Pseudomonas aeruginosa

Pediatrics . 2000 Dec;106(6):E89.
Technical report: precautions regarding the use of aerosolized antibiotics . Committee on Infectious Diseases and Committee on Drugs; Prober CG et al.; In 1998, the Food and Drug Administration (FDA) approved the licensure of tobramycin solution for inhalation (TOBI) . Although a number of additional antibiotics, including other aminoglycosides, beta-lactams, antibiotics in the polymyxin class, and vancomycin, have been administered as aerosols for many years, none are approved by the FDA for administration by inhalation . TOBI was approved by the FDA for the maintenance therapy of patients 6 years or older with cystic fibrosis (CF) who have between 25% and 75% of predicted forced expiratory volume in 1 second (FEV(1)), are colonized with Pseudomonas aeruginosa, and are able to comply with the prescribed medical regimen . TOBI was not approved for the therapy of acute pulmonary exacerbations in patients with CF nor was it approved for use in patients without CF . Currently, no other antibiotics are approved for administration by inhalation to patients with or without CF . The purpose of this statement is to briefly summarize the data that supported approval for licensure of TOBI and to provide recommendations for its safe use . The pharmacokinetics of inhaled aminoglycosides and problems associated with aerosolized antibiotic treatment, including environmental contamination, selection of resistant microbes, and airway exposure to excipients in intravenous formulations, will be discussed.

J Antimicrob Chemother, 2000 Jan, 45(1), 9 - 13
Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa; Ciofu O et al.; Membrane vesicles were isolated from one beta-lactam-sensitive and three beta-lactam-resistant Pseudomonas aeruginosa clinical isolates from patients with cystic fibrosis . The presence of the chromosomally encoded beta-lactamase in the membrane vesicles was shown by electron microscopy and enzymatic studies . This is the first report of extracellular secretion of beta-lactamase in P . aeruginosa and it seems that the enzyme is packaged into membrane vesicles.

J Pineal Res, 2000 Jan, 28(1), 26 - 33
Ototoxicity caused by aminoglycosides is ameliorated by melatonin without interfering with the antibiotic capacity of the drugs; Lopez-Gonzalez MA et al.; The production of free radicals seems to be involved in the mechanisms of ototoxicity . Aminoglycosides produce ototoxicity, which can be determined through distortion product otoacoustic emissions (OAEs) that measure the activity of the outer hair cells of the organ of Corti . An ototoxic chart was obtained in rats using gentamicin or tobramycin . Together with this treatment, the animals ingested melatonin in the drinking water, or melatonin was injected subcutaneously or intramuscularly . The distortion product OAEs were determined over a prolonged period of time for each of the groups . The effect of melatonin on the antibiotic capacity of the aminoglycosides used was also studied . Antibiograms inoculated with Escherichia coli or Pseudomonas aeruginosa and treated with gentamicin or tobramycin in the presence or absence of melatonin at quantities from pharmacological to physiological doses were performed . The ototoxicity produced by gentamicin and tobramycin was maximal from days 3 to 5 post-treatment, returning to normal values in 2 wk . When melatonin was present, the recovery was at day 5 post-treatment, independently of the means of administration of the pineal product . The antibiograms showed that melatonin had no effect on the antibiotic capacity . It is concluded that the ototoxicity caused by gentamicin and tobramycin is ameliorated by melatonin and that the pineal hormone does not interfere with the antibiotic capacity of these antibiotics.

Pediatr Res, 2000 Jan, 47(1), 121 - 6
Pseudomonas aeruginosa from patients with cystic fibrosis affects function of pulmonary surfactant; Lema G et al.; Patients with cystic fibrosis are severely affected by an infection with Pseudomonas aeruginosa, a microbe known to synthesize phospholipase C . This study was designed to determine whether that enzyme would affect the function of pulmonary surfactant phospholipids . Mucoid and nonmucoid strains of P . aeruginosa, freshly obtained from patients with cystic fibrosis, were cultured for 12 h on agar plates . The bacteria were suspended in saline solution and then pelleted by centrifugation . The supernatant was used to dilute the surfactant preparation, calf lung surfactant extract, from 35 to 2 mg/mL . Surfactant function, before and after incubation, was examined with a capillary surfactometer, an instrument specifically developed for an evaluation of the ability of surfactant to maintain patency of a narrow glass tube, simulating a terminal conducting airway . Phospholipid hydrolysis was also evaluated biochemically by determining the total content of phospholipids in surfactant before and after incubation . In five experiments, the lipids were separated with thin-layer chromatography, and the phosphorus content was determined in the diacylphosphatidylcholine band before and after incubation for 6, 24, and 48 h . Capillary openness and phospholipid concentration decreased as enzyme concentration and time of incubation increased (p<0.0001) . Linear regression showed a significant correlation between time of capillary openness and phospholipid concentration (r = 0.957; p<0.0001) . Calf lung surfactant extract hydrolysis was catalyzed by extracts of the bacteria, particularly the nonmucoid, analogous to the catalysis observed with phospholipase C . Surfactant hydrolysis catalyzed by enzymes from P . aeruginosa might severely affect surfactant function provided enzyme concentration is high and time of incubation is long.

J Immunol, 2000 Jan 15, 164(2), 1037 - 45
Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection; Kernacki KA et al.; Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction . This study examined the role of C-X-C chemokines in PMN infiltration into P . aeruginosa-infected cornea and the contribution of these mediators to disease pathology . After P . aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P . aeruginosa infection . While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells . Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice . To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2 . This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease . Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction . Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P . aeruginosa-infected cornea . These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.

Exp Eye Res, 1999 Dec, 69(6), 595 - 601
Effect of galardin on collagen degradation by Pseudomonas aeruginosa; Hao JL et al.; The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P . aeruginosa) in the presence or absence of keratocytes . Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P . aeruginosa culture broth and/or Galardin for 24 hr . Degradation of collagen fibrils during culture was measured by the release of hydroxyproline . The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P . aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by keratocytes . The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P . aeruginosa were also examined . P . aeruginosa broth by itself induced collegen gel degradation . When keratocytes were present, P . aeruginosa broth increased the amount of degraded collagen even further . Galardin significantly reduced the amounts of collagen degraded by P . aeruginosa culture broth, whether keratocytes were present or absent in the gel . However, the protease inhibitors had no inhibitory effects on collagen degradation . Gelatin zymography and Western blotting revealed that inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P . aeruginosa broth . Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P . aeruginosa . The present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P . aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs .

Am J Respir Crit Care Med, 2000 Jan, 161(1), 271 - 9
Effect of Pseudomonas infection on weight loss, lung mechanics, and cytokines in mice; van Heeckeren AM et al.; Poor growth, Pseudomonas aeruginosa endobronchitis, pulmonary inflammation, and decline of lung function are hallmarks of cystic fibrosis (CF), yet the relationship between these features is poorly understood . Because animal models of chronic bronchopulmonary infection with P . aeruginosa used to study pulmonary inflammation in CF have also been associated with weight loss, we sought to determine whether this weight loss was due to the inflammatory process and/or to changes in lung function . P . aeruginosa-laden agarose beads were instilled into the lungs of mice . Weight loss was greatest 3 d after Pseudomonas infection . Infected mice had a rapid though transient rise in absolute neutrophil counts, mTNF-alpha, mIL-1beta, mIL-6, mip-2, and KC in bronchoalveolar lavage fluid . There was no difference in lung resistance or lung compliance measured by body plethysmography between infected and control mice . Weight loss did correlate with the concentration of proinflammatory cytokine levels 3 d after inoculation of mice with Pseudomonas, and body composition analysis revealed loss of skeletal muscle mass . These results suggest that weight loss in P . aeruginosa-infected mice was associated with the inflammatory process and not with altered pulmonary responsiveness . These findings may provide insights into the cause of cachexia and weight loss seen in patients with CF.

