystic fibrosis; Bosso JA et al.; STUDY OBJECTIVE: To compare the efficacy of constant-infusion ceftazidime (CTZ) with that of traditional intermittent dosing in a pilot trial . DESIGN: Prospective, crossover trial . SUBJECTS: Five adults with cystic fibrosis requiring intravenous antibiotic therapy for pulmonary exacerbations of the disease . INTERVENTIONS: Patients were initially treated with standard CTZ 2 g 3 times/day for 10 days . At the next hospitalization patients were crossed over and CTZ was administered as a constant infusion at a rate determined to achieve a serum concentration 6.6 times the minimum inhibitory concentration (MIC) of the least susceptible Pseudomonas aeruginosa isolate . MEASUREMENTS AND MAIN RESULTS: The pharmacokinetics of CTZ were determined, as were MICs for all P . aeruginosa isolates . Outcome parameters of interest were changes with therapy in white blood cell count, P aeruginosa density in sputum, and pulmonary function test results . Differences in these parameters for the two forms of administration were not significant . With the exception of one patient who received 6 g/day with both regimens, the average reduction in dosage with the constant infusion was 50% . CONCLUSION: These preliminary data suggest that constant-infusion CTZ may be as safe and efficacious as intermittent dosing.

Am J Physiol, 1999 May, 276(5 Pt 1), L715 - 27
Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa; Skerrett SJ et al.; To determine the roles of the type 1 tumor necrosis factor (TNF) receptor (TNFR1) in lung inflammation and antibacterial defense, we exposed transgenic mice lacking TNFR1 {TNFR1(-/-)} and wild-type control mice to aerosolized lipopolysaccharide or Pseudomonas aeruginosa . After LPS, bronchoalveolar lavage fluid (BALF) from TNFR1(-/-) mice contained fewer neutrophils and less macrophage inflammatory protein-2 than BALF from control mice . TNF-alpha, interleukin-1beta, and total protein levels in BALF as well as tissue intercellular adhesion molecule-1 expression did not differ between the two groups . In contrast, lung inflammation and bacterial clearance after infection were augmented in TNFR1(-/-) mice . BALF from infected TNFR1(-/-) mice contained more neutrophils and TNF-alpha and less interleukin-1beta and macrophage inflammatory protein-2 than that from control mice, but protein levels were similarly elevated in both groups . Lung inflammation and bacterial clearance were also augmented in mice lacking both TNF receptors . Thus TNFR1 facilitates neutrophil recruitment after inhalation of lipopolysaccharide, in part by augmenting chemokine induction . In contrast, TNFR1 attenuates lung inflammation in response to live bacteria but does not contribute to increased lung permeability and is not required for the elimination of P . aeruginosa.

Cytokine, 1999 May, 11(5), 366 - 72
Paradoxical synergistic effects of tumour necrosis factor and interleukin 1 in murine gut-derived sepsis with Pseudomonas aeruginosa; Matsumoto T et al.; The authors evaluated the synergistic effect of tumour necrosis factor (TNF) and interleukin 1 (IL-1) in gut-derived sepsis in mice . After colonization of Pseudomonas aeruginosa strain D4 in the gastrointestinal tract, cyclophosphamide was administered to induce bacterial translocation of the P . aeruginosa and thereby to cause gut-derived sepsis . In this model, treatment either with 8 microg/kg of recombinant human TNF-alpha (rhTNF-alpha) or 2 microg/kg of recombinant human interleukin 1alpha (rhIL-1alpha) solely did not affect the mortality, whereas combined administration of the same doses of rhTNF-alpha and rhIL-1alpha significantly increased the mortality rate in comparison with saline-treated mice . Bacterial counts in liver and blood were significantly higher in rhTNF-alpha and rhIL-1alpha treated mice than in saline-treated mice . Endogenous TNF-alpha and IL-1beta productions were stimulated after combined treatment with rhTNF-alpha and rhIL-1alpha . On the contrary to these adverse effects, combined treatment with 500 microg/kg of rhTNF-alpha and 50 microg/kg of rhIL-1alpha on the day before the administration of cyclophosphamide significantly reduced the mortality from septic infection . We conclude that TNF and IL-1 synergistically affect the mortality of mice after gut-derived sepsis due to P . aeruginosa in mice and the timing of treatment with these cytokines causes both extremes in their effects .

Thorax, 1999 Feb, 54(2), 141 - 4
Avidity of anti-P aeruginosa antibodies during chronic infection in patients with cystic fibrosis; Ciofu O et al.; BACKGROUND: In order to study the impact on the lung function of patients with cystic fibrosis of the avidity of antipseudomonal antibodies, the avidity of antibodies against the chromosomal beta-lactamase of Pseudomonas aeruginosa (a beta ab) and against the 60-65 kDa heat shock protein of P aeruginosa (anti-GroEL) were measured in serum samples collected longitudinally during chronic infection with P aeruginosa from a group of patients with poor and good lung function . METHODS: The thiocyanate elution method in which the molarity of potassium thiocyanate required to elute 50% bound antibody under conditions of antigen excess in ELISA was used to measure the relative avidity . RESULTS: All patients developed increasing levels of a beta ab and anti-GroEL antibodies during the follow up period but no maturation of the avidity of these antibodies was observed . In patients with good lung function the avidity of a beta ab was higher than in patients with poor lung function (p = 0.018) . No significant difference in the avidity of the anti-GroEL antibodies was observed between the two groups of patients . CONCLUSION: In patients with cystic fibrosis a high avidity of a beta ab could contribute to a more efficient inhibition of the beta-lactamase by these antibodies, resulting in the better lung function seen in this group . The immunopathological implication of the failure in avidity maturation of antibodies in chronic infection is discussed.

Arch Dis Child, 1999 Feb, 80(2), 125 - 31
Once daily ceftriaxone and gentamicin for the treatment of febrile neutropenia; Tomlinson RJ et al.; AIMS: To evaluate the pharmacokinetics of once daily (OD) gentamicin and its effectiveness as part of an OD regimen for the empirical treatment of febrile neutropenia in children with cancer . SUBJECTS: 59 children aged 6 months to 16 years (mean (SD) 5.7 (4) years) with febrile neutropenia (neutrophil count < 0.5 x 10(9)/l) after chemotherapy . METHODS: Over one year, 113 febrile neutropenic episodes were treated empirically with an OD antibiotic regimen of ceftriaxone (80 mg/kg; maximum 4 g) and gentamicin (7 mg/kg; infused over 60 minutes, no maximum) . The patients were assessed after 48 hours . RESULTS: 86 of the 113 episodes settled with the first line antibiotic regimen . In 29 episodes, blood cultures identified a causative bacterial pathogen; for 17 of these, the first line antibiotic regimen was adequate; in four episodes, although the episode settled, ceftriaxone was replaced by a more appropriate antibiotic and OD gentamicin was continued; in the remaining eight episodes, a glycopeptide antibiotic was deemed necessary . There was no failure of treatment in organisms sensitive to gentamicin, including Pseudomonas aeruginosa . In 27 episodes (24%), resolution was obtained by the empirical introduction of a second line regimen of ceftazidime and a glycopeptide antibiotic, and/or amphotericin . Gentamicin concentrations were measured in 110 episodes and they were all below the 24 hour line indicating that there was no need to change the dosing interval . In two episodes (2%), serum creatinine rose transiently by more than 50% of the baseline concentration . Although there was no vestibular toxicity, three of 30 children who underwent pure tone audiometry reported high frequency hearing loss in one ear . CONCLUSION: OD gentamicin can be used safely and effectively to treat febrile neutropenia in children with cancer . When used for a short period (< 5 days), in children not receiving other nephrotoxic drugs and who have normal serum creatinine, serum gentamicin estimations are unnecessary.

J Clin Microbiol, 1999 Jun, 37(6), 2071 - 3
Epidemiological analysis of sequential Pseudomonas aeruginosa isolates from chronic bronchiectasis patients without cystic fibrosis; Pujana I et al.; PCR fingerprinting was used for the epidemiological investigation of 64 Pseudomonas aeruginosa isolates collected from 16 chronic bronchiectasis patients without cystic fibrosis: 56% of the patients harbored one clone, 12.5% carried a single major type with minor variants, and 31.5% carried two clones . Only a minority of the acquisitions of antibiotic resistance was related to the acquisition of exogenous strains . Mucoid and nonmucoid sets of isolates did not display any consistent differences in their patterns . The genetic similarity among the clones ranged from 10 to 69% . Cross-infection or common-source exposure did not appear to have occurred.

Burns, 1999 May, 25(3), 237 - 41
The efficacy of 5% Sulfamylon solution for the treatment of contaminated explanted human meshed skin grafts; Maggi SP et al.; Large TBSA burns have a deficiency of skin graft donor sites necessitating meshed skin autografts, cultured epithelial autografts or biosynthetic skin substitutes . Because these do not effect immediate complete biological closure of the wound, the burn victim remains at risk for life-threatening infection . Topical antimicrobials can protect colonization of these grafts from becoming invasive sepsis . However, many of these agents are cytotoxic to new partially keratinized epithelial cells . This study using a model of epithelialization kinetics of human meshed skin grafts explanted to athymic 'nude' rats evaluated: (1) the effect of bacterial colonization on the rate of closure of meshed graft interstices; (2) the efficacy of 5% Sulfamylon solution for bacterial control and (3) the effect on interstitial closure rates caused by control of bacterial proliferation . Results showed the rate of interstitial closure was progressive over 7 days in noncontaminated grafts treated with moistened saline dressings . Areas of total closure of a 1:1.5 meshed graft were seen as early as 5 days . When grafts were inoculated with 10(2) or 10(3) Pseudomonas aeruginosa organisms and treated with saline moistened dressings, the resultant bacterial load rose to 10(6) organisms, less than 3% of the interstices closed and grafts were destroyed . With the same organism level of contamination, bacterial levels were eradicated with topical 5% Sulfamylon solution, interstitial closure rates returned to normal and areas of total meshed graft closure were seen by day 4 . These data demonstrate the efficacy of 5% Sulfamylon solution on epithelialization kinetics of contaminated meshed skin grafts.

New Microbiol, 1999 Apr, 22(2), 85 - 9
Pseudomonas aeruginosa antibody detection in cystic fibrosis patients; Trancassini M et al.; Chronic respiratory infection due to Pseudomonas aeruginosa remains the most important prognostic factor in cystic fibrosis patients . One method to lengthen the patient's life is to extend the initial state of the illness with an early diagnosis, before Ps . aeruginosa infection becomes chronic . Often this is difficult because of the young age of the patients . This study tested an immunoenzymatic system to evaluate antibody response against three Ps . aeruginosa purified antigens, alkaline protease, elastase and exotoxin A . We studied 40 patients with cystic fibrosis, 20 affected and 20 unaffected by apparent Ps . aeruginosa infection, also from the bacteriological point of view . Serological and bacteriological results were compared for each patient and showed that serological screening can be useful in young subjects, who often have no bacteriological evidence of Ps . aeruginosa colonization.

Przegl Epidemiol, 1998, 52(4), 427 - 40
{The use of molecular biology in the modeling of Pseudomonas aeruginosa strains recovered from nosocomial infections}; Fiett J et al.; Different methods of molecular typing (ribotyping, genomic DNA RFLP and RAPD) were tested on Pseudomonas aeruginosa strains isolated in Polish hospitals in order to elaborate a reliable typing scheme for epidemiological investigations . The combined RAPD analysis with the use of two different primers, RAPD-4 and RAPD-7, was found to have the highest discriminatory power which considering also the easiness and low time-consumption has suggested its high usefulness in studies of outbreaks caused by P . aeruginosa . Ribotyping was shown to be the least discriminatory, however, especially with the use of the PvuII restriction enzyme, this method can be very useful in revealing the genetic structure of P . aeruginosa populations persisting in hospital environments over longer periods . Clonal relations within populations of strains isolated in four different hospitals were revealed . In two of the hospitals P . aeruginosa populations demonstrated a very high diversity which suggested that infections had been caused by strains of different origins, probably introduced from other environments . P . aeruginosa strains from two remaining hospitals were found to form some clonally related clusters what revealed that in these hospitals epidemic strains of this microorganism have been circulating for prolonged periods and infecting predisposed patients.

Thorax, 1998 Sep, 53(9), 732 - 7
Long-term follow up of changes in FEV1 and treatment intensity during Pseudomonas aeruginosa colonisation in patients with cystic fibrosis; Ballmann M et al.; BACKGROUND: Colonisation with Pseudomonas aeruginosa (PA) is a striking feature of lung involvement in cystic fibrosis . To identify the clinical consequences of the different steps of colonisation with PA under a defined therapeutic regime (no prophylactic antibiotic treatment as long as patients had no severe pulmonary disease), their influence on pulmonary function and on therapeutic intensity was examined . METHODS: Forty patients with cystic fibrosis were followed from first detection of PA (PA1), chronic PA colonisation (PAc), first mucoid PA detection (PAm), to chronic mucoid PA colonisation (PAcm) . Percentage predicted forced expiratory volume in one second (FEV1), the number of intravenous antibiotic treatment courses, and the percentage of patients on inhaled antibiotics were followed retrospectively and longitudinally in relation to the different steps of PA colonisation . The annual changes in FEV1 and therapeutic intensity in the two years preceding each step were compared with the two years following each step . Changes in FEV1 were related to therapeutic intensity . RESULTS: The mean (SD) annual changes in FEV1 (% predicted) worsened significantly only with the transition to the mucoid stages (PAm: 4.6 (13.2) versus -4.3 (8.1); PAcm: 7.3 (12.0) versus -4.8 (7.4)) with a mean difference (95% CI) between before and after the transition of 8.9 (2.6 to 15.2) for PAm and 12.1 (6.4 to 17.6) for PAcm . With non-mucoid PA stages the therapeutic intensity increased in the year of transition and with mucoid PA stages it increased in the years following transition . Therapeutic intensity was unrelated to changes in FEV1 . CONCLUSION: With the treatment regime used an accelerated decrease in FEV1 was successfully prevented in the non-mucoid stages but not in the mucoid stages of PA colonisation.

Blood, 1999 May 15, 93(10), 3467 - 72
Additive effects of human recombinant interleukin-11 and granulocyte colony-stimulating factor in experimental gram-negative sepsis; Opal SM et al.; Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used to promote granulocyte recovery from a variety of pathologic states . Recombinant human interleukin-11 (rhIL-11) has recently become available clinically as a platelet restorative agent after myelosuppressive chemotherapy . Preclinical data has shown that rhIL-11 limits mucosal injury after chemotherapy and attenuates the proinflammatory cytokine response . The potential efficacy of combination therapy with recombinant human forms of rhIL-11 and rhG-CSF was studied in a neutropenic rat model of Pseudomonas aeruginosa sepsis . At the onset of neutropenia, animals were randomly assigned to receive either rhG-CSF at a dose of 200 micrograms/kg subcutaneously every 24 hours for 7 days; rhIL-11 at 200 micrograms/kg subcutaneously every 24 hours for 7 days; the combination of both rhG-CSF and rhIL-11; or saline control . Animals were orally colonized with Pseudomonas aeruginosa 12.4.4 and then given a myelosuppressive dose of cyclophosphamide . rhG-CSF resulted in a slight increase in absolute neutrophil counts (ANC), but did not provide a survival advantage (0 of 12, 0% survival) compared with the placebo group (1 of 12, 8% survival) . rhIL-11 was partially protective (4 of 10, 40% survival); the combination of rhG-CSF and rhIL-11 resulted in a survival rate of 80% (16 of 20; P <.001) . rhIL-11 alone or in combination with rhG-CSF resulted in preservation of gastrointestinal mucosal integrity (P <.001), lower circulating endotoxin levels (P <.01), and reduced quantitative levels of P . aeruginosa in quantitative organ cultures . These results indicate that the combination of rhIL-11 and rhG-CSF is additive as a treatment strategy in the prevention and treatment of experimental Gram-negative sepsis in immunocompromised animals . This combination may prove to be efficacious in the prevention of severe sepsis in neutropenic patients.

Alcohol Clin Exp Res, 1999 Apr, 23(4), 735 - 44
Ethanol inhibits lung clearance of Pseudomonas aeruginosa by a neutrophil and nitric oxide-dependent mechanism, in vivo; Greenberg SS et al.; Pseudomonas aeruginosa (P . aeruginosa) is an opportunistic pathogen that can be found in individuals in which the immune system has been suppressed by HIV/AIDS or chronic alcoholism . We evaluated the role of inducible nitric oxide synthase (NOS II) as a modulator of lung concentrations of P . aeruginosa in normal rats and rats given a single dose of ethanol (ETOH) . Rats were pretreated with either sterile saline (PBS, 0.1 ml/kg, i.v.) or the NOS II inhibitor L-N6-iminoethyl lysine (LNIL, 10 mg/kg, i.v.) 15 min before intraperitoneal administration of either PBS (4.5 ml/kg) or ETOH (4.5 g/kg) . Thirty min after administration of PBS or ETOH the rats were placed in inhalation chambers and exposed to 45 min of an aerosol containing P . aeruginosa (5 x 10(4) colony forming units, CFU) . A group of rats (n = 5-6/treatment/time period) were killed immediately (0 hr) or 4 hr after inhalation of P . aeruginosa . The lungs were homogenized and the P . aeruginosa were grown in nutrient broth to determine the number of viable CFU remaining in the lung . The NOS II and TNFalpha mRNA and protein content lung alveolar macrophages (AM) and neutrophils (PMN) were measured with RT-PCR and Western blot . The concentration of nitrate and nitrite anion in the bronchoalveolar lavage fluid (BALf) and ex vivo incubates of PMN were also measured . The CFU of P . aeruginosa present in the lungs of the four groups of rats at 0 hr did not differ . The CFU of P . aeruginosa in the lung increased (p < 0.05) in rats pretreated with ETOH when compared with that obtained from rats pretreated with PBS . However, pretreatment of rats with LNIL decreased (p < 0.05) the 4 hr lung content of P . aeruginosa . Coadministration of LNIL and ETOH to rats augmented the CFU of P . aeruginosa in lungs to amounts which did not differ from that of rats pretreated with ETOH . Inhalation of P . aeruginosa increased NOS II mRNA and protein in rat AM and PMN . Pretreatment of rats with ETOH alone, or in combination with LNIL, inhibited P . aeruginosa-induced NOS II transcription and translation and AM and PMN nitrate and nitrite generation whereas pretreatment with LNIL alone only inhibited nitrate and nitrite generation . Pretreatment of rats with ETOH suppressed P . aeruginosa stimulated PMN recruitment into the lung whereas LNIL enhanced (p < 0.05) P . aeruginosa-stimulated PMN recruitment into the lung . ETOH-induced increases of the lung content of P . aeruginosa were associated with increased PKC delta isozyme in the membrane of the PMN but could not be explained by altered plasma concentrations of hydrocortisone or ETOH . The data demonstrate that selective inhibition of NOS II-derived NO by LNIL decreases the lung content of P . aeruginosa whereas ETOH inhibits the lung clearance of P . aeruginosa . Speculatively, the difference between these effects of LNIL and ETOH may result from differences in drug-induced changes in lung recruitment of PMN.

Immunol Cell Biol, 1999 Apr, 77(2), 164 - 6
TNF-alpha production in the cornea in response to Pseudomonas aeruginosa challenge; Cole N et al.; Pseudomonas aeruginosa can cause ulcerative bacterial keratitis or contact lens-induced acute red eye (CLARE) in humans . The present study used a mouse model of ocular infection and inflammation to examine the relationship between TNF-alpha and inflammation in the cornea in response to challenge with either a strain of P . aeruginosa causing keratitis or a CLARE strain . Constitutive TNF-alpha mRNA was detected in the epithelium, mainly towards the periphery . After infection with the keratitis-inducing strain (6294), TNF-alpha expression was elevated four-fold by 24 h post-challenge . No detectable induction of TNF-alpha mRNA was seen with CLARE strain (Paer1) challenge at any time point . The TNF-alpha protein production detected by ELISA showed a corresponding pattern to the mRNA expression, which also correlated with pathological changes . These results suggest that invasive strains of P . aeruginosa create greater pathological changes as a result of elevated TNF-alpha production, which contributes to inflammation during keratitis in vivo.

Eur Respir J, 1999 Mar, 13(3), 565 - 70
Reduction in the adherence of Pseudomonas aeruginosa to native cystic fibrosis epithelium with anti-asialoGM1 antibody and neuraminidase inhibition; Davies J et al.; The high incidence of colonization of the cystic fibrosis (CF) airway with Pseudomonas aeruginosa has been attributed to several mechanisms including increased numbers of asialoglycolipid receptors, which may be further increased by exposure to the bacterial exoproduct, neuraminidase . This study examined whether the adherence of P . aeruginosa to fresh CF respiratory epithelial cells can be reduced in vitro by anti-asialoGM1 (anti-aGM1) antibody, neuraminidase inhibition, or the use of asialoGM1 tetrasaccharide as a competitive inhibitor . CF nasal epithelial cells were incubated with a nonmucoid strain of P . aeruginosa, in the presence or absence of a polyclonal anti-aGM1 antibody, the neuraminidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (DANA), or the tetrasaccharide moiety of aGM1 . Adherence of bacteria to the apical surface of ciliated epithelial cells was quantified using scanning electron microscopy . Incubation of the cells with bacteria in the presence of either anti-aGM1 antibody or DANA significantly reduced bacterial adherence by 51(7)%, (p<0.01), and 34(9)%, (p<0.01), respectively . In contrast, no significant effect on P . aeruginosa binding was seen in the presence of aGM1 tetrasaccharide . The data are consistent with previous studies on cultured cells, and suggest that the in vivo effects of such interventions should be explored as potential mechanisms to reduce Pseudomonas aeruginosa colonization in cystic fibrosis.

