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