J Ethnopharmacol, 1999 Nov 1, 67(2), 225 - 8
Screening of some Nigerian medicinal plants for antibacterial activity; Kudi AC et al.; Crude extracts from eight Nigerian medicinal plants, used traditionally in the treatment of infectious and septic diseases in both humans and animals were screened in vitro for antibacterial activity, using the hole-plate diffusion method . Most of the extracts were active against gram-positive bacteria . Two of the plant, Angeiossus schimperi and Anacardium occidentale, had good antibacterial activity against Escherichia coli and Pseudomonas aeruginosa which are gram-negative bacteria.

FEMS Immunol Med Microbiol, 2000 Jan, 27(1), 79 - 85
Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans; Lee N et al.; Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines . In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization . Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different . The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses . In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis . Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma . These data suggest that antibody response to P . aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.

J Biol Chem, 2000 Jan 7, 275(1), 141 - 6
Rac1 and Cdc42 are required for phagocytosis, but not NF-kappaB-dependent gene expression, in macrophages challenged with Pseudomonas aeruginosa; Lee DJ et al.; Macrophages respond to Gram-negative bacterial pathogens by phagocytosis and pro-inflammatory gene expression . These responses may require GTPases that have been implicated in cytoskeletal alterations and activation of NF-kappaB . To determine the role of Rac1 and Cdc42 in signal transduction events triggered by Pseudomonas aeruginosa, we expressed GTP binding-deficient alleles of Rac1 or Cdc42, or Chim-GAP, a Rac1/Cdc42-specific GTPase-activating protein domain, in a subline of RAW 264.7 cells, and challenged the transfected cells with a laboratory strain of P . aeruginosa, PAO1 . Expression of Rac1 N17, Cdc42 N17, or Chim-GAP led to a marked reduction of phagocytosis . In contrast, nuclear translocation of p65 NF-kappaB was unaffected by expression of the same constructs . Incubation of macrophages with PAO1 led to NF-kappaB-dependent expression of inducible nitric-oxide synthase, COX-2, and tumor necrosis factor-alpha, which was unaffected by inhibition of Rac1 or Cdc42 function . Isogenic strains of PAO1 that lacked surface adhesins were poorly ingested; however, they induced pro-inflammatory gene expression with an efficiency equal to that of PAO1 . These results indicate that the signal transduction events leading to phagocytosis and pro-inflammatory protein expression are distinct . Rac1 and Cdc42 serve as effectors of phagocytosis, but not NF-kappaB-dependent gene expression, in the macrophage response to P . aeruginosa.

Bioorg Med Chem Lett, 1999 Dec 20, 9(24), 3447 - 52
Novel synthetic analogs of the Pseudomonas autoinducer; Kline T et al.; Release of virulence factors in Pseudomonas aeruginosa is regulated by two N-acylhomoserine lactones, PAI-1 and PAI-2, that activate the respective transcription factors LasR and RhlR . With the goal of developing novel therapeutic agents, we synthesized constrained analogs of PAI-1 and evaluated them in P . aeruginosa . Two of the novel analogs bound to LasR and showed agonist activity in LasR stimulation of a lasI-lacZ reporter construct.

J Drug Target, 1999, 7(1), 33 - 41
Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by Pseudomonas aeruginosa; Beaulac C et al.; Eradication of mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection has been previously demonstrated following the intratracheal administration of Fluidosomes, a low phase transition temperature (low T(C)) liposomal tobramycin preparation administered in liquid form (Beaulac et al., Antimicrob . Agents Chemother., 40, 665-669, 1996) . In the present work, the same liposomal formulation was administered as a dry powder aerosol to an animal model of chronic pulmonary infection in view of a possible clinical development in cystic fibrosis patients . Chronic infection was established by intratracheal administration of 10(5) cfu of a mucoid variant of P . aeruginosa, PA 508, prepared in agar beads . Sixteen hours after one aerosol treatment, the cfu counts performed on lungs (pair) treated with liposomal tobramycin were of 4.31x10(5) cfu/lungs comparatively to 1.32x10(8) and 3.02x10(8) cfu/lungs respectively in untreated and in lungs treated with free antibiotic . Considering the quantity of liposome-tobramycin that has reached the lungs, the results suggest that aerosolization of low T(C) liposomal tobramycin used as a dry powder preparation could be an effective way of treating chronic pulmonary infection caused by Pseudomonas.

Enferm Infecc Microbiol Clin, 1999 Nov, 17(9), 439 - 44
{Factors affecting the clinical course of bacteremia . Prospective study at a university hospital}; Rojo MD et al.; BACKGROUND: The aim of this study was to identify the risk factors mainly influencing the mortality of a series of bacteremic patients . METHODS: A prospective study of the clinically significant bacteremias detected in the Hospital Clinico Universitario in Malaga (Spain) over the period from June 1994 to May 1995 was performed . Univariate analysis of the results was carried out with the chi 2 test and multivariate analysis was undertaken by logistic regression (Stepwise backward) . RESULTS: The incidence of bacteremia was of 19.5 cases/1,000 admissions and the mortality was of 21.9% . According to the univariate analysis, 11 variables were significantly associated with greater risk death: age > 60 years, stay in the intensive care unit, respiratory diseases as the main diagnosis, etiology by Pseudomonas aeruginosa, absence of fever, septic shock, presence of chronic renal insufficiency, cirrhosis or heart disease with underlying diseases, performance of invasive procedures prior to and hospital stay of less than 10 days . Logistic regression analysis determined the factors which mainly influenced in the prognosis of bacteremia: septic shock (p < 0.0001, odds ratio {OR}: 17.97), cardiovascular diseases (p = 0.004, OR: 9.86), AIDS (p = 0.03; OR: 1.02) . CONCLUSIONS: Although the prognosis of bacteremia is strongly influenced by determined conditions of the patient, it may be improved, overall through the control of possible hemodynamic complications of the patients and to a lesser extent by antibiotic treatment.

J Bacteriol, 2000 Jan, 182(1), 100 - 6
HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris; Egland PG et al.; Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation . The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by Rhodopseudomonas palustris have been identified . Here we describe a regulatory gene, hbaR, that is part of the 4-HBA degradation gene cluster . An hbaR mutant that was constructed was unable to grow anaerobically on 4-HBA . However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation . The effect of the HbaR protein on expression of hbaA encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using hbaA::'lacZ transcriptional fusions . HbaR was required for a 20-fold induction of beta-galactosidase activity that was observed with a chromosomal hbaA::'lacZ fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA . HbaR also activated expression from a plasmid-borne hbaA-'lacZ fusion when it was expressed in aerobically grown Pseudomonas aeruginosa cells, indicating that the activity of this regulator is not sensitive to oxygen . The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins . It is most closely related to transcriptional activators that are involved in regulating nitrate reduction . Previously, it has been shown that R . palustris has an FNR homologue, called AadR, that is also required for 4-HBA degradation . Our evidence indicates that AadR activates expression of hbaR in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.

FEMS Microbiol Lett, 2000 Jan 1, 182(1), 111 - 7
Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli; Antonio RV et al.; In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type . The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R . eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase . The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase . In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester . Only in recombinant E . coli fad mutants harboring plasmid pBHR68, the R . eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively . Coexpression of phaC1 from P . aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E . coli strain . These data strongly suggested that R . eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.