Eur Respir J, 1999 Mar, 13(3), 552 - 9
Construct and longitudinal validity of a modified Huang clinical scoring system in adult cystic fibrosis patients; Matouk E et al.; This study reports on the evaluation of a modified Huang scoring system in adult cystic fibrosis patients for construct and longitudinal validity . Two studies were performed . In the first study, the scoring system was applied to 59 adult cystic fibrosis patients prospectively followed at the Montreal Chest Institute . The total score and all the subscores distinguished between patients with the expected mild degree of disease severity seen in patients colonized with only Staphylococcus aureus, compared to the more advanced disease severity seen in patients colonized with Pseudomonas aeruginosa or multiple resistant pseudomonads . The relationship between disease severity assessed by forced expiratory volume in one second per cent predicted and the nonpulmonary function subscores was significant and linear (for the radiological subscore, r2=0.694, p<0.0001) and curvilinear (for the clinical and complications subscores, r2=0.622, p=0.0192 and r2=0.508, p=0.0009 respectively) . In the second study, 20 patients retrospectively recorded were added to the prospective group . There was a good association between changes in nonpulmonary function subscores and changes in spirometry over a mean follow-up period of 779+/-204 days, at all levels of disease severity . The contribution of changes in clinical and complications subscores to the changes in total score became progressively more significant with more advanced disease severity . In conclusion, significant evidence for the construct validity of the scoring system as a discriminative instrument and for the longitudinal validity as an evaluative instrument was demonstrated . It may prove of value in assessing outcome of therapeutic interventions in clinical trials in patients with cystic fibrosis.

J Wildl Dis, 1999 Apr, 35(2), 297 - 300
Mycobacterium avium-related epizootic in free-ranging lesser flamingos in Kenya; Kock ND et al.; An epizootic in free-ranging lesser flamingos (Phoeniconaias minor) in Kenya resulted in more than 18,500 deaths from August through mid-November 1993 . Disease was concentrated along the shores of Rift Valley Lakes Bogoria and Nakuru (Kenya) and did not involve any of the other avian or mammalian species frequenting the lakes . Coincidental to the outbreak was a bloom of algae on Lake Bogoria, toxins from which were first suspected to be causative . Discrete necrotic and granulomatous lesions were often noted in spleen and liver, and Mycobacterium avium serovar I was isolated from both organs . Escherichia coli and Pseudomonas aeruginosa also were often recovered in pure culture from liver . Gross and histopathological evaluation of the cases disclosed signs of acute sepsis and also chronic, potentially life-threatening lesions of mycobacteriosis, primarily involving the spleen and liver . Lesions typical for algae toxicosis were not seen in any birds . Deaths were attributed to septicemia complicated in those affected, by mycobacteriosis.

Biochemistry, 1999 May 4, 38(18), 5858 - 63
Expression of FAS-independent ADP-ribosyltransferase activity by a catalytic deletion peptide of Pseudomonas aeruginosa exoenzyme S; Knight DA et al.; Earlier studies reported that Pseudomonas aeruginosa exoenzyme S (ExoS) possessed an absolute requirement for the eukaryotic protein factor activating exoenzyme S (FAS) for expressing ADP-ribosyltransferase activity . During the characterization of a serum-derived FAS-like activity, we observed the ability of a catalytic deletion peptide of ExoS (DeltaN222) to ADP-ribosylate target proteins in the absence of FAS . Characterization of the activation of DeltaN222 by FAS provided an opportunity to gain insight into the mechanism of ExoS activation by FAS . Under standard enzyme assay conditions, the initial rate of FAS-independent ADP-ribosyltransferase activity of DeltaN222 was not linear with time and rapidly approached zero . Dilution into high-ionic strength buffers stabilized DeltaN222 so it could express FAS-independent ADP-ribosyltransferase activity at a linear rate . This stabilization was a general salt effect, since dilution into a 1.0 M solution of either NaCH3COOH, NaCl, or KCl stabilized the ADP-ribosyltransferase activity of DeltaN222 . Kinetic analysis in a high-ionic strength buffer showed that FAS enhanced the catalytic activity of DeltaN222 by increasing the affinity for NAD and stimulating the turnover rate . Velocity experiments indicated that the stabilization of DeltaN222 by high salt was not functionally identical to stabilization by FAS . Together, these data implicate a dual role for FAS in the allosteric activation of ExoS, involving both substrate binding and catalysis.

Biochemistry, 1999 May 4, 38(18), 5677 - 83
Structural basis of electron transfer modulation in the purple CuA center; Robinson H et al.; The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution . Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms) . The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed . The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other . The high-resolution structure reveals the detailed environment of the delocalized mixed-valence {Cu(1.5).Cu(1.5)} binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology . The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes . A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand . This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.

Mol Microbiol, 1999 Apr, 32(2), 393 - 401
The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins; Pederson KJ et al.; Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway . Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway . Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS . A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (DeltaC234) . Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner . Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells . Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234 . Cytoskeleton rearrangement elicited by DeltaC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1) . CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation . This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by DeltaC234 . Together, these data identify ExoS as a cytotoxin that possesses two functional domains . Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.

Presse Med, 1999 Apr 10, 28(14), 729 - 33
{Antibiotic therapy of febrile neutropenic patients: prospective study at a hematologic department}; Limat S et al.; OBJECTIVES: Many antibiotics have been studied in clinical trials in neutropenic patients with fever . Few have been evaluated in the everyday clinical setting . The aim of our study was to analyze the antibiotic strategy used in an adult hematology unit after guidelines had been set up . METHODS: A prospective study was conducted by a pharmacy team not directly working with the prescribing unit . Parameters recorded were drug use, treatment duration and reasons for changing successive drugs . Bacterial ecology data were analyzed . RESULTS: Seventy-four patients were included in the study . Mean treatment duration was 14.8 days and was related to degree of neutropenia: 10 days in case of short neutropenia and 16.2 days for prolonged neutropenia . The most frequent first intention prescription was for non-anti-pseudomonas fl-lactams plus tobramycin, modifications made were: substitution with an anti-pseudomonas fl-lactam and introduction of a glycopeptide as second or third intention drugs . Imipenem and fluoroquinolones were used little . Mean duration of each regimen was 4 days . Initial treatment was generally empirical (7% of the prescriptions were based on documented susceptibility data) . The highest rate of bacteriological data (20%) was obtained during the first intention regiment (42% coagulase-negative staphylococci, 14% Pseudomonas aeruginosa) . The main reason for changing antibiotics was persistent or renewed fever . No bacterial caused deaths were recorded . No multiresistant Gram negative bacilli were selected . DISCUSSION: Reasonable use of antibiotics is possible in neutropenic patients with fever . First intention anti-pseudomonas fl-lactams and glycopeptides are not indispensable in most of these patients . An analysis of practice in the everyday clinical setting is required for optimal use of antibiotics.

J Infect Dis, 1999 Jun, 179(6), 1449 - 58
The effects of aerosolized dextran in a mouse model of Pseudomonas aeruginosa pulmonary infection; Bryan R et al.; Airway infections initiated by the interaction of bacterial adhesins with carbohydrate receptors can be potentially prevented by nontoxic carbohydrate inhibitors . Intranasal inoculation of neonatal mice with Pseudomonas aeruginosa PAO1 caused pneumonia in 55% of control mice but in only 13% of mice inoculated 2 h after dextran inhalation (P<.001) and in 28% inoculated 4 h after dextran inhalation (P=.02) . PAO1 adherence to epithelial cells was inhibited by 50% in the presence of dextran . Dextran was well distributed throughout the airways and stimulated tumor necrosis factor-alpha production in murine lungs but not interleukin-8 production by human epithelial cell lines . Phagocytosis of PAO1 was not affected by dextran nor was killing by human neutrophils diminished . Administration of dextran by aerosol may prevent murine pneumonia by impeding bacterial access to epithelial receptors and by stimulation of the immune functions of the epithelium.

J Med Microbiol, 1999 May, 48(5), 471 - 7
Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung; Hirakata Y et al.; The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated . Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P . aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not . LPS induced TNF production in BALF in a dose-dependent manner, whereas the P . aeruginosa exo-enzymes did not . When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner . ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro . Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs . ETA also depressed partially the expression of TNF-alpha mRNA in AMs . These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.

Am J Respir Crit Care Med, 1999 May, 159(5 Pt 1), 1464 - 8
HLA class II polymorphism in cystic fibrosis . A possible modifier of pulmonary phenotype; Aron Y et al.; Evolution of lung damage is highly variable in cystic fibrosis (CF) even in patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) mutations . The analysis of genetic factors other than CFTR may help our understanding of genotype-phenotype relationships in CF . As human leukocyte antigen (HLA) class II polymorphism has been associated with a number of diseases including autoimmune and inflammatory diseases, asthma, and allergy, we investigated the possibility that HLA polymorphism contributes to CF-associated pulmonary inflammation . Among the 98 adult CF patients tested, the genotypic frequencies of DR4 and DR7 alleles (serologic group DR53) and DR7/ DQA*0201 haplotype were higher than in 39 selected control subjects without atopy (p </= 10(-)6, relative risk {RR} = 22, and p </= 5.10(-)4, RR = 27, respectively) and in a random population . No significant difference of these allelic distributions was found according to the CFTR genotype . In the CF patients, the DR7 allele was significantly associated with an increase in total IgE and with chronic Pseudomonas aeruginosa colonization (100% of DR7 versus 83% of non-DR7 patients being colonized, p < 0.05) . Our results suggest that genetic factors known to modulate the immune response might contribute to chronic infection with Pseudomonas, increased total IgE, and pulmonary outcome in CF.

Am J Respir Cell Mol Biol, 1999 May, 20(5), 1073 - 80
Reduced interleukin-8 production by cystic fibrosis airway epithelial cells; Massengale AR et al.; The acquisition of Pseudomonas aeruginosa in the airways of patients with cystic fibrosis (CF) is the initial event leading to bronchiectasis and lung disease . Although the host factors that permit initial airway colonization are largely unknown, recent studies suggest that secretion of interleukin (IL)-8 by airway epithelia and local recruitment of neutrophils is the final pathway in a pulmonary cytokine network . To determine whether differences in cytokine production exist between normal and CF airway epithelia, secretion of immunoreactive IL-8 and IL-10 as well as specific messenger RNA (mRNA) abundance were compared in airway epithelia expressing normal and mutant CF transmembrane regulator . After induction with IL-1beta, a CF airway cell line engineered to express the wild-type CF gene (CFT1-LCFSN) secreted significantly more immunoreactive IL-8 than did its isogenic parent that expressed the mutant CF gene (CFT1) or an isogenic vector control line (CFT1-LC3) . Further studies with the three related cell lines demonstrated that expression of CFT1-LCFSN was associated with a significant increase in uninduced secretion of immunoreactive IL-8 as well as a 10- to 20-fold increase in IL-8 mRNA abundance when compared with the isogenic lines expressing the mutant gene . IL-1beta induction and intracellular accumulation of IL-8 appeared to be unaffected by CF genotype . These studies suggest that IL-8 secretion by CF airway epithelial cells is defective and may contribute to Pseudomonas persistence in the CF airway . Further studies are needed to confirm this difference in other cell lines and determine the linkage between IL-8 production and CF gene expression.

J Immunol, 1999 May 1, 162(9), 5601 - 8
Defective self-reactive antibody repertoire of serum IgM in patients with hyper-IgM syndrome; Lacroix-Desmazes S et al.; We have analyzed the self-reactive repertoires of IgM and IgG Abs in the serum of 19 patients with hyper-IgM syndrome (HIM) by means of a quantitative immunoblotting technique that allows for a quantitative comparison of Ab repertoires in health and disease by multiparametric statistical analysis . Normal tissue extracts of liver, lung, stomach, and kidney were used as sources of self Ags . Extracts of Pseudomonas aeruginosa and Staphylococcus epidermidis were used as sources of nonself Ags . We demonstrate a significant bias in repertoires of reactivities of IgM of patients with HIM with self Ags . Ab repertoires of IgM toward nonself Ags did not differ, however, between patients and controls . No difference was found between IgM repertoires of untreated patients and those of patients receiving substitutive treatment with i.v . IgG . IgG in the serum of HIM patients lacked reactivity with self Ags, although it exhibited a pattern of reactivity with nonself Ags that was similar to that of IgG of healthy controls . The data demonstrate that functional CD40-CD40 ligand interactions are essential for the selection of natural self-reactive B cell repertoires.

Am J Respir Cell Mol Biol, 1999 May, 20(5), 880 - 90
Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity; Plotkowski MC et al.; Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein . Because we have observed intracellular (IC) P . aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity . Internalization of piliated P . aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay . Cells cultured on thick gels were differentiated, polarized, and tight . They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein . In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes . Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein . Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films . Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P . aeruginosa internalization . Our results suggest that P . aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

Int J Infect Dis, 1998-99 Winter, 3(2), 99 - 104
Improved prognosis of Pseudomonas aeruginosa bacteremia in 127 consecutive neutropenic patients with hematologic malignancies; Todeschini G et al.; OBJECTIVES: Although decreasing in frequency, Pseudomonas aeruginosa bacteremia is still a major challenge for neutropenic cancer patients . In patients with hematologic malignancies, the prognosis of P . aeruginosa bacteremia is particularly poor due to the prolonged and severe neutropenia, mucosal damage, and other defects in immunity related both to the underlying disease and to the cytotoxic therapy . METHODS: To verify the outcome of P . aeruginosa bacteremia and to try to define possible prognostic factors, the authors reviewed the medical records of 127 consecutive episodes of P . aeruginosa bacteremia observed in the hematologic unit of the Verona University School of Medicine . RESULTS: Presence of pneumonia and septic shock, persistence and severity of neutropenia, delayed and inappropriate antibiotic therapy, and unresponsive underlying disease had negative impact on clinical outcome of P . aeruginosa bacteremia . CONCLUSIONS: With recognition of the risk factors and more careful management, the prognosis of P . aeruginosa bacteremia in neutropenic patients with hematologic malignancies has improved in recent years.

Infect Immun, 1999 May, 67(5), 2497 - 502
Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa; Cole N et al.; Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither . In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P . aeruginosa . The number of IL-6-positive cells was determined by image analysis . IL-6 protein levels were measured by an enzyme-linked immunosorbent assay . BALB/c mice were challenged by use of the wounded-cornea model with P . aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU) . At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA . IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206 . At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA . In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge . Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P . aeruginosa . The data obtained for mRNA expression in the cornea for all three strains of P . aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates . Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.

Infect Immun, 1999 May, 67(5), 2371 - 6
Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity; Terada LS et al.; Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host . We hypothesized that the secreted hemolytic phospholipase C (PLC) of P . aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity . We found that while intact wild-type P . aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (DeltaHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils . In contrast, a second pseudomonas mutant (DeltaN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2.- production . Readdition of purified PlcHR to the DeltaHR strain suppressed neutrophil O2.- production to levels stimulated by wild-type bacteria . Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway . Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient DeltaHR bacterial mutant . We conclude that expression of PlcHR by P . aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.

J Antimicrob Chemother, 1999 Mar, 43 Suppl A, 129 - 34
Clinical and economic evaluation of subsequent infection following intravenous ciprofloxacin or imipenem therapy in hospitalized patients with severe pneumonia; Caldwell JW et al.; A recent multicentre clinical study evaluated the safety and efficacy of i.v . ciprofloxacin therapy compared with imipenem-cilastatin in hospitalized patients with severe pneumonia . Monotherapy with i.v . ciprofloxacin was at least equivalent to imipenem in terms of bacteriological eradication and clinical response . In a single-centre, retrospective, post-therapy evaluation of persistent and subsequent infection, the incidence of gram-negative infections and associated costs were compared . The main elements of the economic analysis included costs of additional antimicrobial therapy and hospitalization . Thirty-two patients were randomized into the study, of whom 27 were efficacy-valid . The 13 patients randomized into the ciprofloxacin group were not significantly different from the 14 patients in the imipenem group in terms of clinical parameters . Clinical cure occurred in ten of 13 patients (77%) in the ciprofloxacin group and in seven of 14 (50%) in the imipenem group . Bacteriological eradication was achieved in 11 of 13 (85%) ciprofloxacin-treated and eight of 14 (57%) imipenem-treated patients . Five of 13 (38%) patients in the ciprofloxacin group and nine of 14 (64%) in the imipenem group experienced persistent or subsequent infection requiring post-treatment antimicrobials . In these five ciprofloxacin patients, three had cultures with gram-positive organisms only and two had cultures with both gram-positive and gram-negative organisms . In the nine imipenem-treated patients requiring post-study antimicrobials, all had gram-negative bacteria and three also had gram-positive organisms . The incidence of subsequent gram-negative infection in the two groups (15% vs 64%) was significantly different (P < 0.05) . Pseudomonas aeruginosa was isolated from seven patients in the imipenem group but only one in the ciprofloxacin group (P < 0.05) . Subsequent costs for post-therapy antimicrobials and hospital stay while receiving study and post-study drug therapy were evaluated; the cost per patient cure was US$29,000 for ciprofloxacin and US$76,000 for imipenem . Initial treatment of severe pneumonia with ciprofloxacin resulted in significantly less subsequent gram-negative infection and was associated with substantially lower curative costs.

Appl Environ Microbiol, 1999 May, 65(5), 2163 - 9
Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls; Hrywna Y et al.; Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source . The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs) . Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA . The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs . The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively . Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs . Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.

Appl Environ Microbiol, 1999 May, 65(5), 2151 - 62
Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates; Tsoi TV et al.; We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142 . Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol . A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P . putida PB2440 the ability to grow on 2-CBA as a sole carbon source . Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA . Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E . coli and P . aeruginosa parental strain 142 . The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts . ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases . A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes . An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found . The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E . coli . The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563 . The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

Appl Environ Microbiol, 1999 May, 65(5), 2065 - 71
Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830; Zago A et al.; Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations . To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P . aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively . The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro . Gene replacement was used to construct a PPK-negative strain of P . aeruginosa 8830 . Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism . Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses . However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.

Antimicrob Agents Chemother, 1999 May, 43(5), 1301 - 3
Resistance to beta-lactam antibiotics in Pseudomonas aeruginosa due to interplay between the MexAB-OprM efflux pump and beta-lactamase; Nakae T et al.; We evaluated the roles of the MexAB-OprM efflux pump and beta-lactamase in beta-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from beta-lactamase-negative, -inducible, and -overexpressed strains . We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to beta-lactam resistance in both beta-lactamase-negative and beta-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed beta-lactamase . Overexpression of the efflux pump alone contributes to the high level of beta-lactam resistance in the absence of beta-lactamase.

Antimicrob Agents Chemother, 1999 May, 43(5), 1085 - 90
Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids; Ochs MM et al.; Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem, a carbapenem antibiotic . Resistance to imipenem due to the loss of OprD is an important mechanism for the loss of clinical effectiveness . To investigate the negative regulatory mechanisms influencing oprD expression, a gene upstream of the coregulated mexEF-oprN efflux operon, designated mexT, was cloned . The predicted 304-amino-acid mature MexT protein showed strong homology to LysR-type regulators . When overexpressed it induced the expression of the mexEF-oprN efflux operon while decreasing the level of expression of OprD . The use of an oprD::xylE transcriptional fusion indicated that it acted by repressing the transcription of oprD . Salicylate, a weak aromatic acid known to reduce porin expression and induce low levels of multiple antibiotic resistance in Escherichia coli, was able to induce imipenem resistance and reduce the expression of OprD but not multiple antibiotic resistance or OprN expression in P . aeruginosa . This was also demonstrated to occur at the level of transcription . Acetyl salicylate and benzoate, but not catechol, were also able to reduce the levels of OprD in the P . aeruginosa outer membranes . These OprD-suppressing compounds increased imipenem resistance even in a mexT-overexpressing and nfxC mutant backgrounds, suggesting that such resistance is independent of the MexT repressor and that oprD is influenced by more than a single mechanism of repression.

Hum Gene Ther, 1999 Apr 10, 10(6), 957 - 64
Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy; Gropp R et al.; Epithelial cells have been to participate actively in host defense by producing small cationic peptides called defensins . To investigate the biological activity of epithelial defensins in more detail, we expressed two defensins, hBD-1 and HD-5, in eukaryotic cell lines . Defensins were localized in the cytoplasm and in cell culture medium and exhibited strong microbicidal activity toward Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus . Moreover, our data indicate that the presence of defensins protected the cells from adenoviral infection . The presence of HD-5 or hBD-1 reduced the infectivity of Av1CF2 three- to fivefold . These results imply that defensins must be considered a serious obstacle whenever adenovirus is used to deliver genes to epithelial cells.

J Antimicrob Chemother, 1999 Mar, 43(3), 415 - 8
A large outbreak of multiresistant Pseudomonas aeruginosa strains in north-eastern Germany; Panzig B et al.; Multiply-resistant Pseudomonas aeruginosa were first detected in north-eastern Germany at the end of 1996; since then they have been isolated predominantly from patients in intensive care units . Colonization/infection, especially of the respiratory tract, has been demonstrated in 80 patients, with strains resistant to beta-lactams, carbapenems, aminoglycosides and quinolones . Amikacin showed in-vitro synergy with cefepime, ceftazidime or piperacillin/tazobactam . Horizontal transfer of strains was followed by PFGE and identical strains were detected in the environment, but the source of infection was not established . Rigorous infection control and restricted clinical use of carbapenems limited further dissemination of this outbreak.

J Antimicrob Chemother, 1999 Mar, 43(3), 389 - 97
Piperacillin/tazobactam plus tobramycin versus ceftazidime plus tobramycin for the treatment of patients with nosocomial lower respiratory tract infection . Piperacillin/tazobactam Nosocomial Pneumonia Study Group; Joshi M et al.; An open-label, randomized, comparative, multi-centre study was conducted at 25 centres in the USA and Canada to compare the safety and efficacy of piperacillin/tazobactam plus tobramycin with ceftazidime plus tobramycin in patients with lower respiratory tract infections . Piperacillin/tazobactam (3 g/375 mg) every 4 h or ceftazidime (2 g) every 8 h were administered i.v . for a minimum of 5 days . Tobramycin (5 mg/kg/day) given in divided doses every 8 h was administered to all patients . Patients with Pseudomonas aeruginosa isolated from respiratory secretions at baseline were to continue tobramycin for the duration of the study . Tobramycin could be discontinued in other patients after the baseline culture results were known . A total of 300 patients was randomized, 155 into the piperacillin/tazobactam group and 145 into the ceftazidime group . Of these, 136 patients (78 in the piperacillin/tazobactam group and 58 in the ceftazidime group) were considered clinically evaluable . Both groups were comparable for age, sex, duration of treatment and other demographic features . The clinical success rate in evaluable patients was significantly greater (P = 0.006) in the piperacillin/tazobactam treatment group (58/78; 74%) than in the ceftazidime group (29/58; 50%) . Eradication of the baseline pathogen was significantly greater (P = 0.003) in the piperacillin/tazobactam group (66%) than in the ceftazidime group (38%) . The clinical and bacteriological responses of those patients with nosocomial pneumonia were similar to the overall results . Twelve (7.7%) piperacillin/tazobactam-treated patients and 24 (17%) ceftazidime-treated patients died during the study (P = 0.03) . Seven of the 24 deaths in the ceftazidime treatment group but only one of the 12 deaths in the piperacillin/tazobactam treatment group were directly related to failure to control infection . The majority of adverse events were thought by the investigator to be attributable to the patients' underlying disease and not drug related . In this study, piperacillin/tazobactam plus tobramycin was shown to be more effective and as safe as ceftazidime plus tobramycin in the treatment of patients with nosocomial LRTI.