Electrophoresis, 1999 Dec, 20(18), 3580 - 8
The microbial proteome database--an automated laboratory catalogue for monitoring protein expression in bacteria; Cordwell SJ et al.; Laboratories devoted to high-throughput characterisation of purified proteins arrayed via two-dimensional (2-D) gel electrophoresis face an arduous task in maintaining a centralised and constantly evolving record of information relating to the characterisation of proteins and their responses following biological challenges . The Microbial Proteome Database (MPD) has been conceived as an in-house resource for complementing the plethora of genomic databases available for such organisms . The database utilises commercially available software to provide an electronic 'lab book' of information obtained daily from 2-D electrophoresis gels, image analysis packages, protein characterisation methodologies, and biological experimentation . The MPD begins from a single 2-D gel image (a 2-D 'reference map') with clickable spots that link to a 'protein catalogue' (ProtCat) with spot information including protein identity, changes in expression determined under experimental conditions, cellular location, mass, and pI . The entry for each protein then contains further links to gel images corresponding to the presence of the particular protein within different subproteomes (as defined by the pH of narrow- and wide-range immobilised pH gradients or from differential extraction methods used to determine the location of the protein within a functional cell) . The database currently contains information from strains of three microbial species (Escherichia coil, Pseudomonas aeruginosa and Staphylococcus aureus) and 32 master gel images . The rapid accessibility of information obtained from microbial proteomes is an essential step towards the integrated analysis of these organisms at the gene, transcript, protein and functional levels and will aid in reducing turnaround times between sample preparation and the discovery of molecules of biological significance.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15202 - 7
Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa; Darby C et al.; Identification of host factors that interact with pathogens is crucial to an understanding of infectious disease, but direct screening for host mutations to aid in this task is not feasible in mammals . The nematode Caenorhabditis elegans is a genetically tractable alternative for investigating the pathogenic bacterium Pseudomonas aeruginosa . A P . aeruginosa toxin, produced at high cell density under control of the quorum-sensing regulators LasR and RhlR, rapidly and lethally paralyzes C . elegans . Loss-of-function mutations in C . elegans egl-9, a gene required for normal egg laying, confer strong resistance to the paralysis . Thus, activation of EGL-9 or of a pathway that includes it may lead to the paralysis . The molecular identity of egl-9 was determined by transformation rescue and DNA sequencing . A mammalian homologue of EGL-9 is expressed in tissues in which exposure to P . aeruginosa could have clinical effects.

Plasmid, 2000 Jan, 43(1), 59 - 72
Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains; Hoang TT et al.; An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed . The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2 . These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the φCTX attachment site . The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase . In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI(q) also contained on the plasmid . Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively . The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants . In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants . Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P . aeruginosa chromosome, and used to express the hexahistidine-tagged P . aeruginosa AI synthase RhlI .

J Cataract Refract Surg, 1999 Dec, 25(12), 1615 - 9
Early- versus late-onset infectious keratitis after radial and astigmatic keratotomy: clinical spectrum in a referral practice; Heidemann DG et al.; PURPOSE: To compare the clinical characteristics of early- versus late-onset keratitis after radial keratotomy (RK) and astigmatic keratotomy (AK) . SETTING: Referral subspecialty practice . METHODS: This retrospective review comprised 19 patients with infectious keratitis after RK and AK . Early- versus late-onset groups were analyzed for predisposing conditions; infiltrate location, size, and depth; microbiologic data; and final visual outcome . RESULTS: Ten patients in the early-onset group developed keratitis within a mean of 7.4 days after surgery (range 3 to 14 days) . Nine patients in the late-onset group developed keratitis a mean of 5.4 years after surgery (range 1.5 to 15.0 years) . Staphylococcus aureus was the predominant organism in the early-onset group and Pseudomonas aeruginosa in the late-onset group . In the early-onset group, most infiltrates occurred in the paracentral aspect of the RK incision and extended to the middle or posterior stroma . In the late-onset group, most infiltrates occurred in the peripheral portion of the RK incision and were localized to the superficial stroma . A hypopyon was present in 7 of 10 ulcers in the early group and in 1 of 9 in the late group . Two patients in the early group developed endophthalmitis . Most patients in the late-onset group had incisional pseudocysts; 2 had other risk factors for keratitis . Final visual acuity was 20/40 or better in 7 of 10 patients in the early group and in 8 of 9 patients in the late group . CONCLUSIONS: Early-onset corneal ulcers after incisional refractive keratotomy were usually paracentral and deep, whereas late-onset ulcers were usually peripheral and superficial . Despite the predominance of Staphylococcus and Pseudomonas in the early- and late-onset groups, respectively, a variety of organisms may be responsible for infections in keratotomy incisions.

J Trauma, 1999 Dec, 47(6), 1052 - 7; discussion 1057-9
Burn injury with infection alters prostaglandin E2 synthesis and metabolism; Hahn EL et al.; OBJECTIVE: The aim of this study was to examine the relationship between prostaglandin synthesis and prostaglandin degradation in a model of burn injury with infection . METHODS: Male B2D6F1 mice were assigned to control, burn (16% dorsal scald burn), or burn with infection (burn with topical application of 1,000 colony forming units of Pseudomonas aeruginosa) groups . Lung tissue was harvested at 1, 2, and 3 days after burn injury and subsequently processed for total RNA and protein . Northern and Western blot analyses were used to examine differences in cyclooxygenase 2 (COX-2) and prostaglandin 15-OH dehydrogenase (PGDH) protein and mRNA expression . Total RNA was probed with the riboprobe for murine PGDH and COX-2 and the 100,000g protein fraction was immunoblotted by using an rabbit anti-murine PGDH and anti-murine COX-2 antibody . RESULTS: COX-2 expression was elevated in the burn with infection animals on day 1 and day 2 after burn injury . At these time points in the burn + infection group, PGDH was significantly depressed . Burn injury increased COX-2 expression on day 1, but by day 2, COX-2 expression had decreased to control values . A corresponding increase in PGDH expression was observed on day 2 in the burned mice . The mRNA expression of COX-2 was followed by a similar increase in COX-2 protein expression at all time points in the injured animals . This was not the case with PGDH expression . On day 1, PGDH mRNA expression was depressed in the burn with infection mice with no change in PGDH protein expression . This finding indicates that PGDH is subject to regulation at both the transcriptional and posttranscriptional levels . CONCLUSION: Burn wound infection depressed both PGDH mRNA and protein expression and increased COX-2 mRNA and protein expression . Therefore, increases in circulating prostaglandin E2 levels during septic injury are derived from alterations in synthesis and degradation of prostaglandin E2.

Salud Publica Mex, 1999, 41 Suppl 1, S38 - 43
{Epidemic of pneumonia associated with mechanical ventilation in Mérida, Yucatán}; Zaidi M et al.; OBJECTIVE: To determine the main epidemiological, clinical, and microbiologic characteristics of an outbreak of ventilator-associated pneumonia at an intensive care unit in Yucatan . MATERIAL AND METHODS: An 11-month prospective and observational study was designed to determine incidence, mortality, potential reservoirs, etiologic agents and antibiotic susceptibility patterns . RESULTS: The incidence of ventilator-associated pneumonia was 74% . The crude mortality rate was 88% compared to a 19.5% expected-mortality rate . Gram-negative bacteria were isolated from 98% of the cultures, of which 46% were susceptible to third generation cephalosporins, 59% to fourth generation cephalosporins, 70% to ciprofloxacin and 100% to imipenem . Klebsiella pneumoniae and Pseudomonas aeruginosa were isolated from some of the ventilator circuits and the sink . CONCLUSIONS: The high incidence of pneumonia and associated mortality in our intensive care unit may be attributed to the absence of infection control measures and the high prevalence of multiresistant organisms which is related to antibiotic abuse.

Presse Med, 1999 Nov 27, 28 Suppl 3, 9 - 10
{Neutropenia with fever}; Ben Ali A; CAUSAL GERMS: Over the last 10 years, hemotology units have seen many changes in the epidemiological pattern of infections in neutropenic patients, with a growing number of infections caused by Gram-positive germs . The number of septicemias due to multiresistant Gram-negative germs (Pseudomonas aeruginosa, Strenotrophomonas maltophilia) has however remained high . IMPACT OF THERAPEUTIC REGIMENS: Improved prognosis in neutropenic patients depends for a large part on careful management of septic episodes . As the risk of septicemia is strongly correlated with the degree of efficacy of antibiotic regimens, susceptibility data must always be examined when developing antibiotic protocols in units caring for neutropenic patients.

J Pharmacol Exp Ther, 2000 Jan, 292(1), 88 - 95
Mercaptoethylguanidine inhibits the inflammatory response in a murine model of chronic infection with Pseudomonas aeruginosa; Wilmott RW et al.; Chronic airway inflammation induced by Pseudomonas aeruginosa is the eventual cause of respiratory failure in most people affected by cystic fibrosis . Recent evidence implicates the involvement of free radical and oxidant stress in the pathogenesis of the inflammatory injury . Here we report the efficacy of a novel experimental therapeutic, mercaptoethylguanidine (MEG), which has combined actions as a selective inhibitor of the inducible nitric oxide synthase and as a scavenger of peroxynitrite, a potent oxidant formed in the reaction of nitric oxide and superoxide radical . Chronic pulmonary infection was established in FVB/N mice by intratracheal administration of 10(5) colony-forming units of P . aeruginosa in agar beads . Treatment with MEG (10 mg/kg/dose every 8 h i.p.) inhibited weight loss in the first 3 days and reduced histologic injury at 8 days postinfection . MEG also reduced myeloperoxidase activity, a marker of neutrophil infiltration, at 8 days and concentrations of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and macrophage inflammatory protein 2 in whole lung homogenates . MEG-treated animals and controls had similar perioperative mortality and comparable colony counts of P . aeruginosa at 8 days, indicating that MEG did not exacerbate infection . Our data suggest that MEG may be an effective immunomodulatory therapy of pulmonary inflammation induced by chronic infection.

Infect Immun, 2000 Jan, 68(1), 403 - 6
ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells; Cowell BA et al.; The presence of invasion-inhibitory activity that is regulated by the transcriptional activator ExsA of cytotoxic Pseudomonas aeruginosa has previously been proposed . The results of this study show that both ExoT and ExoS, known type III secreted effector proteins of P . aeruginosa that are regulated by ExsA, possess this activity . Invasion was reduced 94.4% by ExoT and 96.0% by ExoS . Invasion-inhibitory activity is not linked to ADP-ribosylation activity, at least for ExoS, since a noncatalytic mutant also inhibits uptake by an epithelial cell line (invasion was reduced 96 . 0% by ExoSE381A).

Antimicrob Agents Chemother, 2000 Jan, 44(1), 205 - 6
Morphological change in Pseudomonas aeruginosa following antibiotic treatment of experimental infection in mice and its relation to susceptibility to phagocytosis and to release of endotoxin; Yokochi T et al.; The relationship between morphological changes in Pseudomonas aeruginosa following antibiotic treatment of experimental infection in mice, susceptibility to phagocytosis, and release of endotoxin was studied . The intraperitoneal administration of P . aeruginosa with imipenem or ceftazidime into mice induced morphological changes in the cells 2 h after injection . Round P . aeruginosa cells with imipenem treatment became susceptible to phagocytosis by peritoneal cells, whereas long filamentous cells with ceftazidime treatment were hardly phagocytized by peritoneal cells . The morphological changes also affected the plasma endotoxin level in the circulation.

Immunopharmacology, 1999 Nov, 44(3), 223 - 31
Accelerated recovery from cyclophosphamide-induced leukopenia in mice administered a Japanese ethical herbal drug, Hochu-ekki-to; Kaneko M et al.; The effect of a Japanese ethical herbal drug, Hochu-ekki-to (HOT), on recovery from leukopenia induced by cyclophosphamide (CY) was investigated . Daily oral administration of 1000 mg/kg HOT into CY-treated mice significantly prevented decrease of leukocyte numbers in the peripheral blood and accelerated recovery from leukopenia . Ginsenoside Rgl extracted from Ginseng radix, a major herb of HOT, was one of the active ingredients . HOT increased numbers of neutrophils and monocytes in the peripheral blood compared with CY-treated control . Moreover, HOT augmented the resistance against Pseudomonas aeruginosa infection . The number of colony-forming units in the spleen (CFU-S) also increased in HOT-treated mice . The frequencies of IL-3-, GM-CSF- and IFN-gamma-producing cells increased in the spleen, bone marrow, liver and IEL on HOT treatment, and HOT clearly augmented the expressions of IL-3, GM-CSF and IFN-gamma mRNA in the spleen, bone marrow, liver and IEL except IL-3 and IFN-gamma mRNA in the IEL . These results suggest that HOT enhances the production of hematopoietic lymphokines, stimulates the proliferation of hematopoietic progenitor cells and consequently accelerates recovery from leukopenia in CY-treated mice . Additionally, IFN-gamma which HOT-augmented the production may contribute the protective effect against the bacterial infection by activating of phagocyte cells.

Eur Respir J, 1999 Nov, 14(5), 1145 - 9
Human neutrophil lipocalin, a highly specific marker for acute exacerbation in cystic fibrosis; Eichler I et al.; Cystic fibrosis (CF) is characterized by the production of abnormally thick secretions in the airways, chronic bacterial endobronchial infections and a chronic, predominantly neutrophilic inflammatory response . Therefore, myeloperoxidase (MPO) and lactoferrin are frequently used as inflammatory markers . Recently, a new protein in the neutrophil granules, human neutrophil lipocalin (HNL) has been discovered . The aim of the present study was to investigate HNL in sera of patients with CF and its relation to MPO and lactoferrin as well as to acute pulmonary exacerbation . Serum concentrations of HNL, MPO and lactoferrin were determined in 42 patients with CF and in 25 healthy subjects . Patients with CF were divided into groups with and without acute pulmonary exacerbation (APE) and also with and without colonization with Pseudomonas aeruginosa (Pa) . Median serum levels of HNL (200.5 microg x L(-1)), MPO (595 microg x L(-1)) and lactoferrin (1,356.5 microg x L(-1)) were significantly increased in patients with CF compared to control subjects (57.7, 178 and 478 microg x L(-1), respectively; p<0.0001) . CF patients with APE had significantly increased serum concentrations of HNL (321 versus 97.7 microg x L(-1); p<0.0001), MPO (1,125 versus 300 microg x L(-1); p<0.005) and lactoferrin (4,936 versus 980 microg x L(-1); p<0.001) compared with patients in stable clinical condition . Similarly, patients colonized with Pa had significantly higher concentrations of HNL, MPO and lactoferrin than Pa negative patients . These results indicate that in patients with cystic fibrosis, serum concentrations of human neutrophil lipocalin are markedly increased with a strong relationship to myeloperoxidase and lactoferrin . Thus, determination of serum human neutrophil lipocalin concentrations may be another useful diagnostic tool to monitor neutrophil inflammation in cystic fibrosis . The more marked difference in human neutrophil lipocalin compared with myeloperoxidase concentrations with no overlap between patients with acute pulmonary exacerbation and those in stable condition even suggests that human neutrophil lipocalin may be a more sensitive and specific discriminator.

J Antimicrob Chemother, 1999 Nov, 44(5), 689 - 91
In-vitro effects of a combination of antipseudomonal antibiotics against multi-drug resistant Pseudomonas aeruginosa; Oie S et al.; We evaluated the in-vitro effects of various combinations of five types of widely used antipseudomonal antibiotics (piperacillin, meropenem, ceftazidime, aztreonam and amikacin) against six Pseudomonas aeruginosa strains that were resistant to each of these antibiotics . Among two-drug combinations, the combinations of two beta-lactam antibiotics inhibited growth of one to three P . aeruginosa strains, while those of one beta-lactam antibiotic and amikacin inhibited growth of two to four strains . Among three-drug combinations, the combinations of three beta-lactam antibiotics inhibited growth of four to five strains, and those of two beta-lactam antibiotics and amikacin inhibited growth of five strains . These results suggest the potential usefulness of a combination of two beta-lactam antibiotics and amikacin or that of three beta-lactam antibiotics in treating multi-drug resistant P . aeruginosa infections.