J Antimicrob Chemother, 1999 Mar, 43(3), 339 - 44
Laboratory mutants of OXA-10 beta-lactamase giving ceftazidime resistance in Pseudomonas aeruginosa; Danel F et al.; Several extended-spectrum beta-lactamases (ESBLs) belonging to molecular Class D have been described from Pseudomonas aeruginosa isolates collected in Turkey . Four of these, OXA-11, -14, -16 and -17, are derivatives of OXA-10 beta-lactamase . We tried to select similar mutants in vitro from OXA-10-producing transconjugants of P . aeruginosa, using a multistep method on ceftazidime-containing agars . Forty-four such mutants were obtained; all had increased resistance to ceftriaxone, cefsulodin, cefepime, cefpirome, latamoxef, aztreonam and, especially, ceftazidime whereas MICs of piperacillin, carbenicillin, cefotaxime, cefoperazone and carbapenems were little altered . Genes related to blaOXA-10 were sequenced from five mutants . One mutant enzyme had aspartate instead of glycine at position 157, and corresponded exactly to natural OXA-14 beta-lactamase . Another mutant strain appeared to have both OXA-14 and a new pI 6.2 enzyme, designated OXA-M102, with serine instead of alanine at position 124 and aspartate instead of glycine at position 157 . This latter variant resembled natural OXA-16 enzyme, which has threonine at position 124 and aspartate at position 157 . The remaining three mutant enzymes differed from any so far found in wild-type isolates . Two had leucine replacing tryptophan at position 154 (this enzyme was named OXA-M101) while the third (OXA-M103) had a pI of 7.6, and had lysine instead of asparagine at position 143 . A different mutation at this position was previously found in OXA-11, a wild-type OXA-10 mutant . Thus, some of the ESBL mutants selected (OXA-14 and OXA-M102) correspond exactly or almost exactly to ESBLs found in wild-types, whereas others (OXA-M101 and OXA-M103) were totally new.

Med Dosw Mikrobiol, 1998, 50(3-4), 269 - 75
{Bactericidal properties of copper (II) complexes with 1-alkylimidazole}; Radzyminska-Lenarcik E et al.; The copper(II) complexes with 1-alkylimidazole were prepared . Bactericidal properties of the obtained complexes against Staphylococcus aureus, Micrococcus luteus and Pseudomonas aeruginosa were studied . Particularly high activity against microbial strains was shown by complex {Cu(1-C4H9Im)4(CIO4)2}.

Zhonghua Yi Xue Za Zhi (Taipei), 1999 Mar, 62(3), 175 - 8
Acute necrotizing otitis media in an infant: a case report; Shen KH et al.; Acute necrotizing otitis media (ANOM), an uncommon but severe form of bacterial otitis media, frequently causes distressing sequelae if not properly diagnosed and treated . A four-month-old female infant initially became ill with intermittent fever, followed by left facial nerve paralysis and left otorrhea four days later . Microscopic examination of the left ear revealed congestion and swelling of the external ear canal, perforation of the eardrum and erosions on the malleus . Culture of pus from the otic lesion grew Pseudomonas aeruginosa . The patient's condition did not improve despite systemic administration of antibiotics; thus, surgical intervention was arranged . During the operation, near-total perforation of the eardrum, a dislodged incus, cholesteatoma-like matrix around the stapes, and granulation tissue occupying the middle ear and mastoid cavities were noted . Radical mastoidectomy was conducted and pathologic examination of the surgical specimen disclosed necrotic changes in both soft and bony tissues . The patient recovered soon after surgery . Her fever subsided one day after surgery and the patient was discharged in a stable condition 12 days later . However, she still had left facial nerve paralysis six months later.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 35 - 9
The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated; Evans K et al.; Intrinsic antibiotic resistance in Pseudomonas aeruginosa is attributed to low outer membrane permeability and drug efflux mediated by the products of mexAmexBoprM efflux operon . Using a mexA-phoA fusion, expression of the efflux genes was assessed as a function of growth in a variety of strains . The efflux operon was growth-phase regulated in both wild-type and nalB strains, being minimally expressed in lag phase and increasing in log to late log phase . MexR, the only known regulator of MexAMexBOprM and target of mutation in nalB strains, was not involved in the growth-phase regulation . The las cascade regulates genes based on increased cell-density, but a deletion in lasR had no effect on mexAmexBoprM expression . Putative recognition sequences for AlgT/U and RpoN were identified upstream of mexA, but algT/U and rpoN null mutants also had no effect on mexAmexBoprM expression.

Shock, 1999 Apr, 11(4), 283 - 90
L-arginine and endothelin receptor antagonist bosentan counteract hemodynamic effects of modified hemoglobin; Fischer SR et al.; Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a nitric oxide scavenger, causes systemic and pulmonary vasoconstriction in normal and septic sheep . We studied the effect of L-arginine and the endothelin-1 (ET-1) antagonist bosentan on the PHP response to determine whether the PHP-induced vasoconstriction resulted predominantly from the action of ET-1 or solely from removal of NO . After 24 h of carrier solution (nonseptic sheep), sheep received PHP (20 mg/kg/h; n = 5), PHP plus L-arginine (at 28 h, 100 mg/kg bolus and 500 mg/kg for 1 h) plus bosentan (at 32 h, 10 mg/kg; n = 6), and only L-arginine and bosentan (n = 5) . These protocols were repeated after 24 h of Pseudomonas aeruginosa (S, 6x10(6) colony-forming units/kg/h) . PHP induced vasoconstriction in septic and nonseptic sheep for the duration of its infusion . In nonseptic sheep, neither L-arginine nor bosentan significantly lowered systemic (SVRI) and pulmonary (PVRI) vascular resistance and did not antagonize the PHP-induced vasoconstriction . During sepsis, SVRI fell and cardiac index (CI) rose . L-arginine and bosentan further decreased SVRI (L-arginine: 34+/-2%*, p<.05; bosentan: 35+/-5%*, p<.05) and PVRI (L-arginine: 28+/-2%*, p<.05; bosentan: 33+/-7%*, p<.05) and increased CI (L-arginine: 29+/-4%*, p<.05; bosentan: 11+/-5%, NS) . Both agents antagonized the PHP-induced vasoconstriction lowering SVRI (L-arginine: 29+/-3%*, p<.05; bosentan: 26+/-5%*, p<.05) and PVRI (L-arginine: 27+/-4%*, p<.05; bosentan: 32+/-4%*, p<.05) to levels before PHP administration . Plasma ET-1 levels increased during sepsis (from 9.8+/-.2 to 15.6+/-.7* pg/mL, p<.05) and fell during PHP infusion (to 9.7+/-1.6* pg/mL, p<.05) . In nonseptic sheep, ET-1 levels decreased during PHP (from 8.5+/-.6 pg/mL to 5.9+/-.6*, p<.05) . Bosentan increased ET-1 levels 2.7 times higher in septic than in nonseptic sheep . We conclude that during sepsis, the NO scavenger PHP unmasks an underlying ET-1 mediated vasoconstriction, and its effect is antagonized by L-arginine and bosentan.

Microbiology, 1999 Apr, 145 ( Pt 4), 845 - 53
ThrH, a homoserine kinase isozyme with in vivo phosphoserine phosphatase activity in Pseudomonas aeruginosa; Patte JC et al.; Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis . In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy . A new gene, named thrH, was isolated that, when expressed in E . coli thrB mutant strains, results in complementation of the mutant phenotype . In P . aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated . Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity . Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E . coli and P . aeruginosa serB mutants . These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis . Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms . This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced . ThrH differs from the protein encoded by the serB gene which was identified in P . aeruginosa . Thus, two SerB-like proteins co-exist in P . aeruginosa, a situation also found in Mycobacterium tuberculosis.

Microbiology, 1999 Apr, 145 ( Pt 4), 835 - 44
RpoS-dependent stress tolerance in Pseudomonas aeruginosa; Jorgensen F et al.; Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans . The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species . To determine if the P . aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated . Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells . The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation . However, after 25 d starvation the remaining population of culturable cells was not significantly different . Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P . aeruginosa . Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

Ophthalmic Surg Lasers, 1999 Apr, 30(4), 315 - 6
A case of bacterial endophthalmitis following perforating injury caused by a cat claw; Doi M et al.; A case of bacterial endophthalmitis following a perforating ocular injury caused by a cat claw is reported . The scleral wound was sutured immediately following the injury and systemic antibiotics were administered . Despite this treatment, endophthalmitis occurred 3 days after the injury . The endophthalmitis was resolved by pars plana vitrectomy, however preretinal reproliferation and retinal detachment subsequently occurred . After reoperation the retina was reattached and the corrected visual acuity improved from 10 cm/HM to 20/200 . Pseudomonas aeruginosa was detected in cultured vitreous humor that was collected during surgery . This case illustrates the possibility of endophthalmitis being caused by gram negative bacillus in cases of perforating injuries caused by animal claws . Perforating ocular injuries caused by animal claws are relatively rare . Here we report a case of endophthalmitis due to Pseudomonas aeruginosa that occurred after a perforating injury caused by a cat claw . The eye was treated by pars plana vitrectomy.

J Bacteriol, 1999 May, 181(9), 2789 - 96
PhhB, a Pseudomonas aeruginosa homolog of mammalian pterin 4a-carbinolamine dehydratase/DCoH, does not regulate expression of phenylalanine hydroxylase at the transcriptional level; Song J et al.; Pterin 4a-carbinolamine dehydratase is bifunctional in mammals . In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1alpha . A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC) . Using complementation of tyrosine auxotrophy in Escherichia coli as a functional test, we have found that the in vivo function of PhhA requires PhhB . Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans . Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB on phhA expression . Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E . coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative . However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function . PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation of phhB in P . aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB in Escherichia coli . Experiments using constructs having transcriptional and translational fusions with a lacZ reporter indicated that PhhB activates PhhA at the posttranscriptional level . Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of either L-tyrosine or L-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA . Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

J Pharm Pharmacol, 1999 Feb, 51(2), 201 - 6
Electron-microscopic study of the bactericidal effect of OPB-2045, a new mono-biguanide disinfectant produced from biguanide group compounds, against Pseudomonas aeruginosa; Sakagami Y et al.; The bactericidal activity of OPB-2045 (1-(3,4-dichlorobenzyl)-5-octylbiguanide monohydrochloride hemihydrate) at several concentrations against Pseudomonas aeruginosa IFO 13275 was investigated morphologically by transmission and scanning electron microscopy . The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of OPB-2045 against P . aeruginosa were the same, at 12.5 microg mL(-1), suggesting that it may be a suitable disinfectant for use in the medical field . Test bacteria were treated at concentrations of one half the MIC value (6.25 microg mL(-1)), the MIC value (12.5 microg mL(-1)), twice the MIC value (25 microg mL(-1)) or ten times the MIC value (125 microg mL(-1)) at 37 degrees C for 30 min or 6 h and the cells were then examined by transmission and scanning electron microscopy . The cell damage evident after 6h incubation was greater than observed after 30 min incubation . Especially, at one half the MIC, no cell damage was evident after 30 min incubation, but damaged cells were observed after 6 h incubation . The proportion of empty cells of P . aeruginosa increased as the concentration of added disinfectant was increased, and the release of intracellular components was also recognized . These results suggest that OPB-2045 acts on the cell membrane and cell wall of P . aeruginosa, and destroys their integrity at the level of the MIC (MBC) . With the increase in OPB-2045 concentration and the increase in reaction time, the bactericidal effect increased markedly . Agglutination of the cells was observed at high concentrations of OPB-2045 . This indicates that the bactericidal effect at high concentrations of OPB-2045 differs from that at low concentrations . A clear cell-damaging effect against the test strain was recognized which was dependent on the OPB-2045 concentration and the incubation time . From experiments concerning the relationship between the number of surviving bacteria and MIC values in soybean casein digest broth, the decrease in bacterial numbers was found to be dependent on the OPB-2045 concentration . We conclude that it would be a useful contribution to the medical field to supply a new disinfectant to be employed in preventive countermeasures against infection caused by pathogenic bacteria.

Hepatology, 1999 May, 29(5), 1352 - 7
Lipopolysaccharide from Escherichia coli stimulates mucin secretion by cultured dog gallbladder epithelial cells; Choi J et al.; Biliary infection is associated with mucin hypersecretion by the biliary epithelium . Mucins have been identified as potent pronucleators of cholesterol in bile . The aim of the present study was to determine whether lipopolysaccharides (LPS) from different bacteria are capable of stimulating mucin secretion by cultured dog gallbladder epithelial (DGBE) cells, and to investigate the mechanism by which LPS stimulate mucin secretion . Mucin secretion by confluent monolayers of DGBE cells was quantified by measuring the secretion of {3H}-N-acetyl-D-glucosamine-labeled glycoproteins . Cell viability was evaluated by measuring the leakage of the enzyme, lactate dehydrogenase (LDH), into the culture medium . LPS, derived from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (200 microg/mL), all caused an increase in mucin secretion by the DGBE cells, without causing concomitant cell lysis . LPS from E . coli was found to be the most potent stimulator of mucin secretion, and increased mucin secretion by the DGBE cells to 252% +/- 14% of control . LPS from E . coli had no effect on intracellular cyclic adenosine monophosphate (cAMP) levels in the DGBE cells . Addition of the nitric oxide (NO)-releasing compound, NOR-4 (0.125-1 mmol/L), to the cells did not result in increased mucin secretion, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (4 or 10 mmol/L), did not inhibit the LPS-stimulated mucin secretion . Exogenous tumor necrosis factor alpha (TNF-alpha) (1-10 ng/mL) did cause a minor increase in mucin secretion by the DGBE cells, but the effect of LPS from E . coli on mucin secretion could not be inhibited by preincubation with a TNF-alpha antibody (10 microg/mL) . We conclude that LPS stimulates mucin secretion by the gallbladder epithelium . Whether this stimulation is mediated by TNF-alpha remains to be determined.

Blood, 1999 May 1, 93(9), 3096 - 105
CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge; Lekstrom-Himes J et al.; Targeted mutation of CCAAT/enhancer binding protein (C/EBP) epsilon in mice results in early death, primarily due to spontaneous infection with Pseudomonas aeruginosa . Functional analysis of C/EBPepsilon-deficient neutrophils, in an in vivo model of peritoneal inflammation, shows multiple defects . Reduction of phagocytotic killing by C/EBPepsilon-deficient neutrophils is a result of decreased uptake of opsonized bacteria as well as little to no expression of secondary granule proteins . Abnormalities in neutrophil migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on C/EBPepsilon-deficient neutrophils . Alterations in neutrophil cytokine expression in response to inflammation show decreased levels of interleukin-1 receptor antagonist (IL-1Ra) and increased levels of tumor necrosis factor-alpha (TNF-alpha) expression by C/EBPepsilon-deficient neutrophils . Additionally, TNF-alpha expression is increased in nonactivated, circulating C/EBPepsilon-deficient neutrophils . Overall, C/EBPepsilon-deficient neutrophils are severely functionally impaired, evoking an abnormal microenvironment, which may contribute to the loss of normal responses to inflammatory stimuli . Similarities between the C/EBPepsilon-deficient mouse model and the human disease, specific granule deficiency, will be discussed.

Eur J Biochem, 1999 Apr, 261(2), 500 - 8
Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4; Sanchez Carballo PM et al.; Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure . Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS . Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable . Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography . The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc . Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion {M-H}- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide . The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9 . The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: {equation: see text} Two unusual structural features in the core oligosaccharide of P . aeruginosa were identified for the first time, i.e . the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.

West J Med, 1999 Mar, 170(3), 143 - 7
Cluster of postinjection abscesses related to corticosteroid injections and use of benzalkonium chloride; Olson RK et al.; Benzalkonium chloride (BC) is an unreliable disinfectant . A matched case-control study and environmental investigation were conducted to determine the cause of and risk factors for a cluster of postinjection abscesses at a private medical clinic where BC was used as a disinfectant . Twenty-eight case-patients who had an abscess at the injection site were matched with 126 control patients who had received an intramuscular injection at the clinic on the same day . Risk factors for abscess development in a multivariable logistic model were corticosteroid injection and being female . All case-patients had received a corticosteroid injection from a multidose vial . Cultures of abscesses from 20 of 23 case-patients grew Pseudomonas aeruginosa . Cultures of BC prepared at the clinic also grew P aeruginosa, suggesting that BC was the source of infection . Injection site cleaning with BC did not appear to be the route of infection since use of BC at the time of injection was not associated with abscess development . A more likely route of infection was injection of contaminated corticosteroid from multidose vials that could have been inoculated with pseudomonads via needle puncture after vial septa were wiped with contaminated BC . Benzalkonium chloride should not be used to clean injection vial septa or injection sites.

Pediatr Pulmonol, 1999 Mar, 27(3), 174 - 9
Pseudomonas aeruginosa alginate is a potent secretagogue in the isolated ferret trachea; Kishioka C et al.; Airway mucus hypersecretion is in part a response to infection and inflammation . Pseudomonas aeruginosa infection is nearly universal in advanced cystic fibrosis (CF) lung disease . Mucoid strains of P . aeruginosa produce an exopolysaccharide product called alginate . The purpose of this study was to determine whether P . aeruginosa alginate stimulates secretion from mucous or serous cells in the ferret trachea exposed to alginate at concentrations reported to be present in the CF airway . We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin secretion and spectrophotometry to measure lysozyme secretion from isolated ferret tracheal segments . Purified Pseudomonas aeruginosa alginate stimulated mucin and lysozyme secretion in a dose-dependent fashion (mucin = +111%: P = 0.003; lysozyme = +20%: P = 0.024 at 200 microg/mL) . This stimulated secretion was not due to proteolytic activity, and alginate exposure did not produce ultrastructural damage to the trachea . We conclude that alginate may contribute to mucus hypersecretion and respiratory morbidity associated with P . aeruginosa infection in patients with CF.

Biochemistry, 1999 Apr 20, 38(16), 5216 - 21
Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa; Masters SC et al.; The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis . It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs . Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa . By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro . The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM . ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry . No detectable phosphoproteins were found . A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove . It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.

Anaesth Intensive Care, 1999 Apr, 27(2), 213 - 5
Subcutaneous nodules with pseudomonas septicaemia in an immunocompetent patient; Asumang A et al.; Pseudomonas septicaemia presenting with subcutaneous nodules, though rare, is well described in immunocompromized populations . It is, however, very uncommon in immunocompetent patients . We describe a case of a 42-year-old woman who presented with community-acquired . Pseudomonas aeruginosa septicaemia and subcutaneous nodules . No precipitating cause or immune dysfunction was found . She was successfully treated with appropriate antibiotics, respiratory and cardiovascular support in the Intensive Care Unit . The difficulty in eradicating the organism from the skin lesion and the need for investigating the immune function of septicaemia patients are discussed.

Thorax, 1999 May, 54(5), 380 - 3
Systematic review of antistaphylococcal antibiotic therapy in cystic fibrosis; McCaffery K et al.; BACKGROUND: The respiratory tract in patients with cystic fibrosis is frequently colonised with Staphylococcus aureus . There is great diversity of clinical practice in this area of cystic fibrosis . A systematic review was conducted to study the evidence relating antistaphylococcal therapy to clinical outcome in patients with cystic fibrosis . METHODS: A search strategy already evaluated for the study of the epidemiology of cystic fibrosis clinical trials was used . This yielded 3188 references from which 13 clinical trials of antistaphylococcal therapy were identified . RESULTS: Substantial heterogeneity was observed between trials . In the 13 clinical trials a total of 19 antibiotics were used to assess a wide variety of outcome measures (11 clinical, six laboratory) . Both intermittent and continuous treatment strategies were used . Sputum clearance of S aureus was more frequently achieved than any other beneficial outcome . A beneficial effect on pulmonary function was rarely measured or observed . Although five randomised clinical trials were identified, the extent of heterogeneity precluded the use of meta-analysis for further synthesis of information . CONCLUSIONS: Antistaphylococcal treatment achieves sputum clearance of S aureus in patients with cystic fibrosis . Prophylactic antistaphylococcal treatment in young children with cystic fibrosis is likely to be of clinical benefit . It remains to be determined whether the use of "prophylactic" versus "intermittent" antistaphylococcal therapy in cystic fibrosis is associated with improved lung function and/or chest radiographic scores, an increase in bacterial resistance, or earlier acquisition of Pseudomonas aeruginosa . A large randomised clinical trial lasting approximately two years is urgently required to address this problem.

Res Microbiol, 1999 Mar, 150(2), 105 - 16
Pattern of changes in the activity of enzymes of GDP-D-mannuronic acid synthesis and in the level of transcription of algA, algC and algD genes accompanying the loss and emergence of mucoidy in Pseudomonas aeruginosa; Tavares IM et al.; The low activity levels of the four GDP-D-mannuronic acid-forming enzymes, even in highly alginate-producing strains of Pseudomonas aeruginosa, have made it difficult to compare enzyme activities accompanying the loss/acquisition of mucoidy . Using optimized conditions, we compared the specific activity of these enzymes in three different mucoid P . aeruginosa cystic fibrosis isolates, in their nonmucoid spontaneous variants, and in mucoid variants that emerged during extended incubation of these nonmucoid forms in acetamide broth . A correlation was established between the promptness of emergence of the mucoid forms and the differing sensitivity to nutrient-limitation-induced death of the nonmucoid compared with the isogenic mucoid population . Consistent with the undetectable levels of algD mRNA in nonmucoid forms and with the concept that the step catalyzed by the algD-encoded GDP-mannose dehydrogenase (GMD) is a key step in control of the alginate pathway, GMD activity was undetectable or showed negligible values in nonmucoid variants and correlated with alginate production . However, phosphomannose isomerase (PMI), phosphomannomutase (PMM), and GDP-mannose pyrophosphorylase (GMP) activities in the nonmucoid forms were only slightly (40-70%) below the values in the mucoid forms . Nevertheless, no transcripts homologous to algA (encoding a bifunctional enzyme that possesses both PMI and GMP activities) were detected in the nonmucoid form, and the levels of algC (encoding PMM) transcripts, although detectable in the nonmucoid variants, were, in general, much higher in the mucoid forms . These apparently intriguing observations were cleared up by the identification of two algA functional homologues in P . aeruginosa, recently reported by others, and by the identification of one algC homologue, in contig225 of the PAO1 genome sequence, defining a polypeptide with a deduced amino acid sequence that showed significant homology with that of enzymes of the phosphohexomutase family found in databases . Results are also consistent with the requirement of PMI, GMP and PMM activities for the supply of GDP-D-mannose to (at least) A-band lipopolysaccharide synthesis, while GMD channels this precursor into the alginate pathway.