J Appl Microbiol, 1999 Nov, 87(5), 782 - 6
A kinetic study of the effect of hydrogen peroxide and peracetic acid against Staphylococcus aureus and Pseudomonas aeruginosa using the bioscreen disinfection method; Lambert RJ et al.; Hydrogen peroxide and peracetic acid at pH 4 were examined against Staphylococcus aureus and Pseudomonas aeruginosa using the published 'Bioscreen' technique of biocide analysis . The data were examined using either classical Chick-Watson (CW) log-linear disinfection kinetics or the empirical, non-linear time Hom model . In some cases, modelling the data with the classical CW method gave good linear correlations, in others, however, deviations from this model were observed . In such cases the Hom model proved an adequate descriptor of the data . The Bioscreen technique therefore gives data which can be analysed using the normal mechanistic and empirical models currently available.

J Appl Microbiol, 1999 Nov, 87(5), 735 - 42
Specific variations of fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by quaternary ammonium compounds and relation with resistance to bactericidal activity; Guerin-Mechin L et al.; The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of Quaternary Ammonium Compounds (QACs) was investigated . The strain was grown in a medium with increasing concentrations of a QAC, benzyldimethyltetradecylammonium chloride (C14) and two non-QACs, sodium dichloroisocyanurate and tri-sodium phosphate . In the presence of C14 only, the strain was able to grow in concentrations higher than the minimal inhibitory concentration . As the strain adapted to C14, resistance to bactericidal activity of the same biocide increased . For the non-QACs, no change was noted when cells were grown in the presence of biocides . The C14-adapted cells showed variations in membrane fatty acid composition . A hierarchical clustering analysis was used to compare all fatty acid compositions of cultures in the presence, or not, of the three biocides used here and another QAC studied previously . The clusters obtained underlined specific variations of membrane fatty acids in response to the presence of QACs . Furthermore, with a simple linear regression analysis, a relationship was shown between the membrane fatty acids and the resistance developed by the strain against the bactericidal activity of C14.

J Appl Microbiol, 1999 Nov, 87(5), 718 - 25
A comparison of the bactericidal efficacy of 18 disinfectants used in the food industry against Escherichia coli O157:H7 and Pseudomonas aeruginosa at 10 and 20 degrees C; Taylor JH et al.; A number of proprietary disinfectant products (18) used in the food industry were tested for their bactericidal efficacy against Pseudomonas aeruginosa and Escherichia coli O157:H7 at 20 and 10 degrees C according to the BS EN 1276 (1997) quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas . At 20 degrees C, 13 products passed at their in-use concentration (under clean and dirty conditions) against Ps . aeruginosa and 15 passed against E . coli O157:H7 . The number of products passing the test at 10 degrees C was 11 and 14 for Ps . aeruginosa and E . coli O157:H7, respectively . The products exhibiting reduced efficacy at the lower temperature were amphoterics and quaternary ammonium compounds although some of these types of products were effective at both temperatures . Products that passed against Ps . aeruginosa generally also passed against E . coli O157:H7 . Taking all the results together, only 11 of the total of 18 products achieved a pass result under all the parameters tested . This work demonstrates the need for final verification of disinfectant efficacy by undertaking field trials in the food-processing environment in which the product is intended for use.

J Biol Chem, 1999 Dec 17, 274(51), 36616 - 22
Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa . Cloning of a ceramidase homologue from Mycobacterium tuberculosis; Okino N et al.; We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M . (1998) J . Biol . Chem . 273, 14368-14373) . Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain . Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction . DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17 . The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme . The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence . Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence . Recombinant ceramidase was purified to homogeneity from the lysate of E . coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties . No significant sequence similarities were found in other known functional proteins including human acid ceramidase . However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana . The homologue of the ceramidase gene was thus cloned from an M . tuberculosis cosmid and expressed in E . coli, and the gene was demonstrated to encode an alkaline ceramidase . This is the first report for the cloning of an alkaline ceramidase.

J Biol Chem, 1999 Dec 17, 274(51), 36369 - 72
The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases; Goehring UM et al.; Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin . The ADP-ribosyltransferase domain is located within the C terminus part of ExoS . Recent studies showed that the N terminus part of ExoS (amino acid residues 1-234, ExoS(1-234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells . Here we studied the effects of ExoS(1-234) on nucleotide binding and hydrolysis by Rho GTPases . ExoS(1-234) (100-500 nM) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis . A similar increase in GTPase activity was stimulated by full-length ExoS . Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10-11 nM ExoS(1-234), respectively . We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins . These data identify ExoS as a GTPase-activating protein for Rho GTPases.

Nihon Kokyuki Gakkai Zasshi, 1999 Sep, 37(9), 739 - 42
{Primary ciliary dyskinesia treated with living-donor lobar lung transplantation}; Yamamoto H et al.; We report a case of primary ciliary dyskinesia in which a living-donor lobar lung transplant was performed . A 24-year-old woman with a diagnosis of primary ciliary dyskinesia and bronchiectasis was admitted to Shinshu University Hospital because of persistent dyspnea and pyrexia over a period of 4 months . Although she was given various antibiotics, neutrophilia, elevated plasma C-reactive protein (CRP) levels, and respiratory failure persisted . Chest roentgenograms and computed tomography disclosed severe bronchiectasis and diffuse infiltrative shadows in both lung fields . Pseudomonas aeruginosa was detected in a sputum culture . Although a variety of conventional therapies were administered, the patient's oxygenation progressively deteriorated . She was intubated and assisted by mechanical ventilation . The patient and her family proposed lung transplantation, and we concluded that a living-donor lobar lung transplant would be a suitable treatment for her disease . We transported the patient to Okayama University Hospital by helicopter 10 days after intubation . A living-donor lobar lung transplant was successfully performed with lung tissues donated by the patient's mother and sister for each transplant site.

Nihon Kokyuki Gakkai Zasshi, 1999 Sep, 37(9), 699 - 703
{A case of common variable immunodeficiency that responded to long-term erythromycin chemotherapy}; Maeda K et al.; A 32-year-old woman with common variable immunodeficiency (CVID) accompanied by sinopulmonary infection was evaluated for purulent sputum, cough, and nasal obstruction that did not respond to regular intravenous immunoglobin (IVIG) infusion . Chest X-ray films revealed bronchiectasis affecting both lung bases, and a bacteriological examination of sputum was positive for Pseudomonas aeruginosa . Long-term chemotherapy with erythromycin (EM) was started, and the patient's respiratory symptoms gradually subsided . Sinopulmonary infection is the dominant clinical complication in patients with CVID . This case suggested that long-term EM chemotherapy is useful for the treatment of IVIG-refractory sinopulmonary infection associated with CVID.

Nihon Kokyuki Gakkai Zasshi, 1999 Sep, 37(9), 673 - 9
{Interleukin-8 and airway inflammation}; Inoue H; Airway inflammation is a prominent feature of chronic obstructive diseases of the airways, including asthma, bronchiectasis, chronic bronchitis, and diffuse panbronchiolitis . Neutrophils are implicated in the pathogenesis of these diseases . The present review discusses the role of interleukin-8 (IL-8), a neutrophil chemo-attractant, in neutrophil accumulation in the airways, and the mechanisms of inducing IL-8 expression . IL-8 presents in the sputum of patients with inflammatory airway diseases, and accounts in large part for the chemo-attractant activity present . Focusing on Pseudomonas aeruginosa as the stimulus, it was discovered that when a supernatant of bacterial culture is introduced into the airways in vivo, bacterial products induce IL-8 expression in surface airway epithelial cells and the recruitment of neutrophils into the airways . The neutrophil chemotactic activity of the airway fluid was inhibited by an IL-8 antibody . The luminal IL-8 concentration increased in response to instillation of bacteria, and an inhibitor of neutrophil recruitment markedly reduced the IL-8 levels . From these results, it was speculated that bacteria-induced neutrophil accumulation in the airways involves a cascade of events, and that early neutrophil recruitment in response to bacteria is due to epithelium-derived IL-8, while the amplification of the response is due, at least in part, to IL-8 induction in the neutrophils themselves.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1996 May, 29(2), 90 - 9
Comparison of three typing methods for Pseudomonas aeruginosa; Chen CH et al.; Fifty-seven independent isolates of Pseudomonas aeruginosa from blood specimens were typed with 3 different methods: ribotyping, random amplified polymorphic DNA (RAPD) typing, and pyocin typing . Ribotyping was performed by probing the rRNA genes of genomic DNA that was digested separately with 4 different restriction enzymes . Digestion of DNA from 57 P . aeruginosa isolates with BamHI, ClaI, EcoRI, and PstI produced 4, 4, 6, and 7 patterns, respectively . As a result, ribotyping classified the 57 isolates into 22 types . Six new ribotypes that had not been described previously were found . One BamHI, 1 ClaI, 2 EcoRI, and 2 PstI patterns were novel . RAPD typing was performed with two different polymerase chain reaction (PCR) primers (RAPD1 and RAPD2) . Both primers classified the 57 isolates into 15 RAPD types and produced identical patterns . The pyocin typing method classified the 57 isolates into 10 types . According to the results obtained in this study, the ribotyping has a discriminatory index of 0.865, RAPD, 0.785, and pyocin typing, 0.676, respectively . The ribotyping method was the most effective among the 3 methods compared for typing P . aeruginosa isolates.

Rinsho Byori, 1999 Nov, 47(11), 1064 - 9
{Clinical analysis of Pseudomonas aeruginosa bacteremia}; Kumasaka K et al.; To determine the outcome of Pseudomonas aeruginosa bacteremia and to identify risk factors for these infections in our University hospital, 46 cases (65 episodes) of Pseudomonas aeruginosa bacteremia were retrospectively investigated . The most frequent underlying diseases or cases were from Emergency and Critical care center (18 cases, including 11 case of cerebrovascular accident and head injury) followed by hematologic malignancies (11 cases) but none of the HIV infection was included in this study . The overall crude mortality rate was 50% and mortality rate within the first 1 week was 17% . Clinical analysis of those cases revealed that possible risk factors were neutropenia, sever sepsis and prior use of antibiotics (antipseudomonal antibiotics were administered before positive blood culture episodes in 90% cases) . But these factors were not statistically significant between dead and survived cases . CONCLUSION: To improve the prognosis of Pseudomonas aeruginosa bacteremia, we must change the management of the hospital infection, such as the more rational use of new antipseudomonal antibiotics and the more clean and reasonable management of central venous catheters.

Am J Respir Crit Care Med, 1999 Dec, 160(6), 2040 - 7
IL-10 attenuates excessive inflammation in chronic Pseudomonas infection in mice; Chmiel JF et al.; Cystic fibrosis (CF) lung disease is characterized by an excessive inflammatory response associated with chronic Pseudomonas aeruginosa endobronchial infection . Compared with bronchoalveolar lavage fluid from healthy subjects, lavage fluid from patients with CF contains elevated proinflammatory cytokines but negligible amounts of the anti-inflammatory cytokine interleukin-10 (IL-10) . We sought to determine whether IL-10 deficiency results in increased local and systemic morbidity in mice with chronic endobronchial infection with P . aeruginosa embedded in agar beads and to determine if exogenous IL-10 might reduce these effects . Infected IL-10 knockout mice had more severe weight loss (p = 0.04) and increased area of lung inflammation (28 +/- 4 versus 10 +/- 2%, p < 0.002) but no alterations in bacterial burden compared with wild-type mice . Infected CD-1 mice treated with IL-10 had improved survival (p = 0 . 035), less severe weight loss (p < 0.005), fewer bronchoalveolar lavage neutrophils (3 x 10(5)/ml versus 5 x 10(6)/ml, p < 0.02), and decreased area of lung inflammation (11 +/- 2 versus 35 +/- 7%, p < 0.01) but no alterations in bacterial burden compared with placebo-treated mice . These data suggest that IL-10 is an important regulator of the inflammatory response to P . aeruginosa endobronchial infection and that further investigation into the use of IL-10 in CF is warranted.

Tohoku J Exp Med, 1999 Jul, 188(3), 271 - 3
Usefulness of procalcitonin in Pseudomonas burn wound sepsis model; Nakae H et al.; Procalcitonin (PCT), a precursor of calcitonin, and endotoxin were determined in the burn wound sepsis model in which 21 Sprague-Dawley rats were scalded approximately 30% on their back . On day 2 post burn, the wounds were inoculated 1 x 10(8) colony-forming units of Pseudomonas aeruginosa . On day 5 post burn P . aeruginosa was detected by blood culture in 10 of the 21 rats (47.6%) . The mortality rate 7 days after burn was 90.5% . Significant correlations were observed between serum endotoxin levels and serum PCT levels on day 5 post burn (r = 0.860, p<0.001) . It was suggested that endotoxin may induce the release of PCT and that measuring the levels of PCT may be useful in diagnosing burn wound sepsis.

Biochem J, 1999 Dec 15, 344 Pt 3, 845 - 9
Identification of an anti-mycobacterial domain in NK-lysin and granulysin; Andreu D et al.; NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively . The solution structure of NK-lysin comprises five amphipathic alpha-helices . To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin . CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of alpha-helical peptides . The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin . One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive . Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them . Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity . These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.

J Agric Food Chem, 1999 Oct, 47(10), 4297 - 300
Antibacterial activity of turmeric oil: a byproduct from curcumin manufacture; Negi PS et al.; Curcumin, the yellow color pigment of turmeric, is produced industrially from turmeric oleoresin . The mother liquor after isolation of curcumin from oleoresin contains approximately 40% oil . The oil was extracted from the mother liquor using hexane at 60 degrees C, and the hexane extract was separated into three fractions using silica gel column chromatography . These fractions were tested for antibacterial activity by pour plate method against Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa . Fraction II eluted with 5% ethyl acetate in hexane was found to be most active fraction . The turmeric oil, fraction I, and fraction II were analyzed by GC and GC-MS . ar-Turmerone, turmerone, and curlone were found to be the major compounds present in these fractions along with other oxygenated compounds.

Mol Gen Genet, 1999 Oct, 262(3), 552 - 8
Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c; Teramoto M et al.; Comamonas testosteroni strain R5 is a phenol-degrading bacterium which expresses a phenol-oxygenating activity that is characterized by low Ks (the apparent half-saturation constant in Haldane's equation) and low K(SI) (the apparent inhibition constant) values . We have now cloned the gene cluster encoding a phenol hydroxylase (phcKLMNOP) and its cognate regulator (phcR) from strain R5 . Transformation of Pseudomonas aeruginosa PAO1c (Phenol Catechol+) with pROR502, a derivative of pRO1614 containing the cloned genes, confers the ability to grow on phenol as the sole carbon source . The Ks and K(SI) values for the phenol-oxygenating activity of PAO1c(pROR502) are almost identical to those of strain R5, suggesting that the phcKLMNOP genes encode the major phenol hydroxylase in strain R5 . A phylogenetic analysis shows the phenol hydroxylase from strain R5 to be more closely related to toluene/benzene-2-monooxygenase (Tb2m) from Pseudomonas sp . JS150 than to the phenol hydroxylases from P . putida CF600 and BH, or to the phenol hydroxylase from Ralstonia eutropha E2 . Analysis of the substrate specificity of PAO1c(pROR502) and PAO1c derivatives expressing phenol hydroxylase from P . putida BH or from R . eutropha E2 indicates that these phenol hydroxylases catalyze the oxidation not only of phenol and cresols but also of toluene and benzene.