Chest, 1999 Apr, 115(4), 1107 - 14
The potential for dissemination of Mycobacterium tuberculosis through the anesthesia breathing circuit; Langevin PB et al.; BACKGROUND: Respiratory pathogens that pass through the anesthesia breathing system potentially can infect other patients . This study was designed to determine if bacteria can pass through contemporary anesthesia breathing systems and if the environment within the machine is hostile to these organisms . METHODS: Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis were nebulized into the expiratory limb of an anesthesia breathing circuit and collected from the inspiratory and expiratory limbs in an impinger system that provided a quantitative determination of the number of organisms entering the circuit and the number that would reach the patient in the inspiratory gas . Bacteria were collected before, during, and after nebulization . A second experiment determined if a saturated solution of soda lime was bactericidal . RESULTS: When the gas flow through the circuit was interrupted for < 1 h following the nebulization period, large numbers of microorganisms (1 x 10(3) to 1 x 10(5), around 100% of the nebulized organisms) were collected from the inspiratory gas . Soda lime itself was not bactericidal for any of the organisms tested, but solutions of this material with a pH of 12 were bactericidal . CONCLUSION: Cross contamination between patients may occur unless the gas flow through the anesthesia breathing system is interrupted for > 1 h.

J Clin Invest, 1999 Apr, 103(8), 1113 - 7
Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model; Bals R et al.; Recent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity . We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model . The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus . Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels . Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer . The increase of LL-37 was sufficient to restore bacterial killing to normal levels . The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

Microbiology, 1999 Jan, 145 ( Pt 1), 211 - 20
Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus; Brennan FR et al.; A synthetic peptide (peptide 10) representing a surface-exposed, linear B cell epitope from outer-membrane (OM) protein F of Pseudomonas aeruginosa was shown previously to afford protection in mice from P . aeruginosa infection . This peptide was expressed in tandem with the protein F peptide 18 on each of the two coat proteins of cowpea mosaic virus (CPMV) . The chimaeric virus particles (CVPs) expressing the peptides on the S (small) coat protein (CPMV-PAE4) and L (large) coat protein (CPMV-PAE5) were used to immunize mice . Following subcutaneous immunization in Freund's and QuilA adjuvants, CPMV-PAE4 induced antibodies predominantly against peptide 18, whereas CPMV-PAE5 produced antibodies exclusively against peptide 10, indicating that the site of peptide expression on CPMV influences its immune recognition . The anti-peptide antibodies elicited by CPMV-PAE5 were predominantly of the IgG2a isotype, indicating a highly polarized TH1-type response . The peptide-specific IgG2a strongly recognized the whole F protein, but more importantly, recognized protein F in all seven Fisher-Devlin immunotypes of P . aeruginosa . Furthermore, the peptide-specific IgG2a in CVP/QS-21 adjuvant-immunized mice was shown to bind complement and to augment phagocytosis of P . aeruginosa by human neutrophils in vitro . The ability of CPMV-PAE5 to induce P . aeruginosa-specific opsonic IgG2a gives it potential for further development as a protective vaccine against P . aeruginosa.

J Leukoc Biol, 1999 Apr, 65(4), 508 - 14
Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity; Fierro IM et al.; Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro . Both dexamethasone and L-NMMA, added in vitro to neutrophil cultures, inhibited the production of NO . On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5-lipoxygenase or with a platelet-activating factor (PAF) antagonist . The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin-8) and different cytokines {interleukin-1, tumor necrosis factor alpha, and interferon-gamma (IFN-gamma)} showed that only IFN-gamma was able to induce the production of high amounts of NO . This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN . The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN-gamma, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase . We also showed that NO produced by IFN-gamma activated rat blood PMN involved in the killing of Pseudomonas aeruginosa.

Jpn J Antibiot, 1999 Jan, 52(1), 41 - 56
{Effects of isepamicin and beta-lactam antibiotics on the release of endotoxin from Pseudomonas aeruginosa}; Yamaguchi S et al.; Antibiotic-induced release of endotoxin (lipopolysaccharide, LPS) from Pseudomonas aeruginosa was investigated in in vitro with different antibiotic concentrations and in the pharmacokinetic autosimulation system . We compared the effect of isepamicin (ISP) with those of 3 beta-lactam antibiotics, piperacillin (PIPC), ceftazidime (CAZ) and imipenem (IPM) . ISP showed a strong bactericidal activity, but the amount of free LPS did not increase by 6 hrs (28 +/- 2 ng/ml at 1MIC) . PIPC and CAZ caused a gradual killing and a large amount of LPS release at 4 hrs (515 ng/ml and 493 ng/ml, respectively, at 1 MIC) . At 1/4 x MIC, PIPC and CAZ did not reduce colony forming counts and induced more release of free LPS . The organism treated with IPM released less LPS, while it was killed rapidly . The viable cell counts decreased dramatically after administration of ISP in the pharmacokinetic autosimulation system . ISP inhibited the bacterial regrowth and the following release of free LPS by 8 hrs . Great amounts of free LPS were released 4 hrs after the administration of PIPC and CAZ in the simulation system, associated with morphological changes; elongation, cell lysis or regrowth . IPM showed a strong bactericidal activity and less liberation of free LPS, but the free LPS level increased at 8 hrs, accompanied by the regrowth of the organism . The total amounts of LPS released by P . aeruginosa PAO1 in 8 hours of the pharmacokinetic simulation system were as follows; ISP < IPM < CAZ < PIPC.

Can Respir J, 1999 Jan-Feb, 6 Suppl A, 20A - 2A
A review of the mechanisms of action and resistance of antimicrobial agents; Wise R; Over the past 60 years, the introduction of new antibiotics has been matched by the development of new mechanisms of resistance by the bacteria . Current antibiotics act at a variety of sites within the target bacteria, including the cross-linking enzymes in the cell wall (beta-lactams), various ribosomal enzymes (macrolides, tetracyclines and aminoglycosides), nucleic acid polymerases (quinolones and rifampin) and folate synthesis (sulphas and trimethoprim) . Four major mechanisms of resistance have been shown . Target site alterations, such as changes to the penicillin-binding protein, are common . Inactivation of antimicrobials, as by penicillinases or the new carbapenemases, is often seen . Some bacteria such as Pseudomonas aeruginosa can produce alterations in cell wall permeability that deny access to antimicrobials during the course of therapy . Finally, newly described efflux mechanisms pump the antimicrobial out of the cell before it can reach its target site.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4360 - 5
Acyl homoserine-lactone quorum-sensing signal generation; Parsek MR et al.; Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression . These signals are synthesized by members of the LuxI family of proteins . To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme . Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine . An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism . Because pathogenic bacteria such as P . aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules.

Aust N Z J Med, 1999 Feb, 29(1), 15 - 21
Are antipseudomonal antibiotics really beneficial in acute respiratory exacerbations of cystic fibrosis?
Wolter JM, Bowler SD, McCormack JG.
BACKGROUND: Although anti-pseudomonal antibiotics are routinely used in the treatment of acute respiratory exacerbations of adult cystic fibrosis (CF), the specific efficacy of such treatment remains uncertain, the mechanism of action of these agents is not fully understood, and the role of sputum susceptibility testing in clinical decision making is controversial . AIMS: We investigated the relationship between susceptibility testing and clinical outcome in adult CF patients colonised with Pseudomonas aeruginosa . METHODS: Sputum samples were collected before, during and after respiratory exacerbations from 31 admissions (17 patients) . Sputum colony counts and MIC of P . aeruginosa were performed . RESULTS: Sputum colony counts did not change significantly during or after intravenous (IV) antibiotic therapy . Clinical outcome parameters (lung function, 12-minute walking distance, sputum weight and quality of life) were compared with susceptibility of P . aeruginosa colonies isolated at admission to the antibiotics used, and no correlation was evident . There was no evidence for the development of cross-resistance and there was no change in the proportion of mucoid colonies with therapy . CONCLUSIONS: While this study has a small patient sample size, it highlights the inconsistency of the role of antipseudomonal drugs also shown in other similar studies . The presence of P . aeruginosa in sputum of acutely ill CF patients prompts us to prescribe i.v . antipseudomonal drugs . If this approach was valid, we would expect to find a reduction in sputum colony counts and improvement in clinical parameters with the use of antibiotics to which the organisms were sensitive . The fact that such a relationship cannot be consistently demonstrated in this and other studies suggests that the use of antipseudomonal therapy in these patients requires more critical bacteriological and clinical evaluation.

Klin Monatsbl Augenheilkd, 1999 Jan, 214(1), 44 - 9
{Ocular bacteriosis in a patient with aplastic anemia}; Spraul CW et al.; BACKGROUND: For more than a century it has been recognized that bacteria may infect the eye by way of the blood stream . Until fifty years ago, eyes so afflicted nearly always were blinded and a majority of the victims died from overwhelming sepsis . Although effective antibiotics are available today the prognosis is still guarded . CASE REPORT: A 39-year-old physician died 6 years after he was diagnosed for idiopathic aplastic anemia . He had developed pseudomonas aeruginosa bacteremia and later a therapy refractive pneumonia caused by aspergillus which had led to respiratory failure . The patient had experienced a marked loss of visual acuity in his left eye ten days before he died . Ophthalmic examination at that point displayed signs of endophthalmitis, hyphaema, and heterochromic irises . The eyes were obtained postmortem and histologic examination of the left eye displayed numerous bacilli forming cuffs around blood vessels and a sheet between the necrotic retinal pigment epithelium and Bruch's membrane . No inflammatory response to the bacilli was present . The bacilli could be demonstrated with a Giemsa stain and could also be identified with electron microscopic examination . DISCUSSION: The histologic results in our patient displayed numerous bacilli without inflammatory reaction which was a result of the aplastic state . Therefore, this disease was classified as "ocular bacteriosis" . The blood-ocular barrier undergoes direct assault with intraocular inflammation and the impermeability to blood-borne antibiotics is lost . However, with the lack of an inflammatory reaction the permeability to antibiotics may be too low and the achieved concentration insufficient.

J Cataract Refract Surg, 1999 Apr, 25(4), 540 - 5
Pseudomonas aeruginosa endophthalmitis caused by contamination of the internal fluid pathways of a phacoemulsifier; Zaluski S et al.; PURPOSE: To report 4 cases of Pseudomonas aeruginosa endophthalmitis caused by internal contamination of the internal pathways of a phacoemulsifier . SETTING: Ophthalmology Center, Perpignan, France . METHODS: Four clinical cases of postoperative endophthalmitis occurred after phacoemulsification . An investigation was necessary to prove the cause of the bacteriological contamination . RESULTS: Serotyping and ribotyping of the Pseudomonas aeruginosa strains obtained from the vitreous samples and from the phacoemulsifier showed that all these strains were identical and that the initial site of the contamination was the phacoemulsifier . CONCLUSIONS: The profession should be cognizant of this cause of endophthalmitis, although its occurrence is rare . Cataract surgeons should test samples from the collection bags of their phacoemulsifiers to ensure there is no bacteriological contamination.

J Bacteriol, 1999 Apr, 181(8), 2459 - 64
The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa; Lu CD et al.; Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon . Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start . ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine . This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter . Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting . This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P . aeruginosa . Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites . In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter . According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

HNO, 1999 Feb, 47(2), 107 - 11
{Infection frequency and type of bacteria after tympanostomy tube drainage in childhood: gilded-silver tubes versus silicone tubes}; Schmal F et al.; Otorrhea is the most common complication after tympanostomy tube insertions . In Germany there are currently two commonly used types of tympanostomy tubes: silicon tubes (ST) and gilded silver tubes (GT) . Previously published in vitro studies by Tajima uncovered a positive correlation between the silicon concentration in culture fluid and the rate of growth of Staphylococcus aureus . Our study retrospectively evaluates the types of bacteria and rates of otorrhea after ST and GT insertions . The present study was undertaken to determine which of these tubes had a higher incidence of otorrhea and then whether silicon tubes stimulated the growth of certain types of bacteria, such as Staphylococcus aureus . In all, 186 ST and 59 GT were placed in 245 ears of 144 children . Both ST and GT were separated into three groups: first insertion of a tympanostomy tube, second implantation and insertion of a tympanostomy tube in an infected ear in the course of a mastoidectomy . No differences between ST and GT in causing otorrhea were found in the three groups . Nevertheless, ST in comparison to GT was associated with a higher incidence of infections with Pseudomonas aeruginosa . In contrast, a higher incidence of Staphylococcus aureus related to ST could not be proved . Twenty percent of the ears with mastoiditis were found to have Pseudomonas aeruginosa, but none of these ears implanted with a GT developed postoperative otorrhea . Our findings show that GT should be used when a ventilation tube is used during a mastoidectomy . Further, it is tenable to implant only GT because postoperative otorrhea in many cases is caused by insufficient water protection and water is frequently polluted with Pseudomonas aeruginosa.

J Appl Microbiol, 1999 Mar, 86(3), 537 - 43
Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa; Hirst PH et al.; It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition . In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied . Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6 . Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased . Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process . The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps . aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.

Am J Orthop, 1999 Mar, 28(3), 156 - 60
Sequential irrigation with common detergents: a promising new method for decontaminating orthopedic wounds; Burd T et al.; This investigation sought to determine the capacity of irrigation solutions in decontaminating orthopedic wounds challenged with a polymicrobial inoculum . Rats were divided into two groups, a control group and a treatment group . After creation of a dorsolumbar incision and placement of a wire through the spinous process, rats were inoculated with Staphylococcus aureus and Pseudomonas aeruginosa . Wounds were irrigated with control or treated solutions . At 2 weeks, cultures were obtained . There were statistically significant differences between groups regarding total number of culture positive sites (P < 0.001), culture-positive animals (P = 0.02), and quantitative cultures (P < 0.02) . Sequential irrigation with surfactants lowers bacteria counts recovered from polymicrobial wounds.

J Pept Res, 1999 Feb, 53(2), 170 - 6
Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase; Rival S et al.; Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides . The identity of the products was confirmed by FAB(+)-MS . After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured . With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position . This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2) . The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position . Furthermore, synthesis initial rates were at least 100 times faster with the elastase . To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy . We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups . It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine . Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position . A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

Thorax, 1998 Dec, 53(12), 1053 - 8
Cross-colonisation with Pseudomonas aeruginosa of patients in an intensive care unit; Bergmans DC et al.; BACKGROUND: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa is usually preceded by colonisation of the respiratory tract . During outbreaks, colonisation with P aeruginosa is mainly derived from exogenous sources . The relative importance of different pathways of colonisation of P aeruginosa has rarely been determined in non-epidemic settings . METHODS: In order to determine the importance of exogenous colonisation, all isolates of P aeruginosa obtained by surveillance and clinical cultures from two identical intensive care units (ICUs) were genotyped with pulsed field gel electrophoresis . RESULTS: A total of 100 patients were studied, 44 in ICU 1 and 56 in ICU 2 . Twenty three patients were colonised with P aeruginosa, seven at the start of the study or on admission and 16 of the remaining 93 patients became colonised during the study . Eight patients developed VAP due to P aeruginosa . The incidence of respiratory tract colonisation and VAP with P aeruginosa in our ICU was similar to that before and after the study period, and therefore represents an endemic situation . Genotyping of 118 isolates yielded 11 strain types: eight in one patient each, two in three patients each, and one type in eight patients . Based on chronological evaluation and genotypical identity of isolates, eight cases of cross-colonisation were identified . Eight (50%) of 16 episodes of acquired colonisation and two (25%) of eight cases of VAP due to P aeruginosa seemed to be the result of cross-colonisation . CONCLUSIONS: Even in non-epidemic settings cross-colonisation seems to play an important part in the epidemiology of colonisation and infection with P aeruginosa.

Vaccine, 1999 Mar 26, 17(13-14), 1663 - 6
A recombinant hybrid outer membrane protein for vaccination against Pseudomonas aeruginosa; Knapp B et al.; Among the numerous targets which can be used for the development of vaccines against Pseudomonas aeruginosa we focused on the outer membrane proteins OprF and OprI . The C-terminal part of OprF from aa 190 to aa 350 was investigated for its conservation and its localization of B-cell epitopes . A hybrid protein which combines the protective epitopes of OprF and OprI was expressed in E . coli and was proven to be highly protective against an intraperitoneal challenge with P . aeruginosa by active immunization of immunocompromised mice as well as by passive immunization of SCID mice with specific antisera . A purification procedure of the N-terminal His-tagged hybrid antigen was established using immobilized-metal-affinity chromatography . To evaluate its safety and immunogenicity the recombinant protein was purified for the immunization of human volunteers . The OprF/OprI hybrid protein is considered to be a candidate for a vaccine against P . aeruginosa.

J Pediatr, 1999 Apr, 134(4), 413 - 21
Comparison of a beta-lactam alone versus beta-lactam and an aminoglycoside for pulmonary exacerbation in cystic fibrosis; Smith AL et al.; We determined whether a beta-lactam and an aminoglycoside have efficacy greater than a beta-lactam alone in the management of a pulmonary exacerbation in patients with cystic fibrosis.Study design: Azlocillin and placebo or azlocillin and tobramycin were administered to 76 patients with a pulmonary exacerbation caused by Pseudomonas aeruginosa in a randomized double-blind, third-party monitored protocol . Improvement was assessed by standardized clinical evaluation, pulmonary function testing, sputum bacterial density, sputum DNA content, and time to the next pulmonary exacerbation requiring hospitalization . RESULTS: No significant difference was seen between the 2 treatment groups in clinical evaluation, sputum DNA concentration, forced vital capacity, forced expiratory volume in second 1, or peak expiratory flow rate at the end of treatment (33 receiving azlocillin alone and 43 both antibiotics); adverse reactions were equivalent in each group . Sputum P . aeruginosa density decreased more with combination therapy (P =.034) . On follow-up evaluation, an average of 26 days after the end of treatment, all outcome indicators had worsened in both groups . Time to readmission for a new pulmonary exacerbation was significantly longer in the group receiving azlocillin plus tobramycin (P <.001) . Treatment-emergent tobramycin resistance occurred in both groups and was more frequent with combination therapy . CONCLUSION: We conclude that the combination of a beta-lactam and an aminoglycoside produces a longer clinical remission than a beta-lactam alone and slightly better initial improvement.

Cutis, 1999 Mar, 63(3), 161 - 3
Subcutaneous nodules caused by Pseudomonas aeruginosa without sepsis; Aleman CT et al.; Pseudomonas aeruginosa infection can cause a wide array of skin manifestations . While some infections are mild, as are the cases with hot tub folliculitis and toe web or nail infection, others are a result of sepsis and can be fatal without prompt treatment . The classic skin finding of P . aeruginosa sepsis is Ecthyma gangrenosum, but other signs such as papules, petechiae, and hemorrhagic bullae can also be seen . Suppurative panniculitis can also be caused by P . aeruginosa sepsis and clinically manifests as solitary or multiple subcutaneous nodules . Reports in the literature describe these nodules in the setting of clinical sepsis or with positive blood cultures . We report a case of localized subcutaneous nodules on the leg caused by P . aeruginosa in a patient without sepsis or positive blood cultures . The source of the infection was thought to be from a traumatic inoculation . This raises the possibility that P . aeruginosa can cause subcutaneous nodules from a localized infection, perhaps via lymphangitic spread without the manifestations of sepsis.

J Biol Chem, 1999 Apr 9, 274(15), 10517 - 22
Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa; Guan L et al.; The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein . The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively . Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments . To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method . We obtained the following unique characteristics . MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane . Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively . This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM . Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively . This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model . This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly . This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.

Respir Care, 1991 Feb, 36(2), 104 - 9
Clinical evaluation of ColdSpor, a glutaraldehyde-phenolic disinfectant; Miner NA et al.; Disinfecting solutions may vary in concentration and bactericidal activity with use, may have subjectively unpleasant characteristics, and may affect the appearance and physical condition of the equipment processed . We evaluated the high-level disinfectant ColdSpor (0.5% glutaraldehyde, 0.025% ortho-phenylphenol, and 0.005% para-tertiary amylphenol) by using it as the disinfecting agent in the processing of equipment for a large respiratory care service . MATERIALS & METHOD: The type and quantity of equipment disinfected and the physical condition of the equipment were observed and recorded . Samples of the disinfectant solution were analyzed each week, and the antimicrobial activity of the solution was tested against clinical isolates and test cultures of Pseudomonas aeruginosa . The odor of the solution and ease of use were subjectively evaluated by two equipment-processing technicians . RESULTS: More than 2,400 pieces of respiratory equipment were passed through the disinfectant solution during the 30-day study period . No changes in the appearance of the equipment were noted . Analysis revealed that 71% or more of the antimicrobial chemicals remained in the solution . Clinical isolates and test cultures of P aeruginosa showed no growth when cultured with samples of the solution in use up to 30 days . The two technicians subjectively judged the solution to have no noticeable odor and to produce no burning of the eyes . CONCLUSION: The concentration of components of the glutaraldehyde-phenolic solution maintained bactericidal activity for as long as 30 days . The solution produced no apparent physical change in equipment and was subjectively acceptable to those processing equipment.