J Antimicrob Chemother, 1999 Oct, 44(4), 537 - 40
Contribution of the MexAB-OprM multidrug efflux system to the beta-lactam resistance of penicillin-binding protein and beta-lactamase-derepressed mutants of Pseudomonas aeruginosa; Srikumar R et al.; Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the beta-lactam resistance of beta-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant . This highlights the multifactorial nature of beta-lactam resistance in this organism . Moreover, the contribution of efflux to the net resistance seen in some beta-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming beta-lactam resistance attributed to efflux as well as to other mechanisms of beta-lactam resistance.

Folia Microbiol (Praha), 1999, 44(2), 187 - 90
Antibacterial effect of some substituted tricyclic quinazolines and their synthetic precursors; Jantova S et al.; Eleven substituted tricyclic quinazolines and their synthetic precursors were tested for antibacterial effects . 3-Chloromethylcarbonyl-2-methylquinazolin-4-thione and 5-phenyl-2,3-dihydro-1,2,4-triazolo{4,3-c}quinazolin-3-one had the highest antibacterial effect against Bacillus subtilis, the MIC values being 50 mg/L . Two tested derivatives were more active against Pseudomonas aeruginosa than ampicillin, the IC50 values being 80 and 100 mg/L . The most effective derivatives contained in the structure generally pharmacologically active chromophores--methyl group in position 2 and a chloromethyl configuration on the carbonyl group in position 3.

Pediatr Pulmonol, 1999 Dec, 28(6), 449 - 50
If you can't stand the rash, get out of the kitchen: an unusual adverse reaction to ciprofloxacin; Jaffe A et al.; Ciprofloxacin, a quinolone antibiotic, is used to treat a wide variety of infections including Pseudomonas aeruginosa in patients with cystic fibrosis (CF) . Photosensitivity is a well-known complication of treatment with this group of antibiotics, and it is more common in patients with CF . We report on a case of photosensitivity induced by indoor fluorescent strip-lighting (spectral range, 295-760 nm) in a 12-year-old girl with CF treated with ciprofloxacin . This type of lighting emits UVA rays (320-400 nm) which cause skin damage in the presence of sensitizing agents . Patients taking ciprofloxacin are usually advised to protect their skin from direct sunlight . We suggest that more attention should be paid to indoor sources of UV light .

Invest Ophthalmol Vis Sci, 1999 Dec, 40(13), 3168 - 76
Evidence for TIMP-1 protection against P . aeruginosa-induced corneal ulceration and perforation; Kernacki KA et al.; PURPOSE: To determine the biological significance of individual endogenous tissue inhibitors of metalloproteinases (TIMPs) in protection against tissue destruction using a Pseudomonas aeruginosa-induced model of corneal ulceration . METHODS: Corneal TIMP-1, -2, and -3 mRNA levels were compared between young adult (resistant) and aged (susceptible) mice challenged with P . aeruginosa . Resistant mice that demonstrated greater amounts of an individual TIMP were treated with polyclonal antibody (pAb) to that TIMP . To determine whether TIMP neutralization exacerbated P . aeruginosa-induced corneal disease, TIMP pAb- and normal rabbit serum (NRS)- (control) treated mice were examined macroscopically and histopathologically after infection . Corneal neutrophil (PMN) myeloperoxidase (MPO) levels also were examined in these mice . RESULTS: Greater amounts of TIMP-1 mRNA only were found in corneas of resistant versus suscep tible mice after P . aeruginosa challenge . Systemic treatment of resistant mice with TIMP-1 pAb resulted in corneal perforation by 5 to 7 days after infection (PI) . Histopathologic evaluation of corneal tissues from TIMP-1 pAb- versus NRS-treated mice confirmed that TIMP-1 pAb treatment resulted in extensive stromal dissolution . This treatment also was associated with loss of epithelium within the central cornea . Both the histopathology and PMN MPO enzyme assays also showed an increase in corneal PMN number following TIMP-1 pAb treatment . CONCLUSIONS: These studies provide evidence that, after P . aeruginosa infection, adequate endogenous expression of TIMP-1 in cornea protects against extensive corneal tissue destruction . The protective effects of TIMP-1 may be multifactorial . In addition to directly protecting extracellular matrix components from active matrix metalloproteinases, TIMP-1 may either directly or indirectly influence recruitment of PMNs into infected cornea . Finally, TIMP-1 also may affect wound healing and resurfacing of the corneal epithelium.

Cytobios, 1999, 99(392), 183 - 9
Pseudomonas aeruginosa (GRC1) as a strong antagonist of Macrophomina phaseolina and Fusarium oxysporum; Gupta CP et al.; A plant growth promotory bacterial strain, isolated from the potato rhizosphere, was characterized as Pseudomonas aeruginosa (GRC1) . The isolate produced an hydroxamate type of siderophore after 48 h of incubation on tryptic soy medium under iron deficient conditions . The in vitro antifungal activity of P . aeruginosa was tested against two soil-borne plant pathogens, Macrophomina phaseolina and Fusarium oxysporum . The antagonistic behaviour of the isolate was tested by dual culture technique . The growth inhibition of M . phaseolina and F . oxysporum was 74.1% and 70.5%, respectively, after 5 days of incubation . The production of hydrocyanic acid and indole acetic acid was also recorded under normal growth conditions.

Eur J Biochem, 1999 Dec, 266(3), 986 - 96
Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAO1; Quadroni M et al.; A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two-dimensional electrophoresis and MS . Thirteen proteins were found to be induced de novo or upregulated in P . aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source . Protein spots excised from the two-dimensional gels were analysed by N-terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments . The coding sequences for 11 of these proteins were unambiguously identified in the P . aeruginosa genome sequence . Expression of these genes was investigated by reverse transcription-PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level . Three classes of sulfur-regulated proteins were found . The first class (five proteins) were high-affinity periplasmic solute-binding proteins with apparent specificity for sulfate and sulfonates . A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins) . The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein . This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription-PCR significantly increases the conclusions that can be drawn from the proteomic study.

Br J Surg, 1999 Nov, 86(11), 1433 - 6
Improved management of infrainguinal bypass graft infection with methicillin-resistant Staphylococcus aureus; Chalmers RT et al.; BACKGROUND: There is considerable debate over the management of infected infrainguinal grafts . This report describes recent experience in this field and documents the change in clinical practice needed to deal with methicillin-resistant Staphylococcus aureus (MRSA) . METHODS: All infected infrainguinal grafts between January 1991 and July 1997 were reviewed . In the light of the findings, clinical practice was modified considerably . A further 1 year was audited prospectively up to August 1998 . RESULTS: Twenty-six patients were treated for 27 infrainguinal graft infections (25 prosthetic, two vein) . Twenty were treated by complete graft excision as the initial therapy; graft preservation was attempted in six patients . Before 1995, the infecting organisms were predominantly Pseudomonas aeruginosa or methicillin-sensitive staphylococci . Subsequently all 14 patients treated up to 1997 had infection with MRSA . The overall amputation rate was 17 of 26; ten amputations were in patients with MRSA . Four patients died, all with MRSA sepsis . As a result of this experience a policy of complete isolation was adopted for all patients infected with MRSA . In the 12 months since this policy was introduced, 77 infrainguinal grafts (61 vein, 16 prosthetic) have been inserted . Two grafts (3 per cent) have become infected, necessitating graft excision and amputation . CONCLUSION: MRSA infection of an infrainguinal graft is a serious complication with high associated amputation and mortality rates . Isolation and barrier nursing appeared to contain the problem.