Hosp Formul, 1994 May, 29(5), 392 - 4, 399, 402-4
Concurrent ceftazidime DUE with clinical pharmacy intervention; Okpara AU et al.; Ceftazidime use at our institution, a 580-bed county teaching hospital, has steadily increased since its addition to the formulary in 1986 . In response to this increased use and because the institutional antibiogram showed increased resistance by Pseudomonas aeruginosa (from 10 to 28% resistant), the P & T Committee requested that a drug use evaluation (DUE) of ceftazidime be conducted . Analysis of this retrospective pilot study showed that 87% of ceftazidime use was inappropriate . To further evaluate ceftazidime use, to identify problems not assessed during retrospective review, and to correct problems while patients were receiving the drug, a concurrent ceftazidime use evaluation was conducted . The methods and results of the concurrent review are presented below.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 981 - 2
Pseudomonas aeruginosa: a survey of resistance in 136 hospitals in Spain . The Spanish Pseudomonas aeruginosa Study Group; Bouza E et al.; We carried out a nationwide study with all of the isolates of Pseudomonas aeruginosa collected in a week in 136 hospitals in Spain . The data on 1,014 isolates included resistance to the following antimicrobials: piperacillin-tazobactam, 7%; meropenem, 8%; amikacin, 9%; tobramycin, 10%; piperacillin, 10%; ticarcillin, 13%; imipenem, 14%; ceftazidime, 15%; cefepime, 17%; ciprofloxacin, 23%; aztreonam, 23%; ofloxacin, 30%; gentamicin, 31% . The most frequent serotypes were O:1 (25.1%), O:4 (21.6%), and O:11 (11.3%).

Antimicrob Agents Chemother, 1999 Apr, 43(4), 960 - 2
Integron-mediated rifampin resistance in Pseudomonas aeruginosa; Tribuddharat C et al.; A new rifampin resistance gene, arr-2, has been found in Pseudomonas aeruginosa . The ARR-2 protein shows 54% amino acid identity to the rifampin ADP-ribosylating transferase encoded by the arr gene from Mycobacterium smegmatis . This arr-2 gene is located on a gene cassette within a class I integron.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 902 - 6
Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-beta-lactamase IMP-1 produced by Escherichia coli; Laraki N et al.; The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture . The structural and functional properties of the enzyme purified from E . coli were identical to those of the enzyme produced by P . aeruginosa . The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems . Only monobactams escape its action . The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient . Two zinc-binding sites were found . The zinc content of the P . aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 882 - 9
Clavulanate induces expression of the Pseudomonas aeruginosa AmpC cephalosporinase at physiologically relevant concentrations and antagonizes the antibacterial activity of ticarcillin; Lister PD et al.; Although previous studies have indicated that clavulanate may induce AmpC expression in isolates of Pseudomonas aeruginosa, the impact of this inducer activity on the antibacterial activity of ticarcillin at clinically relevant concentrations has not been investigated . Therefore, a study was designed to determine if the inducer activity of clavulanate was associated with in vitro antagonism of ticarcillin at pharmacokinetically relevant concentrations . By the disk approximation methodology, clavulanate induction of AmpC expression was observed with 8 of 10 clinical isolates of P . aeruginosa . Quantitative studies demonstrated a significant induction of AmpC when clavulanate-inducible strains were exposed to the peak concentrations of clavulanate achieved in human serum with the 3.2- and 3.1-g doses of ticarcillin-clavulanate . In studies with three clavulanate-inducible strains in an in vitro pharmacodynamic model, antagonism of the bactericidal effect of ticarcillin was observed in some tests with regimens simulating a 3.1-g dose of ticarcillin-clavulanate and in all tests with regimens simulating a 3.2-g dose of ticarcillin-clavulanate . No antagonism was observed in studies with two clavulanate-noninducible strains . In contrast to clavulanate . No antagonism was observed in studies with two clavulanate-noninducible strains . In contrast to clavulanate, tazobactam failed to induce AmpC expression in any strains, and the pharmacodynamics of piperacillin-tazobactam were somewhat enhanced over those of piperacillin alone against all strains studied . Overall, the data collected from the pharmacodynamic model suggested that induction per se was not always associated with reduced killing but that a certain minimal level of induction by clavulanate was required before antagonism of the antibacterial activity of its companion drug occurred . Nevertheless, since clinically relevant concentrations of clavulanate can antagonize the bactericidal activity of ticarcillin, the combination of ticarcillin-clavulanate should be avoided when selecting an antipseudomonal beta-lactam for the treatment of P . aeruginosa infections, particularly in immunocompromised patients . For piperacillin-tazobactam, induction is not an issue in the context of treating this pathogen.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 794 - 801
Nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells; Sar B et al.; We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of Pseudomonas aeruginosa, could induce interleukin-8 (IL-8) generation in a variety of respiratory cells, including bronchial epithelial cells (K . Oishi et al . Infect . Immun . 65:2648-2655, 1997) . In this report, we examined the mode of nitrite reductase (PNR) release from a serum-sensitive strain of live P . aeruginosa cells during in vitro treatment with four different antimicrobial agents or human complement . Bacterial killing of P . aeruginosa by antimicrobial agents induced PNR release and mediated IL-8 production in human bronchial epithelial (BET-1A) cells . Among these agents, imipenem demonstrated rapid killing of P . aeruginosa as well as rapid release of PNR and resulted in the highest IL-8 production . Complement-mediated killing of P . aeruginosa was also associated with PNR release and enhanced IL-8 production . The immunoprecipitates of the aliquots of bacterial culture containing imipenem or complement with anti-PNR immunoglobulin G (IgG) induced twofold-higher IL-8 production than did the immunoprecipitates of the aliquots of bacterial culture with a control IgG . These pieces of evidence confirmed that PNR released in the aliquots of bacterial culture was responsible for IL-8 production in the BET-1A cells . Furthermore, the culture supernatants of the BET-1A cells stimulated with aliquots of bacterial culture containing antimicrobial agents or complement similarly mediated neutrophil migration in vitro . These data support the possibility that a potent inducer of IL-8, PNR, could be released from P . aeruginosa after exposure to antimicrobial agents or complement and contributes to neutrophil migration in the airways during bronchopulmonary infections with P . aeruginosa.

J Biochem (Tokyo), 1999 Apr, 125(4), 746 - 9
Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide; Tani M et al.; A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab . The radioactive substrate was used more preferentially by the acid ceramidase . It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide . For the alkaline and neutral enzymes, the Vmax and k (Vmax/Km) with C12-NBD-ceramide were much higher than those with 14C-ceramide . In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate . It is noteworthy that the labeling of ceramide with NBD did not itself reduce the Km of the alkaline enzyme, but did that of the neutral enzyme . It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines . This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes . Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases.

Biotechnol Bioeng, 1999 May 20, 63(4), 401 - 9
Kinetic model of biosurfactant-enhanced hexadecane biodegradation by Pseudomonas aeruginosa; Sekelsky AM et al.; Many sites of environmental concern contain groundwater contaminated with nonaqueous phase liquids (NAPL) . In such sites interfacial processes may affect both the equilibrium and kinetic behavior of the system . In particular, insoluble hydrocarbon partitioning and microbial biodegradation of insoluble hydrocarbon are influenced by the physicochemical and interfacial characteristics of the system . A mechanistic model describing the influence of biological surfactants on microbial biodegradation of liquid-phase insoluble hydrocarbon and subsequent reduction of nonaqueous-phase liquid hydrocarbon is presented . The model consists of six coupled differential equations which use lumped kinetic parameters to describe surfactant micelle formation and diffusion to the microbial cell, nonlinear kinetic expressions for microbial growth and degradation of insoluble hydrocarbon, kinetic spatial descriptions of the change in NAPL-phase droplet size and the organic phase volume fraction with time, as well as equilibrium partitioning expressions for hydrophobic organic contaminant partioning into the surfactant micelle . The model is validated by comparison to data obtained for hexadecane degradation in a well-mixed batch system by the biosurfactant producing microorganism Pseudomonas aeruginosa strain PG201 as well as for nonproducing mutants' growth and hexadecane biodegradation in the presence of exogenously added biosurfactant . Experimentally determined biological growth parameters, as well as physical parameters such as hydrocarbon droplet size, were applied in the kinetic model . Parameter sensitivity analysis was performed on the physical and biological parameters in the model . The parameter sensitivity analysis indicates that for the biological system examined the rate of hydrocarbon solubilization and micellar transport to the cell controls the rate at which cellular uptake and biodegradation of insoluble hydrocarbon occurs . Practical aspects relating to use of the model for support of surfactant-based bioremediation efforts are discussed .

Biotechnol Bioeng, 1998 Feb 20, 57(4), 462 - 70
Development of microbial mercury detoxification processes using mercury-hyperresistant strain of Pseudomonas aeruginosa PU21; Chang JS et al.; A mercury-hyperresistant strain of Pseudomonas aeruginosa PU21 harboring plasmid Rip64 was utilized to develop bioprocesses able to detoxify and recover soluble mercuric ions in aquatic systems . The kinetics of mercury detoxification was investigated to determine the parameters needed for the design of the bioprocesses . Batch, fed-batch, and continuous bioreactors were utilized to evaluate the efficiency and feasibility of each mode of operation . The results showed that the specific mercury detoxification rate was dependent on cell growth phases, as well as the initial mercury concentrations . Cells at the lag growth phase exhibited the best specific detoxification rate of approximately 1.1 x 10(-6) microg Hg/cell/h, and the rate was optimal at an initial mercury concentration of 8 mg/L . In batch operations with initial mercuric ions ranging from 2 to 10 mg/L, the mercuric ions added were rapidly volatilized from the media in less than 2-3 h . With periodic feeding of 3 or 5 mg Hg/L at fixed time intervals, the fed-batch processes had mercury removal efficiencies of 2.9 and 3.3 mg Hg/h/L, respectively . For continuous operations, the effluent cell concentration (Xe) was essentially invariant at 527 and 523 mg/L with the dilution rates (D) of 0.18 and 0.325 h-1, respectively . The increase in mercury feeding concentrations (Hgf) from 1.0 to 6.15 mg Hg2+/L did not affect the steady-state cell concentration (Xe) but forced the effluent mercury concentration (Hge) to increase . The decrease in the dilution rate, however, resulted in lower Hge values . It was also found that sequential mercury vapor absorption columns recovered over 80% of the Hg degrees released from the bioreactor while the residual mercury vapor was subsequently immobilized by an activated carbon trap in the down stream of the absorption column .

Am J Respir Cell Mol Biol, 1999 Apr, 20(4), 729 - 36
Proteinase 3, a potent secretagogue in airways, is present in cystic fibrosis sputum; Witko-Sarsat V et al.; We evaluated the roles of proteinase 3 (PR3) and human neutrophil elastase (HNE), two neutrophil serine proteinases in the mechanisms leading to airway inflammation and hypersecretion in cystic fibrosis (CF) . Using specific enzyme-linked immunosorbent assay (ELISA), we found higher levels of PR3 than HNE in sputum from CF patients . Using two inhibitors, ICI (Imperial Chemical Industries) 200,355 (which inhibits both HNE and PR3) and secretory leukoproteinase inhibitor (SLPI) (which inhibits only HNE), we showed that PR3 was enzymatically active in sputum, and its activity, as assessed by SLPI-resistant serine proteinase activity, correlated highly with its antigenic concentration measured by ELISA . Interestingly, sputum pellet-associated serine proteinase activity was mostly due to HNE . PR3 purified from neutrophil azurophil granules triggered airway gland secretion, as measured by the release of radiolabeled molecules from cultured bovine tracheal serous cells pulse-labeled with Na235SO4 . This secretory activity was inhibited by ICI 200,355 . PR3 concentration in CF sputum was highly correlated with taurine concentration, a reliable marker of airway inflammation and respiratory scores (e.g., FEV1%), whereas no significant correlation was observed with HNE . We verified that Pseudomonas aeruginosa proteinases did not interfere with the assessment of PR3 and HNE . Indeed, the PR3/HNE ratio was greatest in patients chronically infected by P . aeruginosa . We suggest that PR3 may play a role in the hypersecretory process that is characteristic of CF.

Am J Respir Cell Mol Biol, 1999 Apr, 20(4), 710 - 9
Characterization of chronic bronchopulmonary Pseudomonas aeruginosa infection in resistant and susceptible inbred mouse strains; Tam M et al.; Chronic bronchopulmonary Pseudomonas aeruginosa infection, initiated by intratracheal instillation of 1 to 2 x 10(5) colony-forming units of a mucoid strain of bacteria trapped in agar beads, was characterized in resistant BALB/c mice and susceptible C57BL/6 (B6) mice through 28 d postinfection . B6 mice experienced a more severe infection than BALB/c mice as evidenced by significantly higher mortality and significantly greater weight loss during the first 14 d . Furthermore, B6 mice had significantly higher numbers of bacteria in the lungs through 21 d after infection . Overall, only 22% of these hosts cleared the infection . In contrast, 67% of BALB/c mice cleared the infection . These differences between resistant and susceptible mice were found to correlate with histopathologic differences in the type of inflammation and the extent of tissue damage . An acute, predominantly neutrophilic inflammation and extensive tissue damage were apparent in the lungs of susceptible B6 mice, whereas chronic, granulomatous inflammation and little or no tissue damage were visible in resistant BALB/c mice . The finding of acute inflammation in the lungs of infected B6 mice was confirmed by fluorescence-activated cell sorter (FACS) analyses, which demonstrated that these mice had significantly greater proportions of polymorphonuclear neutrophils in the lungs on Days 7 and 14 after infection than did BALB/c mice . FACS analyses also revealed significant and similar increases in CD3(+) lung cells in both strains as the infection progressed . The CD4/CD8 ratio was significantly greater in BALB/c mice by 21 d after infection when the majority of these animals, but not B6 mice, had cleared the infection.

Infect Control Hosp Epidemiol, 1999 Mar, 20(3), 205 - 7
Nosocomial infections with ceftazidime-resistant Pseudomonas aeruginosa: risk factors and outcome; Lee SC et al.; Prospective studies were conducted for nosocomial Pseudomonas aeruginosa infections from February 1, 1994, to October 30, 1995 . Of 97 P . aeruginosa isolates from 97 patients, 35 were resistant to ceftazidime . Logistic regression revealed previous cephalosporin or piperacillin use as independent risk factors for nosocomial ceftazidime-resistant P . aeruginosa infection . Pulsed-field gel electrophoresis revealed that four nosocomial ceftazidime-resistant P . aeruginosa infections were caused by cross-infection, probably through medical personnel.

Gene, 1999 Mar 18, 229(1-2), 145 - 53
A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins; Matthey B et al.; Recombinant immunotoxins (rITs) are highly specific anti-tumor agents composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins . A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems . Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs . We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter . Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis . An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography . A major modification resulted from the insertion of a specifically designed multiple cloning site . It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed insertion of single-chain variable fragments (scFv) available from established bacteriophage systems . For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aeruginosa exotoxin A (ETA') . Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution . The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA' . These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.

J Bacteriol, 1999 Apr, 181(7), 2175 - 84
RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa; de Kievit T et al.; As components of a Pseudomonas aeruginosa quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second P . aeruginosa quorum-sensing system . To date, no information exists on negative regulation of the quorum-sensing cascade in P . aeruginosa . Here we describe a novel gene, rsaL, which is located downstream from lasR and transcribed antisense relative to lasR . In P . aeruginosa, overexpression of rsaL results in reduced lasB expression and decreased elastase activity . With the use of a six-His protein fusion system, we demonstrate that rsaL encodes an 11-kDa protein . Direct quantitation of PAI-1 levels in cultures and studies utilizing Escherichia coli lambda lysogens carrying lacZ transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene, lasI . RsaL's repressive effect on lasI and the associated decrease in elastase activity have important implications for the expression of all LasR-PAI-1-dependent virulence genes and the overall pathogenicity of P . aeruginosa.

Mol Microbiol, 1998 Nov, 30(4), 689 - 95
Fatal attraction of mammalian cells to Legionella pneumophila; Kwaik YA; Legionella pneumophila is a protozoan parasite that causes Legionnaires' disease . Its ability to do so is dependent on its capacity to replicate intracellularly within a phagosome that is not trafficked through the endosomal-lysosomal pathway and is surrounded by the rough endoplasmic reticulum . Within this unique niche, the bacterium undergoes alterations in gene expression . In addition, many virulence-related phenotypes that are induced in vitro by starvation are expressed intracellularly as the bacteria exit the logarithmic growth phase . (p)ppGpp appears to signal expression of the virulence-related genes in L . pneumophila upon starvation . This growth phase-dependent phenotypical transition is concomitant with lysis of the host cell, in which both necrosis and apoptosis seem to play roles . Many genetic loci that are required for intracellular replication within mammalian and protozoan cells have been identified, and the majority of them are novel . Two secretion systems have been identified, one of which may be distantly related to type IV secretion systems . The other is a type II secretion system similar to the PilBCD piliation system of Pseudomonas aeruginosa.

Presse Med, 1999 Feb 27, 28(8), 451 - 8
{Current microbiological problems . Antibiotic resistance and therapeutic problems raised by Pseudomonas aeruginosa}; Bert F et al.; RESISTANCE: Pseudomonas aeruginosa is characterized by its low intrinsic susceptibility to many antibiotics and its capacity to acquire additional resistance mechanisms to usually active drugs . Some beta-lactam resistance mechanisms are well known (penicillinase production, cephalosporinase overproduction) and others have been recently identified, such as active efflux systems, which confer coresistance to quinolones, and new beta-lactamases which are limited to a few countries (extended-spectrum beta-lactamases, imipenemase) . Ceftazidime remains the most active beta-lactam agent . ACTIVE DRUGS: Among aminoglycosides, amikacin and isepamicin are the most frequently active drugs . The use of fluoroquinolones is limited by a high incidence of acquired resistance . The percentage of resistant strains is highly variable according to countries, hospitals and wards . CLINICAL PRACTICE: Therapy, usually based on a beta-lactam-aminoglycoside combination, will be empirical at first, according to local epidemiological factors, site of infection and previously administered antibiotics, then re-evaluated according to susceptibility results.

J Biol Chem, 1999 Apr 2, 274(14), 9503 - 8
Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases; Ganesan AK et al.; Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites . One site appeared to be Arg41, but the second site could not be localized . In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined . Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras . Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS . Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128 . ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation . The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128 . Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras . The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases . This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases . These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.

J Mol Biol, 1999 Apr 2, 287(3), 695 - 715
Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps; Johnson JM et al.; Broad-specificity efflux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria . Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate efflux from the cytoplasmic membrane across the outer membrane through an unknown mechanism . The periplasmic efflux proteins (PEPs) and outer membrane efflux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the difficulty of obtaining reasonable multiple alignments . We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples . Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species . We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families . This process was used to identify a probable alpha-helical hairpin in the PEP family and was applied to the detection of transmembrane beta-strands in OEPs . We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions .

Biotechniques, 1999 Mar, 26(3), 473 - 8, 480
Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria; Lehoux DE et al.; We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo . A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids . Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening . Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR . A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P . aeruginosa 5.9-Mb genome . This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.

Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 539 - 41
Crystallization and preliminary X-ray crystallographic analysis of the Pseudomonas aeruginosa cyclohexadienyl dehydratase; Kongsaeree P et al.; The title protein has been crystallized in a new crystal form . The crystals belong to the cubic space group P4132 (or P4332) with unit-cell dimensions a = b = c = 126.1 A at 100 K and typically diffract beyond 1.6 A at the Cornell High Energy Synchotron Source (CHESS) A1 beamline.

Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 379 - 85
Structures of ruthenium-modified Pseudomonas aeruginosa azurin and {Ru(2,2'-bipyridine)2(imidazole)2}SO4 x 10H2O; Faham S et al.; The crystal structure of Ru(2, 2'-bipyridine)2(imidazole)(His83)azurin (RuAz) has been determined to 2.3 A -resolution by X-ray crystallography . The spectroscopic and thermodynamic properties of both the native protein and {Ru(2, 2'-bipyridine)2(imidazole)2}2+ are maintained in the modified protein . Dark-green RuAz crystals grown from PEG 4000, LiNO3, CuCl2 and Tris buffer are monoclinic, belong to the space group C2 and have cell parameters a = 100.6, b = 35.4, c = 74.7 A and beta = 106 . 5 degrees . In addition, {Ru(2,2'-bipyridine)2(imidazole)2}SO4 x 10H2O was synthesized, crystallized and structurally characterized by X-ray crystallography . Red-brown crystals of this complex are monoclinic, space group P21/n, unit-cell parameters a = 13.230 (2), b = 18.197 (4), c = 16.126 (4) A, beta = 108.65 (2) degrees . Stereochemical parameters for the refinement of Ru(2, 2'-bipyridine)2(imidazole)(His83) were taken from the atomic coordinates of {Ru(2,2'-bipyridine)2(imidazole)2}2+ . The structure of RuAz confirms that His83 is the only site of chemical modification and that the native azurin structure is not perturbed significantly by the ruthenium label.

Kaohsiung J Med Sci, 1999 Feb, 15(2), 80 - 6
Stability of self-prepared fortified antibiotic eyedrops; Lin CP et al.; Self-prepared fortified antibiotic eye drops are essential for the severe ocular infection . The relation of potency decay and storage conditions including temperature, concentration and duration were studied by the changes of MIC . 1% and 10% amikin, 10% and 50% pipril, and 5% and 25% vancomycin were diluted from the prarenteral antibiotics with the 5% glucose and storage at 4, -18 and -80 degrees C for 3, 7, 14 and 28 days . MICs of amikin against Pseudomonas aeruginosa, pipril and vancomycin against Staphylococcus aureus were determined by the agar diffusion method . Fluctuations of the MIC were noted during the observation period . Most of the significant changes of MIC were found during the first 7 days . When the potencies between time of zero and 28 days are compared, only 10% pipril and 25% vancomycin stored at -80 degrees C had significant change . Our conclusion is that all of the fortified antibiotic eye drops in this study can be stored in the house refrigerator or freezer for up to 28 days . High concentration may show a negative result of the preservation . Temperature does not influence the preservative effect within four weeks' observation.

Infect Immun, 1999 Apr, 67(4), 2040 - 4
Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells; Vallis AJ et al.; A strain of Pseudomonas aeruginosa that fails to express known type III-secreted effector proteins was constructed as an expression host . Individual effectors were expressed in trans, and their biological effects on CHO cells were assessed in an acute cellular infection model . Intoxication with ExoS, ExoT, or ExoY resulted in alterations in cell morphology . As shown in previous genetic studies, ExoU expression was linked to acute cytotoxicity.

Chest, 1999 Mar, 115(3), 746 - 50
Factors predicting mortality of patients with lung abscess; Hirshberg B et al.; BACKGROUND: The rates of morbidity and mortality associated with lung abscess are still significant despite the introduction of antibiotic treatments . The aim of this work was to identify the factors that predict a poor outcome for patients with lung abscess . METHODS: We retrospectively reviewed the records and the roentgenographic files of adult patients with lung abscess who were hospitalized from 1980 to 1996 at the Hadassah University Hospital, in Jerusalem, Israel . RESULTS: The study population comprised 75 patients, and the mean age was 52 years old (range, 12 to 89 years) . The mean (+/- SD) hospitalization duration was 25.7+/-21.5 days (range, 5 to 94 days) . Fifteen patients (20%) succumbed to the infection . The patients who died had more predisposing factors (+/-SD), such as pneumonia, neoplasm, and altered consciousness, than those who survived, respectively: 2.73+/-1.4 vs 1.9+/-1.3 (p < 0.03) . The patients with anemia on admission (hemoglobin levels of < 10 g/dL) had a higher mortality rate than those with higher hemoglobin levels, respectively: 58.3 vs 12.9% (p = 0.0008) . A higher mortality rate was also associated with infection by Pseudomonas aeruginosa (83%), Staphylococcus aureus (50%), and Klebsiella pneumoniae (44%) . The patients who died had larger abscess volumes (+/-SD) than those who survived (233+/-99 vs 157+/-33 mL), although it did not reach statistical significance . The diameter of the abscess correlated with the hospitalization time (r = 0.5; p < 0.001) . CONCLUSION: High rates of morbidity and mortality are associated with lung abscess despite appropriate antibiotic therapy and better supportive care . In patients with several predisposing factors, such as a large abscess size and a right-lower-lobe location, the prognosis was worse . The patients infected with S aureus, K pneumoniae, and particularly P aeruginosa had an ominous prognosis . As the prognosis for lung abscess has not improved sufficiently since the introduction of antibiotics, other modalities should be considered for patients with poor prognostic signs.

Cytometry, 1999 Mar 1, 35(3), 235 - 41
Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry; Suller MT et al.; BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative . In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively . METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue . At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer . RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed . DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts . CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations . The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure . CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics . The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.

Biochemistry, 1999 Mar 16, 38(11), 3379 - 85
H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin; Dong S et al.; Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer . Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines . The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84) . In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand . We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu . The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu . The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84 . The more stable mutants N38Q and N38T were characterized spectroscopically . The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa . The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter . Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding . These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.

Microbiology, 1999 Feb, 145 ( Pt 2), 471 - 81
Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase; Schobert M et al.; Pseudomonas aeruginosa ATCC 17933 grown aerobically on ethanol produces a soluble cytochrome c550 together with a quinoprotein ethanol dehydrogenase . A 3.2 kb genomic DNA fragment containing the gene encoding cytochrome c550 was cloned and sequenced . Two other complete and two truncated ORFs were also identified . A truncated ORF encoding the quinoprotein ethanol dehydrogenase (exaA) was found upstream of the cytochrome c550 gene (exaB) and in reverse orientation . An ORF encoding a NAD(+)-dependent acetaldehyde dehydrogenase (exaC) was located downstream of the cytochrome c550 gene and in the same orientation . Another ORF showed similarity to the pqqA gene and a truncated ORF similarity to the pqqB gene, both involved in the biosynthesis of the prosthetic group PQQ . The organization of these genes was found to be different from the well-studied methanol oxidation system in methylotrophic bacteria . The deduced amino acid sequence of cytochrome c550 from P . aeruginosa showed some similarity to cytochrome c6 of the alga Chlamydomonas reinhardtii and the haem domain of quinohaemoprotein alcohol dehydrogenases of acetic acid bacteria, but no similarity to the soluble cytochrome cL of the quinoprotein methanol oxidation system of methylotrophs could be detected . A mutant of P . aeruginosa with an interrupted cytochrome c550 gene was unable to grow on ethanol, which proves that cytochrome c550 is an essential component of the ethanol oxidation system in this organism.

Cardiovasc Surg, 1999 Jan, 7(1), 16 - 26
Source of elastin-degrading enzymes in mycotic aortic aneurysms: bacteria or host inflammatory response?
Buckmaster MJ, Curci JA, Murray PR, Liao S, Allen BT, Sicard GA, Thompson RW.
Elastolytic matrix metalloproteinases play a central role in the development of chronic atherosclerotic aortic aneurysms, but mycotic aortic aneurysms are a distinct and unusual form of aneurysm disease caused by bacterial infection . Mycotic aortic aneurysms follow a more rapid and unpredictable course than chronic aneurysm disease and they exhibit a predilection for the suprarenal aorta, further implying unique pathophysiologic mechanisms . The purpose of this study was to examine the nature and source of elastin-degrading enzymes in mycotic aortic aneurysm . Bacterial isolates and aortic tissues were obtained from four consecutive patients undergoing surgical repair of suprarenal mycotic aortic aneurysm . Using an in vitro 3H-labeled elastin degradation assay, elastin-degrading enzyme activity was only observed in the bacteria-conditioned medium from an isolate of Pseudomonas aeruginosa . Elastin-degrading enzyme activity in the aortic tissue homogenate of this patient was abolished by the serine protease inhibitor, phenylmethylsulfonyl fluoride, but it was not suppressed by the metalloproteinase inhibitor, ethylenediamine tetraacetic acid (EDTA) . In contrast, elastin-degrading enzyme activity in the bacterial-conditioned medium was decreased by about half by both phenylmethylsulfonyl fluoride and EDTA . Elastin substrate zymography revealed two phenylmethylsulfonyl fluoride-inhibitable elastin-degrading enzyme activities in the aortic tissue homogenate that corresponded to human neutrophil elastase (approximately 30 kDa) and its stable complex with alpha 1-proteinase inhibitor (approximately 80 kDa), but no activity attributable to Pseudomonas elastase, a 33-kDa metal-dependent enzyme . Human neutrophil elastase was readily detected throughout mycotic aortic aneurysm tissues by immunohistochemistry, but elastolytic metalloproteinases were only occasionally observed . The results of this study suggest that the elastin-degrading enzyme produced in mycotic aortic aneurysm are largely serine proteases of host neutrophil origin, rather than elastases produced by the infecting microorganisms or the macrophage-derived metalloproteinases typically observed in atherosclerotic aneurysm disease . Further studies will be needed to extend these findings to a larger number of patients with mycotic aortic aneurysm and those caused by additional microorganisms.

Curr Microbiol, 1999 Apr, 38(4), 239 - 43
The clinically isolated FIZ15 bacteriophage causes lysogenic conversion in Pseudomonas aeruginosa PAO1; Vaca-Pacheco S et al.; FIZ15 bacteriophage, from a human clinical isolate of Pseudomonas aeruginosa, causes lysogenic conversion in the P . aeruginosa strain PAO1 . The prophage-conferred phenotypes are: (1) increased resistance to phagocytosis by mouse peritoneal macrophages; (2) increased resistance to killing by normal human serum, and (3) increased adhesion to human buccal epithelial cells . These phenotypes are related to the prophage-induced change at the level of its own bacterial receptor, which appears to be the O-antigen.

Curr Microbiol, 1999 Apr, 38(4), 228 - 32
The occurrence of 4-amino-4-deoxy-arabinose in LPS of supersusceptible strain Pseudomonas aeruginosa Z61: noncorrelation with polymyxin resistance; Conrad RS et al.; Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities . The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P . aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC) . Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates . 31P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups . The presence of 4-AraN in P . aeruginosa LPS did not correlate with antimicrobial resistance.

Lett Appl Microbiol, 1999 Feb, 28(2), 103 - 7
Phototoxicity of rose bengal in mycological media--implications for laboratory practice; Chilvers KF et al.; The effects of Rose Bengal (RB) on plate counts of the bacteria Escherichia coli and Pseudomonas aeruginosa, and the yeast Saccharomyces cerevisiae, were studied under natural sunlight and artificial fluorescent lighting . While RB was not inherently toxic in darkness at concentrations found in mycological media, the illumination of media containing RB caused a decrease in colony counts in all cases, and especially for surface spread plates . A negative synergy was observed between chloramphenicol, RB and illumination using a spring water sample containing substantial numbers of Gram-negative bacteria . Exposure of media containing RB to moderate amounts of light during standard laboratory procedures may inhibit microbial growth, with positive benefits in relation to the suppression of contaminant bacteria, or negative implications where fungi are inhibited.

J Antimicrob Chemother, 1998 Dec, 42(6), 697 - 702
Mechanisms of beta-lactam resistance amongst Pseudomonas aeruginosa isolated in an Italian survey; Bonfiglio G et al.; The mechanisms of resistance to beta-lactam antibiotics in 325 isolates of Pseudomonas aeruginosa were examined . These isolates were selected because of their resistance to meropenem and imipenem (breakpoint, >4 mg/L), carbenicillin (>128 mg/L), ceftazidime (>8 mg/L), piperacillin and ticarcillin/clavulanate (>64 mg/L) . The most frequent mechanism of resistance was beta-lactamase-independent, so called 'intrinsic resistance', which was found in 183 isolates and was probably due to impermeability and/or efflux mechanisms . beta-Lactamase-mediated resistance was demonstrated in 111 strains (11.1%) . Derepression of Ambler Class C chromosomal beta-lactamase was detected in 64 isolates, most of which were resistant to ceftazidime and piperacillin but susceptible to meropenem, whereas secondary plasmid-encoded beta-lactamases were found in 34 isolates, all of them resistant to carboxypenicillins and ureidopenicillins and susceptible to carbapenems . Twelve strains showed more than one plasmid-encoded beta-lactamase plus derepression of chromosomal Class C enzyme . Resistance to carbapenems was independent of resistance to other beta-lactam antibiotics, indicating a different mechanism of resistance, probably due to the loss of the D2 porin . In total, 32 strains were resistant to carbapenems: 24 only to imipenem and eight to both imipenem and meropenem.

Eur J Clin Microbiol Infect Dis, 1998 Dec, 17(12), 873 - 6
Pharmacokinetics of meropenem in patients with cystic fibrosis; Christensson BA et al.; The pharmacokinetics of meropenem were studied after single i.v . infusions of 15 mg meropenem/kg body weight in eight subjects with cystic fibrosis (CF) and eight healthy volunteers matched for age, sex, and weight . Significantly shorter terminal half-lives (mean, 0.74 h vs . 0.99 h) and mean residence times (mean, 1.09 h vs . 1.39 h) were noted in CF subjects . Plasma and renal clearances tended to be higher and distribution volumes smaller among the patients, but differences were not statistically significant . The results are consistent with the findings for many other beta-lactam agents used in CF patients . Assuming a MIC90 of 4 mg/l for meropenem against Pseudomonas aeruginosa, the time above the MIC was less than 3.3 h in six of the eight CF patients . This finding should be kept in mind when designing treatment regimens with meropenem in CF subjects.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 155 - 61
Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa; Kato J et al.; Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC3 and PC4, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis . These mutants were not complemented by the P . aeruginosa cheY and cheZ genes, which had been previously cloned (Masduki et al., J . Bacteriol., 177, 948-952, 1995) . DNA sequences downstream of the cheY and cheZ genes were able to complement PC3 but not PC4 . Sequence analysis of a 9.7-kb region directly downstream of the cheZ gene found three chemotaxis genes, cheA, cheB, and cheW, and seven unknown open reading frames (ORFs) . The predicted translation products of the cheA, cheB, and cheW genes showed 33, 36, and 31% amino acid identity with Escherichia coli CheA, CheB, and CheW, respectively . Two of the unknown ORFs, ORF1 and ORF2, encoded putative polypeptides that resembled Bacillus subtilis MotA (40% amino acid identity) and MotB (34% amino acid identity) proteins, respectively . Although P . aeruginosa was found to have proteins similar to the enteric chemotaxis proteins CheA, CheB, CheW, CheY, and CheZ, the gene encoding a CheR homologue did not reside in the chemotaxis gene cluster . The P . aeruginosa cheR gene could be cloned by phenotypic complementation of the PC4 mutant . This gene was located at least 1,800 kb away from the chemotaxis gene cluster and encoded a putative polypeptide that had 32% amino acid identity with E . coli CheR.

Radiol Med (Torino), 1998 Nov, 96(5), 454 - 61
{Role of diagnostic imaging in the assessment of lung complications of burns}; Sergiacomi GL et al.; PURPOSE: We investigated the frequency of pulmonary complications in burn patients and the clinical and prognostic role of chest radiography and CT patterns in these patients . MATERIAL AND METHODS: We examined 203 patients with first- to third-degree burns involving up to 90% of the body surface; the patients were 119 men and 84 women ranging in age 1 to 96 years (mean: 30) . Burns on the face, sooth in the sputum and fire in an unventilated area indicated smoke inhalation in three patients . All patients were submitted to bedside chest radiography on hospitalization and the examination was repeated during the hospital stay in 26 patients . Seven patients with pulmonary complications also underwent chest CT . Five patients with severe, extensive burns (> 70% of the body surface) and with no clinical signs of respiratory complications were submitted to HRCT within 48 hours of admission . RESULTS: Lung complications developed in 16 patients (7.8%), leading to clinical and radiographic signs of adult respiratory distress syndrome (ARDS) in 11 of them (5.4%), namely 5 women and 6 men (age range: 19-96 years, mean: 50) . Only one of the three patients with smoke inhalation developed ARDS . The extension of burn injuries ranged 18-86% of the body surface . ARDS developed within 12 hours-14 days of injury (mean: 8 days) . Four patients (36%) had right lung involvement alone, two (18%) had bilateral, mostly right-lobe, abnormalities, and five patients (46%) had frankly bilateral findings; the latter were associated with pleural effusions in the left lower lobe in one patient . Compared with chest radiography, HRCT always identified the initial signs of interstitial edema and subpleural emphysematous bullae were detected in a patient who subsequently exhibited clinical and radiographic signs of ARDS . Nine (82%) of the 11 ARDS patients died of respiratory insufficiency . Most deaths (6 patients, 67%) occurred within a few hours of the onset of distress; in three patients with unilateral pulmonary edema death occurred within 6, 7 and 8 days, respectively . ARDS patients had significantly larger body surface areas burned and higher incidence of third-degree burns . DISCUSSION: The incidence of radiologically confirmed pneumonia was 1%; the causative pathogens were Pseudomonas aeruginosa and Staphylococcus aureus . HRCT detected a pneumatocele in a patient with Staphylococcus pneumonia . One patient had eosinophilic pleurisy and another a pulmonary microembolization . The overall mortality in our patients with burns and pulmonary complications was 56% versus 2% in the rest of the series, which confirms the importance of an early diagnosis to optimize treatment planning in such cases . For these reasons CT, and particularly HRCT, studies can be best because these techniques can show even minimal parenchymal changes . These examinations will be increasingly feasible also in critically ill and barely movable patients thanks to the latest mobile CT units which permit scanning also in intensive and subintensive care units.

J Am Board Fam Pract, 1999 Jan-Feb, 12(1), 1 - 7
Treatment patterns for otitis externa; Halpern MT et al.; BACKGROUND: Although otitis externa is a common and painful infection of the outer ear canal, there is little specific information available regarding current treatment patterns in the United States . We wanted to examine treatment patterns for otitis externa . METHODS: Data were analyzed from the 1993 National Ambulatory Medical Care Survey (NAMCS) and the 1993 National Hospital Ambulatory Medical Care Survey (NHAMCS) for adults and children treated for otitis externa . Data analyses included the reasons for physician visits, concomitant diagnoses, types of physicians seen, sources of payment, medical procedures administered, drugs prescribed, and patient disposition following a physician visit . RESULTS: Study results suggested that treatment patterns differ substantially for adults and children, as well as by physician specialty . Although otitis externa is frequently painful, few cases are classified as severe, and the data indicated that less than 20 percent of patients have concomitant diagnoses treatable by medication . Nevertheless, 40 percent of patients received both topical and systemic medication, and many of the oral antibiotics prescribed are not active against Staphylococcus aureus or Pseudomonas aeruginosa, the most common bacterial pathogens in otitis externa . CONCLUSIONS: Appropriate treatment of localized otitis externa with topical antibiotics should eliminate the need for systemic medications . Addition of systemic medications can unnecessarily increase treatment costs and the likelihood of side effects, and could reduce the likelihood of patient compliance.

Appl Environ Microbiol, 1999 Mar, 65(3), 1228 - 35
Tn5-induced and spontaneous switching of Sinorhizobium meliloti to faster-swarming behavior; Wei X et al.; Tn5 mutants of Sinorhizobium meliloti RMB7201 which swarmed 1.5 to 2 . 5 times faster than the parental strain in semisolid agar, moist sand, and viscous liquid were identified . These faster-swarming (FS) mutants outgrew the wild type 30- to 40-fold within 2 days in mixed swarm colonies . The FS mutants survived and grew as well as or better than the wild type under all of the circumstances tested, except in a soil matrix subjected to air drying . Exopolysaccharide (EPS) synthesis was reduced in each of the FS mutants when they were grown on defined succinate-nitrate medium, but the extent of reduction was different for each . It appears that FS behavior likely results from a modest, general derepression of motility involving an increased proportion of motile and flagellated cells and an increased average number of flagella per cell and increased average flagellar length . Spontaneous FS variants of RMB7201 were obtained at a frequency of about 1 per 10,000 to 20,000 cells by either enrichment from the periphery of swarm colonies or screening of colonies for reduced EPS synthesis on succinate-nitrate plates . The spontaneous FS variants and Tn5 FS mutants were symbiotically effective and competitive in alfalfa nodulation . Reversion of FS variants to wild-type behavior was sporadic, indicating that reversion is affected by unidentified environmental factors . Based on phenotypic and molecular differences between individual FS variants and mutants, it appears that there may be multiple genetic configurations that result in FS behavior in RMB7201 . The facile isolation of spontaneous FS variants of Escherichia coli and Pseudomonas aeruginosa indicates that switching to FS behavior may be fairly common among bacterial species . The substantial growth advantage of FS mutants and variants wherever nutrient gradients exist suggests that switching to FS forms may be an important behavioral adaptation in natural environments.

Appl Environ Microbiol, 1999 Mar, 65(3), 1175 - 9
Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates; Morgan JA et al.; Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains . While P . aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples . A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P . aeruginosa strains are not readily distinguishable . The variation in the central regions of the flagellin genes of seven of the isolates was investigated further . The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp) . The route by which flagellin gene variation has occurred in P . aeruginosa is discussed.

Am J Vet Res, 1999 Feb, 60(2), 181 - 5
Detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts; Howe LM et al.; OBJECTIVE: To determine existence of portal and systemic bacteremia in dogs with induced severe hepatic disease, compared with clinically normal dogs, before and after vena caval banding . ANIMALS: 6 control dogs and 10 dogs with induced severe hepatic disease and multiple portosystemic shunts (PSS) . PROCEDURE: Dogs of the diseased group were given dimethylnitrosamine (2 mg/kg of body weight, PO) twice weekly until multiple PSS developed . Surgery was performed on dogs of both groups, and blood for baseline aerobic and anaerobic bacterial culture was collected from catheters placed in the portal and hepatic veins and caudal vena cava . All dogs underwent vena caval banding, and blood for aerobic and anaerobic bacterial culture was collected from the portal and hepatic venous catheters at 120, 240, and 360 minutes after banding . RESULTS: Compared with control dogs (16% gram-positive and 84% gram-negative bacteria), diseased dogs had significantly higher percentage of gram-positive bacteria (42% of positive culture results, P < or = 0.01) and significantly lower percentage of gram-negative bacteria (58% of positive culture results, P < or = 0.01) isolated . Pseudomonas aeruginosa was isolated most frequently from dogs of both groups; more than 1 organism was isolated from 5 dogs of each group . Antimicrobial susceptibility included that to aminoglycosides (particularly amikacin), fluorinated quinolones, and imipenem . CONCLUSION: Portal and systemic, predominantly gram-negative, bacteremia is present in catheterized, clinically normal dogs and dogs with dimethylnitrosamine-induced hepatic disease and multiple PSS.

J Bacteriol, 1999 Mar, 181(5), 1623 - 9
Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility; Glessner A et al.; Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen . The production of several virulence factors by P . aeruginosa is controlled through two quorum-sensing systems, las and rhl . We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts . Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts . Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2 . Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili . Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type . It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili . The ability of P . aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system . Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells . Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.

J Bacteriol, 1999 Mar, 181(5), 1464 - 73
A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa; Kertesz MA et al.; When Pseudomonas aeruginosa is grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins) . The gene encoding one of these proteins, PA13, was isolated from a cosmid library of P . aeruginosa PAO1 and sequenced . It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have named msuEDC . The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2 for the reaction, and showed highest activity with methanesulfonate . MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme . Expression of the msu operon was analyzed with a transcriptional msuD::xylE fusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates . Growth with methanesulfonate required an intact cysB gene, and the msu operon is therefore part of the cys regulon, since sulfite utilization was found to be CysB independent in this species . Measurements of msuD::xylE expression in cysN and cysI genetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulating msu expression, and sulfonate utilization therefore appears to be tightly regulated.

J Bacteriol, 1999 Mar, 181(5), 1409 - 14
Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme; Pecina A et al.; The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp . This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein . The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location . The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria . The A . chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose . The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

Antimicrob Agents Chemother, 1999 Mar, 43(3), 530 - 6
Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV; Akasaka T et al.; The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe . The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms . The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P . aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E . coli ParC and ParE, respectively . The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined . The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity . So, the cloned genes were identified as P . aeruginosa topoisomerase IV genes . The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared . The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities of P . aeruginosa DNA gyrase . These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-type P . aeruginosa.

Surg Today, 1999, 29(2), 157 - 9
Induction of a critical elevation of povidone-iodine absorption in the treatment of a burn patient: report of a case; Aiba M et al.; A critical elevation of povidone-iodine absorption which occurred in a burn patient who was topically treated with 10% povidone-iodine (PI) gel is herein reported . A 65-year-old man was admitted to our hospital for deep second- and third-degree burns covering 26% of his total body surface area . The intravenous administration with lactated Ringer's solution and topical treatment with silver sulfadiazine were applied in addition to such treatments as debridement and skin grafting . However, wound infection occurred due to Pseudomonas aeruginosa . Topical treatment with PI gel was effective for this condition . Persistent nodal bradycardia with hypotension, metabolic acidosis, and renal failure occurred 16 days after the start of PI gel treatment . Iodine toxicosis caused by PI gel was suspected with a serum iodine level of 20600 microg/dl (normal range 2-9 microg/dl) . The PI gel treatment was therefore discontinued immediately, and hemodialysis was scheduled . However, the patient's family refused hemodialysis and he died 44 days after admission . To our knowledge, only eight patients with iodine toxicosis have been reported in burn patients treated with PI gel.

Lett Appl Microbiol, 1999 Jan, 28(1), 76 - 80
Survival and chromate reducing ability of Pseudomonas aeruginosa in industrial effluents; Ganguli A et al.; The ability of a chromate-reducing Pseudomonas aeruginosa strain, isolated from tannery effluent, to survive and reduce chromate in the effluent of a tannery and an electroplating unit was evaluated . The test strain survived in the native tannery effluent but numbers fell sharply in the native electroplating effluent . Supplementation with a carbon (C), nitrogen (N) and phosphorus (P) source supported bacterial multiplication and chromate reduction in both types of effluents with almost equal efficiency . Chromate reduction, however, was not observed in the absence of C, N or P supplement, or in the chromate-reducing strain.

Lett Appl Microbiol, 1999 Jan, 28(1), 13 - 6
Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa; Ayres HM et al.; Agents which had previously been shown to act as permeabilizers against Pseudomonas aeruginosa or other Gram-negative bacteria were tested to determine whether susceptibility to various antibiotics could be increased . In the absence of a permeabilizer, Ps . aeruginosa was resistant to several hydrophobic antibiotics and vancomycin, but not to gentamicin . Tartaric and gluconic acids had weak potentiating activity, whereas ethylenediamine tetraacetic acid and citric acid were more effective permeabilizers . However, sodium polyphosphate enhanced the activity of erythromycin, fucidin, novobiocin, rifampicin and methicillin; vancomycin was unaffected and the activity of gentamicin was reduced considerably.

Mol Microbiol, 1999 Jan, 31(2), 399 - 419
The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages; Nakayama K et al.; phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa . In this study, we determined the complete nucleotide sequence of the phi CTX phage genome . The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends . Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int . Among them, 15 gene products were identified in the phage particle by protein microsequencing . The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity . The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P . aeruginosa chromosome . In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding . These findings indicate that phi CTX is a P2-like phage well adapted to P . aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages . Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted . They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.

Infection, 1999 Jan-Feb, 27(1), 39 - 41
The use of SDS polyacrylamide gel electrophoresis of periplasmic proteins to subtype Pseudomonas aeruginosa pyocin type 10/b clinical isolates; Adwan K; The periplasmic protein banding patterns (PPBPs) of thirteen strains of Pseudomonas aeruginosa pyocin type 10/b implicated in two nosocomial outbreaks in the neonatal unit of Rafeidia Hospital, Nablus, Palestine, were examined . In addition, five strains from sporadic cases from the same unit occurring in 1996 and 1997 were also studied . Despite different sources of the strains, PPBPs generated by PAGE suggested a clonal nature of the strains obtained during each of the two outbreaks . Although they had very similar PPBPs, the two outbreak clones were not identical . In contrast, sporadic strains of P . aeruginosa pyocin type 10 appeared to be much more heterogeneous than those of the two outbreaks . PPBP analysis appeared to be a useful tool that may be of value for epidemiological purposes.

Infect Immun, 1999 Mar, 67(3), 1461 - 70
Safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers; Mansouri E et al.; A hybrid protein {Met-Ala-(His)6OprF190-342-OprI21-83} consisting of the mature outer membrane protein I (OprI) and amino acids 190 to 342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography . After safety and pyrogenicity evaluations in animals, four groups of eight adult human volunteers were vaccinated intramuscularly three times at 4-week intervals and revaccinated 6 months later with either 500, 100, 50, or 20 microg of OprF-OprI adsorbed onto A1(OH)3 . All vaccinations were well tolerated . After the first vaccination, a significant rise of antibody titers against P . aeruginosa OprF and OprI was measured in volunteers receiving the 100- or the 500-microg dose . After the second vaccination, significant antibody titers were measured for all groups . Elevated antibody titers against OprF and OprI could still be measured 6 months after the third vaccination . The capacity of the elicited antibodies to promote complement binding and opsonization could be demonstrated by a C1q-binding assay and by the in vitro opsonophagocytic uptake of P . aeruginosa bacteria . These data support the continued development of an OprF-OprI vaccine for use in humans.

Glycobiology, 1999 Mar, 9(3), 311 - 21
The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection; Davril M et al.; Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations . The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis . All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa . As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis . However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients . Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one alpha1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope . Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients . Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and alpha1,3 fucosyltransferases in the human bronchial mucosa.

Infect Immun, 1999 Mar, 67(3), 1508 - 10
Pseudomonas aeruginosa exoenzyme S is a biglutamic acid ADP-ribosyltransferase; Radke J et al.; Kinetic analysis of two mutations within Pseudomonas aeruginosa exoenzyme S (ExoS) showed that a E379D mutation inhibited expression of ADP-ribosyltransferase activity but had little effect on the expression of NAD glycohydrolase activity while a E381D mutation inhibited expression of both activities . These data identify ExoS as a biglutamic acid ADP-ribosyltransferase, where E381 is the catalytic residue and E379 contributes to the transfer of ADP-ribose to the target protein.

Infect Immun, 1999 Mar, 67(3), 1481 - 92
Cystic fibrosis transmembrane conductance regulator-mediated corneal epithelial cell ingestion of Pseudomonas aeruginosa is a key component in the pathogenesis of experimental murine keratitis; Zaidi TS et al.; Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for Pseudomonas aeruginosa ingestion into respiratory epithelial cells . In experimental murine keratitis, P . aeruginosa enters corneal epithelial cells . We determined the importance of CFTR-mediated uptake of P . aeruginosa by corneal cells in experimental eye infections . Entry of noncytotoxic (exoU) P . aeruginosa into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117 . Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to P . aeruginosa following corneal cell ingestion . In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (exoU+) or noncytotoxic P . aeruginosa resulted in large reductions in bacteria in the eye and markedly lessened eye pathology . Compared with wild-type C57BL/6 mice, heterozygous DeltaF508 Cftr mice infected with P . aeruginosa had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology . Homozygous DeltaF508 Cftr mice were nearly completely resistant to P . aeruginosa corneal infection . CFTR-mediated internalization of P . aeruginosa by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung, P . aeruginosa uptake by surface epithelial cells enhances P . aeruginosa clearance from this tissue.

Infect Immun, 1999 Mar, 67(3), 1207 - 12
The Pseudomonas aeruginosa secretory product pyocyanin inactivates alpha1 protease inhibitor: implications for the pathogenesis of cystic fibrosis lung disease; Britigan BE et al.; Alpha1 Protease inhibitor (alpha1PI) modulates serine protease activity in the lung . Reactive oxygen species inactivate alpha1PI, and this process has been implicated in the pathogenesis of a variety of forms of lung injury . An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease who are infected with Pseudomonas aeruginosa . P . aeruginosa secretes pyocyanin, which, through its ability to redox cycle, induces cells to generate reactive oxygen species . We tested the hypothesis that redox cycling of pyocyanin could lead to inactivation of alpha1PI . When alpha1PI was exposed to NADH and pyocyanin, a combination that results in superoxide production, alpha1PI lost its ability to form an inhibitory complex with both porcine pancreatic elastase (PPE) and trypsin . Similarly, addition of pyocyanin to cultures of human airway epithelial cells to which alpha1PI was also added resulted in a loss of the ability of alpha1PI to form a complex with PPE or trypsin . Neither superoxide dismutase, catalase, nor dimethylthiourea nor depletion of the media of O2 to prevent formation of reactive oxygen species blocked pyocyanin-mediated inactivation of alpha1PI . These data raise the possibility that a direct interaction between reduced pyocyanin and alpha1PI is involved in the process . Consistent with this possibility, pretreatment of alpha1PI with the reducing agent beta-mercaptoethanol also inhibited binding of trypsin to alpha1PI . These data suggest that pyocyanin could contribute to lung injury in the P . aeruginosa-infected airway of cystic fibrosis patients by decreasing the ability of alpha1PI to control the local activity of serine proteases.

Pediatr Pulmonol, 1999 Jan, 27(1), 62 - 4
Pseudomonal pericarditis complicating cystic fibrosis; Altemeier WA et al.; Patients with advanced cystic fibrosis typically have chronic bacterial infection of the upper and lower respiratory tracts, but rarely develop extrapulmonary sites of infection . We report a case of purulent pericarditis due to Pseudomonas aeruginosa in a patient with cystic fibrosis and no other risk factors for pericarditis . This is a previously unreported complication in cystic fibrosis prior to lung transplantation.

Ann Surg, 1999 Feb, 229(2), 272 - 8
Route and type of nutrition influence mucosal immunity to bacterial pneumonia; King BK et al.; OBJECTIVE: To develop a model of established respiratory immunity against Pseudomonas aeruginosa pneumonia and to investigate the effects of route and type of nutrition on this immunity . SUMMARY BACKGROUND DATA: Diet influences the ability of gut-associated lymphoid tissue (GALT) to maintain mucosal immunity . Complex enteral diets and chow maintain normal GALT populations against established IgA-mediated antiviral respiratory immunity . Both intravenous and intragastric total parenteral nutrition (TPN) produce GALT atrophy, but only intragastric TPN preserves established antiviral immunity . The authors hypothesized that both GALT-depleting diets (intragastric and intravenous TPN) would impair immunity against bacterial pneumonia . METHODS: P . aeruginosa was administered intratracheally to determine the mortality rate at increasing doses, and liposomes containing P . aeruginosa antigens were used to generate effective respiratory immunization . In the final experiment, mice received liposomes containing P . aeruginosa antigens to establish immunity and then were randomized to chow, complex enteral diets, intragastric TPN, or intravenous TPN . After 5 days of diet, mice received live intratracheal P . aeruginosa, and the death rate was recorded at 24 and 48 hours . RESULTS: The LD50 and LD100 were 9 x 10(7) and 12 x 10(7), respectively . Immunization reduced the mortality rate from 66% to 12% . This immunization was maintained in mice fed chow or a complex enteral diet and was lost in animals receiving intravenous TPN . Intragastric TPN partially preserved this respiratory immunity . CONCLUSIONS: Protection against bacterial pneumonia can be induced by prior antigenic immunization . This protection is lost with intravenous TPN, partially preserved with a chemically defined enteral diet, and completely preserved with chow or complex enteral diets . Both route and type of nutrition influence antibacterial respiratory tract immunity.

Rev Esp Quimioter, 1998 Dec, 11(4), 333 - 8
{Relationship between the sensitivity of Pseudomonas aeruginosa and the post-antibiotic effect of sparfloxacin and ciprofloxacin}; Pastor A et al.; The post antibiotic effect (PAE) of 8 strains of Pseudomonas aeruginosa with different susceptibility to sparfloxacin and ciprofloxacin was evaluated by the colony counting method using centrifugation to remove the antibiotic . The bacteria were exposed for 60 min to a quinolone concentration of 1 mg/ml . The MIC of sparfloxacin and ciprofloxacin ranged from 0.25-256 mg/ml and from 0 . 25-128 mg/ml, respectively . PAE values ranged from 46 8.71 to 59.6 2.51 min and from 46.33 15.2 to 62.6 3.70 min, respectively . No significant statistical differences were found in the PAE duration for quinolones nor between strains . No correlation could be established between PAE and susceptibility of P . aeruginosa to quinolones.

Arch Biochem Biophys, 1999 Feb 15, 362(2), 233 - 41
Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound; Rapoport N et al.; The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate . This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells . Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR . In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells . However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized . The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers . It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction . In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction . These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls .

Cell, 1999 Jan 8, 96(1), 47 - 56
Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model; Mahajan-Miklos S et al.; The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans . Using systematic mutagenesis of PA14 to identify mutants that fail to kill C . elegans and a C . elegans mutant that lacks P-glycoproteins, we identified phenazines, secreted P . aeruginosa pigments, as one of the mediators of killing . Analysis of C . elegans mutants with altered responses to oxidative stress suggests that phenazines exert their toxic effects on C . elegans through the generation of reactive oxygen species . Finally, we show that phenazines and other P . aeruginosa factors required for C . elegans killing are also required for pathogenesis in plants and mice, illustrating that this model tackles the dual challenges of identifying bacterial virulence factors as well as host responses to them.

Microb Drug Resist, 1998 Winter, 4(4), 257 - 61
Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa; Jalal S et al.; Principal mechanisms of bacterial resistance to quinolones are modification of target enzymes, DNA gyrase (gyrA) and topoisomerase IV (parC), or reduction of intracellular concentration due to mutations in the regulatory genes for efflux systems, such as mexR and nfxB . We have examined gyrA, parC, mexR, and nfxB genes from 16 quinolone-resistant clinical isolates of Pseudomonas aeruginosa to determine the relation between mutations in DNA replicating enzymes or regulatory genes for efflux systems and to correlate the mutations with minimal inhibitory concentrations (MICs) . The quinolone resistance-determining regions (QRDR) of these genes were amplified by PCR and sequenced by capillary electrophoresis . Fourteen of 16 isolates had mutations in gyrA, and 13/14 strains with MIC to norfloxacin > or = 8 mg/L had threonine at position 83 changed to isoleucine . Seven of 8 strains with MIC > or = 32 mg/L had mutations in parC . One of these strains showed a parC mutation at position 74 without any mutation in gyrA . Four strains had mexR and two strains nfxB mutations . The data indicate that gyrA mutation is the most important component of quinolone resistance, and simultaneous presence of parC mutations is associated with high-level resistance . parC mutation alone may contribute to resistance, and gyrA mutation may not be a prerequisite for parC mutation to express resistance . mexR and nfxB mutations were found mostly in strains with high-level resistance.

Mol Microbiol, 1998 Dec, 30(5), 933 - 41
Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis; Kamath S et al.; Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host . Elastase is secreted in non-mucoid P . aeruginosa . It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P . aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients . We have previously reported that in mucoid P . aeruginosa, an intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form . This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis . The main aim of this study was to purify and characterize this protease . The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P . aeruginosa 8821, a CF isolate . Further analysis using a gelatin containing SDS-polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS-polyacrylamide gel . The first 10 amino acids from the N-terminus of the 33 kDa protease showed 100% identity to the mature form of elastase . An elastase-negative lasB::Cm knock-out mutant in the mucoid 8821 background was constructed, and it showed a non-mucoid phenotype . This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions . We present evidence for the retention of active elastase in the periplasm of mucoid P . aeruginosa and its role in the generation of the 12 kDa form of Ndk . Finally, we demonstrate that elastase, when overproduced in both mucoid and non-mucoid cells, stimulates alginate synthesis . This suggests that the genetic rearrangements that trigger mucoidy in P . aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.

Pediatr Nurs, 1998 May-Jun, 24(3), 258 - 9
Inhaled tobramycin (TOBI); Bonsignore CL; Inhaled tobramycin was recently approved by the FDA in a 300 mg formulation for inhalation . The new product, manufactured by PathoGenesis Corporation, is referred to as TOBI and is indicated for cystic fibrosis patients with Pseudomonas aeruginosa . The advantage of high dose inhaled tobramycin is a greater concentration of the drug delivered to bacteria in the lung with potentially reduced oto- and nephrotoxicity.

Vaccine, 1999 Jan, 17(2), 158 - 68
Human immune response to a Pseudomonas aeruginosa outer membrane protein vaccine; Jang IJ et al.; In order to evaluate in humans the safety and immunogenicity of a Pseudomonas aeruginosa vaccine composed of outer membrane proteins (OMPs), CFC-101, we carried out a phase I/IIa clinical trial in healthy male volunteers . Groups of six volunteers were immunized either subcutaneously (s.c.) or intramuscularly (i.m.) with three dosages of the vaccine three times at 7-day intervals . The vaccine was well tolerated by volunteers . Local reactions in the injection sites were generally mild and transient . Significant increases in OMP-specific antibody were observed in both route groups after vaccinations but was higher in the i.m.-immunized group, where vaccination with 0.5 or 1.0 mg doses yielded 100% seroconversion . The specificity of the induced antibodies to P . aeruginosa OMP was demonstrated by western blot analysis and immunoprecipitation assay . An increase in Clq-binding capacity and ability to confer mice protection from lethal challenges with P . aeruginosa indicated the protective efficacy of the elicited antibodies . Based on these data, we concluded that the P . aeruginosa OMP vaccine is safe and effective in humans with an optimal dose of 0.5 and 1.0 mg and that i.m . is the better route than s.c . for this vaccine.

J Bacteriol, 1999 Feb, 181(4), 1281 - 91
Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor; Ohnishi M et al.; Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis . The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced . The P . aeruginosa frr was mapped at 30 to 32 min of the P . aeruginosa chromosome . The deduced amino acid sequence of RRF showed a 64% identity to that of E . coli RRF . In an assay including E . coli polysome and elongation factor G, purified recombinant RRF of P . aeruginosa released monosomes from polysomes . This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery . The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr . The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E . coli and Bacillus subtilis . Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved . Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF . This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.

J Bacteriol, 1999 Feb, 181(4), 1072 - 8
Physiological characterization of Pseudomonas aeruginosa during exotoxin A synthesis: glutamate, iron limitation, and aconitase activity; Somerville G et al.; Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from Pseudomonas aeruginosa . We tested the possibility that glutamate affects growth during iron restriction . We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate . We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation . Glutamate had no effect on toxA (ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells . In contrast, citrate and isocitrate diminished toxA transcription . Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle . Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control . There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis . We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility . Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo . In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase . We speculate that one or more of the aconitases in P . aeruginosa may contribute to the control of virulence factor synthesis.

Rev Invest Clin, 1998 Sep-Oct, 50(5), 383 - 8
Epidemiology and prognosis of Pseudomonas aeruginosa bacteremia in a tertiary care center; Sifuentes-Osornio J et al.; OBJECTIVE: To describe the epidemiology and prognosis of P . aeruginosa bloodstream infections in a tertiary-care center . DESIGN: Retrospective analysis . SETTING: Tertiary-care teaching hospital in Mexico City . PATIENTS: All cases of P . aeruginosa bacteremia diagnosed from 1981 to 1994 . DATA: Relevant demographic, clinical and therapeutic variables were analyzed . RESULTS: A total of 153 bacteremias were found between 1981 and 1994, with a mean prevalence of 4.1 episodes per 1000 hospital discharges . Twenty-five percent of the infections derived from the biliary tract, and the most frequent underlying diseases were hematologic malignancies . The overall crude mortality was 46% (70/153) whereas mortality in the nosocomially-acquired episodes was 47% (58/124) . Mortality within the first 72 h was 24% (37/153) . A multivariate analysis showed six risk factors associated with a fatal outcome: age > or = 40 years, shock, mechanical ventilation, prior use of antibiotics, splenectomy and inappropriate selection of antibiotics . CONCLUSION: The identification of risk factors, and therefore a prompt instauration of specific antibiotic therapy, improved the prognosis of these severely ill patients.

Crit Care Med, 1999 Jan, 27(1), 162 - 7
Selective inhibition of inducible nitric oxide synthase: effects on hemodynamics and regional blood flow in healthy and septic sheep; Booke M et al.; OBJECTIVES: To investigate the effects of S-ethylisothiourea (S-EITU) on hemodynamics, oxygen transport, and regional blood flow in healthy and septic sheep . DESIGN: Prospective, randomized, controlled experimental study with repeated measures . SETTING: Investigational intensive care unit at a university medical center . SUBJECTS: Eleven healthy, female adult sheep of the Merino breed, divided into a control group (n = 5) and into a group treated with S-EITU (n = 6) . INTERVENTIONS: All sheep were chronically instrumented . After a 5-day recovery period, they were randomly assigned to either control or S-EITU groups . While control sheep received only saline, S-EITU was administered in increasing doses of 1, 3, and 9 mg/kg/hr over 1 hr each (nonseptic phase) . After 2 days of recovery, a continuous infusion of live Pseudomonas aeruginosa (2.5 x 106 colony-forming units/min) was started in all sheep and maintained for the remainder of the experiment . After 24 hrs of sepsis, the sheep again received their assigned treatment (septic phase) . In both the nonseptic and septic phases, the sheep received colored microspheres through a left atrial catheter to allow analysis of regional blood flows . All animals were autopsied at the end of the experiments, and organ probes were removed for blood flow analyses . MEASUREMENTS AND MAIN RESULTS: The administration of S-EITU caused a dose-dependent vasoconstriction in the nonseptic phase . After 24 hrs of Pseudomonas infusion, all sheep developed a hyperdynamic circulatory state, with increased cardiac indices and reduced arterial pressures and systemic vascular resistances . Oxygen extraction decreased significantly, preventing an increase in oxygen consumption, despite an increased oxygen delivery . The hyperdynamic circulation was dose dependently reversed by S-EITU, causing an increase in arterial pressure by peripheral vasoconstriction . Sheep in the control group showed a continuation of the hyperdynamic circulation . The effects of S-EITU on hemodynamics and regional blood flows were comparable under septic and nonseptic conditions . CONCLUSIONS: With the inducible form of nitric oxide synthase expressed under septic, but not under nonseptic conditions, S-EITU was expected to have vasoconstrictive properties only in the septic phase . It produced a comparable vasoconstriction during the nonseptic phase of the experiment . Thus, either S-EITU does not selectively block the inducible nitric oxide synthase in sheep, or other vasodilators besides nitric oxide play an important role in septic vasodilation.

J Burn Care Rehabil, 1999 Jan-Feb, 20(1 Pt 1), 42 - 9
Contribution of the regulatory gene lasR to the pathogenesis of Pseudomonas aeruginosa infection of burned mice; Rumbaugh KP et al.; Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes severe infections in patients with burns . The P aeruginosa regulatory gene, lasR, regulates the expression of several virulence factors . The specific lasR isogenic mutant, PAO-R1, is defective in the synthesis of the 2 elastases (LasB and LasA) and produces low levels of exotoxin A and alkaline proteases . In this study, we used a burned mouse model to examine the role of lasR in the pathogenesis of P aeruginosa infections . We have examined the following aspects of P aeruginosa infections: 1) lethality to the burned mouse, 2) the dissemination within the body of the burned mouse, and 3) the local spread within the burned skin . In comparison with its parent strain, PAO1, PAO-R1 was less lethal . In addition, the numbers of PAO-R1 microorganisms recovered from the livers and spleens of the burned mice were less than those of PAO1 . Furthermore, at 8 hours postinfection, equivalent numbers of PAO1 and PAO-R1 were detected at the inoculation site of the burned skin . However, only PAO1 microorganisms were detected at other sites of the burned skin . These results suggest that the lasR gene contributes (directly and indirectly) to the dissemination of P aeruginosa within the body of burned mice and its horizontal spread within the burned skin.

Clin Exp Immunol, 1999 Jan, 115(1), 103 - 9
Quantitative and qualitative differences in bronchoalveolar inflammatory cells in Pseudomonas aeruginosa-resistant and -susceptible mice; Sapru K et al.; The difference in severity of Pseudomonas aeruginosa-induced chronic lung infection may be determined by differences in host inflammatory responses . In the present study we investigate this possibility using BALB/c and C57Bl/6 mice, resistant and susceptible, respectively, to chronic lung infection with P . aeruginosa . Following intratracheal inoculation of P . aeruginosa-impregnated agar beads, C57Bl/6 mice mounted a stronger inflammatory response with significantly higher total cell numbers in the bronchoalveolar lavage fluid compared with BALB/c mice . While polymorphonuclear leucocytes were the predominant cell in C57Bl/6 mice, macrophages constituted the majority in BALB/c mice at day 7 post-infection . Alveolar macrophages from C57Bl/6 mice showed significantly higher spontaneous production of nitric oxide (NO) at day 7 post-infection compared with BALB/c mice . Following in vitro stimulation with heat-killed Pseudomonas antigen, these cells produced significantly higher NO compared with cells from BALB/c mice at day 21 post-infection . Production of tumour necrosis factor-alpha (TNF-alpha) by alveolar macrophages was significantly higher at day 7 in BALB/c mice compared with C57Bl/6 mice, which showed significantly higher levels at day 28 post-infection . Taken together, these results suggest that defects in the host inflammatory process contribute to the variable outcome of chronic lung infection with P . aeruginosa . An exaggerated inflammatory response dominated by polymorphonuclear cells correlates with susceptibility to infection, whilst a modest inflammatory response dominated by macrophages correlates with resistance . Moreover, the quantity and timing of production of NO and TNF-alpha by alveolar macrophages may modulate the course and outcome of infection.

Perit Dial Int, 1998 Nov-Dec, 18(6), 637 - 40
Pseudomonas exit-site infections in CAPD patients: evolution and outcome of treatment; Lo CY et al.; OBJECTIVE: To examine the natural history of Pseudomonas aeruginosa (PSA) exit-site infections (ESI) in patients treated with antibiotics with or without surgical interventions . DESIGN: Retrospective record review from May 1994 to April 1997 . SETTING: A single dialysis unit in a district hospital . PATIENTS: The review included 353 patients who had undergone continuous ambulatory peritoneal dialysis (CAPD) . OUTCOME MEASURES: The prevalence and etiology of ESI, the treatment regimen for PSA ESI, and the outcome of treatment . RESULTS: The prevalence of ESI was 55% . A total of 131 episodes (range 1-5) of PSA ESI occurred in 78 (40.2%) of the 194 patients who experienced ESI . Among these 78 patients, 4 groups with different outcomes were identified . In group I, 35 patients (44.9%) were treated successfully with antibiotic therapy alone . Among these 35 patients, 4 developed PSA peritonitis at a mean of 5 months (range 2-10 mth) after apparent clinical resolution of PSA ESI . Two of the 4 patients switched to long-term hemodialysis (HD) because of peritoneal failure . In group II, 8 patients (10.3%) responded to a combination of antibiotics and shaving of the external cuff . In group III, 21 patients (26.9%) with recurrent ESI underwent elective Tenckhoff catheter removal and reinsertion . One of the 21 patients had relapse of PSA ESI 14 months after the operation . In group IV, 14 patients (17.9%) had recurrent PSA ESI that failed to respond to multiple courses of antibiotics and shaving of the external cuff . Consent for Tenckhoff catheter removal was not obtained and 4 of these 14 patients subsequently developed PSA peritonitis . One of the 4 patients changed to permanent HD due to peritoneal failure . CONCLUSIONS: Considering the increased risk and the poor outcome of PSA peritonitis in patients with persistent PSA ESI, early Tenckhoff catheter removal is recommended if the patient fails to respond to antibiotics with or without externalization of the external cuff.

Biochem J, 1999 Feb 15, 338 ( Pt 1), 29 - 33
Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect anti-bacterial peptide; Saido-Sakanaka H et al.; Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria . This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus . We identified the active site of beetle defensin by measuring anti-bacterial activity against S . aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini . An LCAAHCLAIGRR-NH2 (19L-30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides . The 19L-30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria . CD spectra showed that the 19L-30R-NH2 fragment formed an alpha-helical structure in the lipidic environment . The anti-bacterial effect of the 19L-30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose . Its anti-bacterial activity was increased when certain amino acid residues were replaced . Truncated peptides having had some amino acids removed from the N-terminus of the 19L-30R-NH2 fragment (8-10-mer peptides) still had strong anti-bacterial activity . Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L-30R-NH2 fragment, i.e . RR-NH2, is important for exerting anti-bacterial activity . The AHCLAIGRR-NH2 (22A-30R-NH2) fragment and its analogues exhibited about 3-fold and 9-12-fold higher activity against S . aureus than did the 19L-30R-NH2 fragment, and these analogues were effective against methicillin-resistant S . aureus and Pseudomonas aeruginosa isolated from patients . These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

Rocz Panstw Zakl Hig, 1998, 49(3), 299 - 312
{Sensitivity of Pseudomonas sp . strain isolated from distilled water to disinfectants}; Krzywicka H et al.; The aim of the study was to compare Pseudomonas sp . strain isolated from distilled water and grown on the sterile filtrate from this water with the referent strain of Pseudomonas aeruginosa NCTC 6749 grown under the standard conditions, with the respect to their sensitivity to eight common disinfectants . Partly modified EN 1040 method was applied . The reduction factor (RF) for at least five concentrations of each of the products were determined . The linear relationship between concentration of the disinfectants and RF was established in a graphic form . Sensitivity of the strains was compared at the RF = 4 . The WS strain was found to be more sensitivity than the referent strain to the disinfectants . The ratio concentrations of the solutions that were effective during 15 minutes against both the strains were; 1.3 for ethyl alcohol; 2.1 for glutaraldehyde; 1.7 for formalin; 1.85 for phenol . In the case of chloramine-T, the sodium salt of dichloroisocyanuric acid, and peracetic acid the ratios of concentrations were 156.0; 96.5 and 21.5 respectively . They indicate much higher sensitivity of the strain isolated from distilled water to these chemicals.

Toxicology, 1998 Nov 16, 131(2-3), 133 - 43
Sarcophytolide: a new neuroprotective compound from the soft coral Sarcophyton glaucum; Badria FA et al.; Bioactivity-guided fractionation of an alcohol extract of the soft coral Sarcophyton glaucum collected from the intertidal areas and the fringing coral reefs near Hurghada, Red Sea, Egypt resulted in the isolation of a new lactone cembrane diterpene, sarcophytolide . The structure of this compound was deduced from its spectroscopic data and by comparison of the spectral data with those of known closely related cembrane-type compounds . In antimicrobial assays, the isolated compound exhibited a good activity towards Staphylococcus aureus, Pseudomonas aeruginosa, and Saccharomyces cerevisiae . Sarcophytolide was found to display a strong cytoprotective effect against glutamate-induced neurotoxicity in primary cortical cells from rat embryos . Preincubation of the neurons with 1 or 10 microg/ml of sarcophytolide resulted in a significant increase of the percentage of viable cells from 33 +/- 4% (treatment of the cells with glutamate only) to 44 +/- 4 and 92 +/- 6%, respectively . Administration of sarcophytolide during the post-incubation period following glutamate treatment did not prevent neuronal cell death . Pretreatment of the cells with sarcophytolide for 30 min significantly suppressed the glutamate-caused increase in the intracellular Ca2+ level ({Ca2+}i) . Evidence is presented that the neuroprotective effect of sarcophytolide against glutamate may be partially due to an increased expression of the proto-oncogene bcl-2 . The coral secondary metabolite, sarcophytolide, might be of interest as a potential drug for treatment of neurodegenerative disorders.

J Surg Res, 1999 Feb, 81(2), 147 - 55
Selective downregulation of neutrophils by a phosphatidic acid generation inhibitor in a porcine sepsis model; Oka Y et al.; Effects of lisofylline (1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine), a functional inhibitor of phosphatidic acid (PA) generation derived from de novo synthesis, on neutrophil function were examined in a porcine sepsis model . Hanford minipigs (18-25 kg) were randomly separated into six groups of six animals each: (1) saline control group; (2) sepsis control group, infused with Pseudomonas aeruginosa (1 x 10(6) colony-forming units/kg/min) for 2 h; (3) lisofylline control group, given a 25 mg/kg bolus of lisofylline 30 min prior to time zero, followed by a continuous infusion of 10 mg/kg/h throughout the study; (4) lisofylline pretreatment sepsis group, given lisofylline 30 min prior to sepsis, (5) lisofylline 1-h post-treatment sepsis group, and (6) lisofylline 2-h post-treatment sepsis group . All animals were studied for 6 h . Neutrophils were isolated at -0.5, 2, and 6 h . In the pretreatment and 1-h post-treatment groups, sepsis-induced neutrophil attachment to fibronectin was significantly attenuated . Sepsis-enhanced phagocytic activity was significantly reduced in the lisofylline pretreatment sepsis group, but not in the post-treatment groups . No treatment affected phorbol 12-myristate 13-acetate-induced chemiluminescence and basal filamentous actin content, which increased in sepsis, and cap formation, which declined in sepsis . Sepsis caused neutropenia, pretreatment produced neutrophilia, and 1-h post-treatment caused the neutropenia to recover to control levels . Interestingly, toward the end of the 6-h period, the neutrophil count was higher in the lisofylline control group than in the saline control groups . Thus, the inhibition of PA generation from de novo synthesis during sepsis not only can selectively downregulate some neutrophil functions but can also reverse neutropenia .

Appl Environ Microbiol, 1999 Feb, 65(2), 489 - 98
Effect of O-side-chain-lipopolysaccharide chemistry on metal binding; Langley S et al.; Pseudomonas aeruginosa PAO1 produces two chemically distinct types of lipopolysaccharides (LPSs), termed A-band LPS and B-band LPS . The A-band O-side chain is electroneutral at physiological pH, while the B-band O-side chain contains numerous negatively charged sites due to the presence of uronic acid residues in the repeat unit structure . Strain PAO1 (A+ B+) and three isogenic LPS mutants (A+ B-, A- B+, and A- B-) were studied to determine the contribution of the O-side-chain portion of LPS to metal binding by the surfaces of gram-negative cells . Transmission electron microscopy with energy-dispersive X-ray spectroscopy was used to locate and analyze sites of metal deposition, while atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry were used to perform bulk quantitation of bound metal . The results indicated that cells of all of the strains caused the precipitation of gold as intracellular, elemental crystals with a d-spacing of 2.43 A . This type of precipitation has not been reported previously for gram-negative cells and suggests that in the organisms studied gold binding is not a surface-mediated event . All four strains bound similar amounts of copper (0.213 to 0.222 micromol/mg {dry weight} of cells) at the cell surface, suggesting that the major surface metal-binding sites reside in portions of the LPS which are common to all strains (perhaps the phosphoryl groups in the core-lipid A region) . However, significant differences were observed in the abilities of strains dps89 (A- B+) and AK1401 (A+ B-) to bind iron and lanthanum, respectively . Strain dps89 caused the precipitation of iron (1.623 micromol/mg {dry weight} of cells) as an amorphous mineral phase (possibly iron hydroxide) on the cell surface, while strain AK1401 nucleated precipitation of lanthanum (0.229 micromol/mg {dry weight} of cells) as apiculate, surface-associated crystals . Neither iron nor lanthanum precipitates were observed on the cells of other strains, which suggests that the combination of A-band LPS and B-band LPS produced by a cell may result in a cell surface which promotes the formation of metal-rich precipitates . We therefore propose that the negatively charged sites located in the O-side chains are not directly responsible for the binding of metallic ions; however, the B-band LPS molecule as a whole may contribute to overall cell surface properties which favor the precipitation of distinct metal-rich mineral phases.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 428 - 31
Italian survey on comparative levofloxacin susceptibility in 334 clinical isolates of Pseudomonas aeruginosa; Segatore B et al.; A national survey on susceptibility patterns of 334 Pseudomonas aeruginosa isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin . Amikacin and imipenem had the best susceptibility profiles . The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime . The effect of levofloxacin in vitro on P . aeruginosa clinical isolates suggests that further clinical investigations are warranted.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 424 - 7
Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems; Kohler T et al.; While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786 . In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance . However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 415 - 7
Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa; Mine T et al.; Two new genes (mexXY) similar to mexAB, mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa . Elevated ethidium extrusion was observed with Escherichia coli cells harboring the plasmid carrying mexXY . This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E . coli ToIC or P . aeruginosa OprM is necessary for the function of the MexXY system.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 406 - 9
Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones; Takenouchi T et al.; gyrA point mutations in 335 clinical Pseudomonas aeruginosa isolates were examined mainly by nonisotopic single-strand conformation polymorphism analysis and direct sequencing . Seven types of missense gyrA mutations were observed in 70 of 335 strains (20.9%), and ciprofloxacin MICs were > or = 3.13 micrograms/ml for 63 of 70 strains (90.0%) . These included two double point mutations and three novel mutations (Ala-67-->Ser plus Asp-87-->Gly, Ala-84-->Pro, and Gln-106-->Leu) . Thr-83-->Ile mutants were predominantly observed (63 of 70 mutants) and showed high-level fluoroquinolone resistance (ciprofloxacin MIC at which 50% of isolates are inhibited, 25 micrograms/ml).

Antimicrob Agents Chemother, 1999 Feb, 43(2), 400 - 2
Interplay between chromosomal beta-lactamase and the MexAB-OprM efflux system in intrinsic resistance to beta-lactams in Pseudomonas aeruginosa; Masuda N et al.; We investigated the role of chromosomal beta-lactamase and the MexAB-OprM efflux system in intrinsic resistance to beta-lactams in Pseudomonas aeruginosa . Determination of the susceptibilities of a series of isogenic mutants with impaired production of the beta-lactamase and the efflux system to 16 beta-lactams including penicillins, cephems, oxacephems, carbapenems, and a monobactam demonstrated that the intrinsic resistance of P . aeruginosa to most of the beta-lactams is due to the interplay of both factors.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 287 - 91
In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM; Ziha-Zarifi I et al.; During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients . In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients . In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts . These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains . Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates . To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs . Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR . Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR . Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics . These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

Hematol Cell Ther, 1998 Dec, 40(6), 269 - 74
Early infectious complications after bone marrow transplantation requiring medical ICU admission; Gruson D et al.; The objective of this study was to define the type, the incidence and the outcome of early infectious complications (mean interval between day 1 post-BMT and the onset of fever was 9+/-3 days) occurring in granulocytopenic bone marrow transplant recipients, requiring medical intensive care unit admission . Over a five-years period, forty-one patients with microbiologically confirmed infection were enrolled, with a statistically significant higher frequency of allogeneic marrow transplant recipients (51%, p < 0.02) . Infectious pneumonia occurred in 24 patients (59%), septicemia with septic shock in ten patients (24%), catheter-related infection in 5 patients (12%) and meningitis in 2 patients (5%) (p < 0.001) . Twenty-six patients died (63%) . Among the patients with confirmed infectious pneumonitis, which occurred most frequently in allogeneic marrow recipients (p < 0.02), 16 died (67%) . This poor outcome was related to the requirement of mechanical ventilation . Eight patients (80%) with septicemia and septic shock and the two patients with meningitis died . Bacteria (Pseudomonas aeruginosa and Staphylococcal species) were the most common isolated in bronchoalveolar lavage fluid and blood cultures . We found a lower incidence of fungal or viral infections compared to previous studies . Empiric antimicrobial therapy in the cases of patients admitted in ICU may be included antibiotics anti-Pseudomonas and anti-Staphylococcus, as the ecology of hematology unit . The requirement of mechanical ventilation is the main adverse prognostic factor in transplanted patients . At ICU admission, patients with hepatic failure combined with respiratory failure represented a subgroup with a dismal prognosis.

Eur J Clin Microbiol Infect Dis, 1998 Nov, 17(11), 754 - 60
Antibacterial activity of meropenem against Pseudomonas aeruginosa, including antibiotic-induced morphological changes and endotoxin-liberating effects; Trautmann M et al.; The in vitro effects of meropenem on Pseudomonas aeruginosa were examined by studying (i) the inhibitory and bactericidal concentrations of meropenem versus those of imipenem for clinical isolates; (ii) changes in bacterial morphology during in vitro culture; and (iii) release of endotoxin induced by meropenem compared with that induced by other antipseudomonal compounds . Meropenem MIC90 and MBC90 values for 108 clinical isolates were 2 and 4.8 mg/l compared to 4.5 and 9.6 mg/l for imipenem . Morphological studies using phase-contrast and scanning electron microscopy showed that meropenem induced the formation of indeterminate bacterial cell forms at drug concentrations of 1-2.5 mg/l (0.5- to 1.25-fold the MIC), while spheroplasts predominated at drug levels exceeding 5 mg/l (2.5-fold the MIC) . Determination of free and EDTA-releasable endotoxin activity by means of the Limulus lysate test showed that both meropenem and imipenem liberated significantly less endotoxin than did ceftazidime . Therefore, although meropenem binds to penicillin-binding proteins (PBPs) 2 and 3 (in contrast to imipenem, which binds to PBP2 only), endotoxin release should not be a cause of concern when treating systemic gram-negative infections with this drug.

J Bacteriol, 1999 Feb, 181(3), 973 - 80
Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5; Burrows LL et al.; The wbp cluster of Pseudomonas aeruginosa O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length . A gene (formerly wbpF), contiguous with wzy in the wbp cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains . This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or "flippase." Insertion of a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production . To our knowledge, this is the first report of the successful generation of chromosomal wzx gene replacement mutations . Surprisingly, inactivation of wzx also led to a marked delay in production of the ATP-binding cassette-transporter-dependent, D-rhamnose homopolymer, A-band LPS . This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans . WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in P . aeruginosa O5 (H . L . Rocchetta, L . L . Burrows, J . C . Pacan, and J . S . Lam, Mol . Microbiol . 28:1103-1119, 1998) . These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm . Without adequate WbpL, A-band LPS synthesis is delayed . A subset of wzx mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely . Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

J Food Prot, 1999 Jan, 62(1), 35 - 9
Quantitative PCR for Listeria monocytogenes with colorimetric detection; Wang C et al.; An enzyme-linked immunosorbent assay (ELISA)-mediated polymerase chain reaction (PCR) technique was developed to detect and quantify Listeria monocytogenes in food products . The bacterial DNA was extracted from artificially contaminated food and co-amplified with a synthetic internal standard (IS) using primers specific for the target gene coding for the invasive-associated protein (i.a.p.), a virulence factor of L . monocytogenes (i.a.p.) or IS in the presence of fluorescein-dUTP PCR products were hybridized with biotinylated probes designed for the i.a.p . or IS, and then the hybrids were bound to a streptavidin-coated ELISA plate . An alkaline phosphatase-conjugated antibody to fluorescein was added to the plate and in the presence of substrate, PCR products were quantitated based on an optical density reading . The detection limit for L . monocytogenes experimentally inoculated into milk samples and channel catfish fillets was 20 CFU/ml and 1-2 CFU/g, respectively . Little or no cross reaction was detected in the presence of other spoilage and pathogenic organisms such as Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus . The ELISA-mediated PCR technique, when compared to traditional methods, is more rapid (2 working days) for detecting and quantifying L . monocytogenes.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 145 - 50
The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of P . aeruginosa PAO1 and P . fluorescens ATCC 13525; Meyer JM et al.; FpvA, the ferripyoverdine outer membrane receptor of Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is not specific to the pyoverdine produced by PAO1, but is also able to recognize the structurally different (ferri)pyoverdine of P . fluorescens ATCC 13525 . The specificity of FpvA was assessed by iron uptake competitions using the wild-type strains P . aeruginosa ATCC 15692 and P . fluorescens ATCC 13525 and their respective ferripyoverdines, and by fpvA gene complementation of a FpvA-deficient mutant of P . aeruginosa ATCC 15692 . The receptor mutant was able to utilize none of the two pyoverdines, while the same but fpvA-complemented mutant recovered simultaneously the ability to incorporate iron thanks to each of the two siderophores . The broad specificity of recognition of FpvA is viewed as an advantage for the strain in iron competition . Moreover, it allows an interesting approach for the understanding of the recognition mechanism between a (ferri)pyoverdine and its cognate outer membrane receptor.

Enferm Infecc Microbiol Clin, 1998 Dec, 16(10), 461 - 4
{Ribotypes of strains of Pseudomonas aeruginosa in a patient with cystic fibrosis}; Ferrus MA et al.; BACKGROUND: Pseudomonas aeruginosa is the major causative agent of respiratory infection in cystic fibrosis (CF) patients, and its presence in sputum is related with important deterioration of lung function in affected patients, thus a methodical monitorization and control of such a microorganism is decisive in the clinical status of the patient . Due to the limited effectiveness of phenotypic subtyping techniques, it is necessary to use molecular characterization methods . MATERIAL AND METHODS: We have studied the respiratory secretions periodically obtained over a period of time of 19 months, screening for total bacterial counts, P . aeruginosa load and genomic analysis of isolates using a ribotyping protocol . RESULTS: This study has showed the chronical and transitory carriage of different strains and the relation of increased bacterial counts with clinical deterioration periods . CONCLUSIONS: We consider that the use of molecular markers can be of great interest in the epidemiological surveillance of P . aeruginosa in CF patientsPublication Types:

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