Antimicrob Agents Chemother, 1999 Dec, 43(12), 3033 - 5
Beneficial effect of adjunctive azithromycin in treatment of mucoid Pseudomonas aeruginosa pneumonia in the murine model; Nicolau DP et al.; While a time-kill methodology noted no appreciable improvement in bactericidal activity with the addition of azithromycin (AZM) to a ceftazidime (CAZ) regimen, data from the murine pneumonia model showed that the addition of AZM significantly improved survival compared to treatment with CAZ alone . These data suggest that AZM might be a useful adjunctive therapy in the management of pneumonia resulting from mucoid isolates of Pseudomonas aeruginosa.

Antimicrob Agents Chemother, 1999 Dec, 43(12), 2975 - 83
Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability; Westbrock-Wadman S et al.; Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P . aeruginosa isolates are of the impermeability phenotype . The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated . A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype . Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system . The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P . aeruginosa is very different from that described for Escherichia coli with mexXY . Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of amrB in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain . Furthermore, transcription of the amrAB genes was shown to be up-regulated in P . aeruginosa clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient . This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance . Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.

Antimicrob Agents Chemother, 1999 Dec, 43(12), 2877 - 80
Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis; Shawar RM et al.; The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation . The tobramycin MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90) were 1 and 8 microg/ml, respectively . Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics . The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%) . For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates . Of 56 isolates for which the tobramycin MIC was > or = 16 microg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as "impermeability."

Diagn Microbiol Infect Dis, 1999 Oct, 35(2), 143 - 51
United States geographic bacteria susceptibility patterns . 1997 ASCP Susceptibility Testing Group; Emerging ciprofloxacin-resistant Pseudomonas aeruginosa; Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33136, USAPURPOSE: To report a clinical series of ciprofloxacin-resistant ocular isolates of Pseudomonas aeruginosa from a tertiary care ophthalmic center . METHODS: Review of in vitro sensitivities of all ocular isolates of P . aeruginosa be tween July 1991 and September 1998 . In vitro resistance was defined as a minimum inhibitory concentration of 4 or more microg per ml . RESULTS: Nine of 423 ocular isolates of P . aeruginosa showed in vitro resistance to ciprofloxacin . From 1991 to 1994, 0.44% (1/227) of ocular isolates were resistant to ciprofloxacin, whereas from 1995 to 1998, 4.1% (8/ 196) of ocular isolates showed in vitro resistance (P = .014) . CONCLUSIONS: Ciprofloxacin-resistant P . aeruginosa has been identified in recent clinical ocular specimens . Ciprofloxacin resistance among ocular isolates of P . aeruginosa is a local and worldwide concern.

Toxicology, 1999 Nov 5, 138(2), 103 - 26
Pathophysiological mechanisms of TNF during intoxication with natural or man-made toxins; Schumann J et al.; Intoxication with different natural toxins or man-made toxicants has been associated with the induction of tumor necrosis factor alpha (TNF) . These include endotoxin, superantigens, Pseudomonas aeruginosa exotoxin A, bacterial DNA, T cell stimulatory agents such as agonistic anti-CD3 mAbs or concanavalin A, alpha-amanitin, paracetamol, ethanol, carbon tetrachloride, dioxin, and dimethylnitrosamine . In this paper we compile and discuss the current knowledge on the pathophysiological role of TNF during intoxication with all mentioned toxins and toxicants . A possible role of gut-derived endotoxin in several TNF-dependent toxic events has been considered . The development of pharmaceuticals that selectively interfere with the detrimental pathways induced by TNF during intoxication with bacteria, viruses, drugs, or other chemicals requires detailed knowledge of the signaling pathways originating from the two TNF receptors (TNFR1 and TNFR2) . Major characteristics of these signaling pathways are described and put together.

Mikrobiologiia, 1999 Jul-Aug, 68(4), 485 - 90
{Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures}; Maksimov VN et al.; The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P . aeruginosa K-2 was studied . The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate . The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate . The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium . The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant . In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.

Genetika, 1999 Sep, 35(9), 1182 - 90
{Natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa}; Mit'kina LN et al.; Bacterial viruses of Pseudomonas aeruginosa assigned to two groups, D3112 and B3, recombine with very low frequencies . Previous study of the genome structure of intergroup hybrids suggested the incompatibility of some genetic modules of these bacteriophages . In this work, several natural hybrid transposable phages that had the genomes largely consisting of modules of phages from group D3112 and B3, were described . The discovery of these phages suggests the continuous genetic exchange in nature of these viruses belonging to different species . This model is considered as promising from the viewpoint of monitoring virus evolution.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13904 - 9
Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa; Whiteley M et al.; Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response . Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically . We have constructed a library of random insertions in the chromosome of a P . aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ . This library was screened for acyl-HSL induction of lacZ . Thirty-nine quorum sensing-regulated genes were identified . The genes were organized into classes depending on the pattern of regulation . About half of the genes appear to be in seven operons, some seem organized in large patches on the genome . Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites . Many of the genes identified showed a high level of induction by acyl-HSL signaling.

Singapore Med J, 1999 Aug, 40(8), 508 - 12
Hospital acquired pneumonia in the medical intensive care unit--a prospective study; Stebbings AE et al.; AIM OF STUDY: The aim of the study was to define the prevalence, risk factors, spectrum of organisms and sensitivity patterns, and the outcome in patients with severe hospital acquired pneumonia (HAP) in the Medical Intensive Care Unit (SCU) in a hospital in Singapore . METHOD: Consecutive patients admitted to the MICU over a 6-month period were studied and assessed daily to determine whether patients had developed HAP based on defined clinical criteria . Sputum or endotracheal aspirate was obtained and results recorded from each patient on admission and every subsequent three days throughout the stay in the MICU . Mortality during hospital stay was the main outcome measure recorded . RESULTS: A total of 136 patients (150 admissions) were studied; 24 patients were identified as having HAP . The prevalence of HAP was 17% {both ventilator-associated pneumonia (VAP) and pneumonia acquired from the ward (WAP)} . Cerebral disease was the main risk factor for VAP (OR 4.94, 95% CI 1.33-18.4) . The spectrum of organisms which caused HAP were polymicrobial, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and coagulase negative Staphylococcus . The mortality of patients with VAP and WAP were 72.7% and 76.9% respectively . CONCLUSION: This study concludes that HAP in the MICU is common with a high mortality . The spectrum of organisms was comparable to previous studies.

J Bacteriol, 1999 Dec, 181(23), 7401 - 4
Negative control of flagellum synthesis in Pseudomonas aeruginosa is modulated by the alternative sigma factor AlgT (AlgU); Garrett ES et al.; Many respiratory isolates of Pseudomonas aeruginosa from cystic fibrosis patients are mucoid (alginate producing) yet lack flagella . It was hypothesized that an alginate regulator inhibits flagellar gene expression . Mutations in algB, algR, and algT resulted in nonmucoid derivatives, yet algT mutants expressed flagella . AlgT-dependent control of flagellum synthesis occurred through inhibition of fliC but not rpoN transcription.

J Bacteriol, 1999 Dec, 181(23), 7398 - 400
Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa; Alvarez AH et al.; Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more (51)CrO(4)(2-) than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain . Chromate uptake showed saturation kinetics with an apparent K(m) of 0.12 mM chromate and a V(max) of 0 . 5 nmol of chromate/min per mg of protein . Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate . The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.

J Bacteriol, 1999 Dec, 181(23), 7221 - 7
Cloning and analysis of the capsid morphogenesis genes of Pseudomonas aeruginosa bacteriophage D3: another example of protein chain mail?
Gilakjan ZA, Kropinski AM.
The terminal DNA restriction fragments (PstI-D and -B) of Pseudomonas aeruginosa bacteriophage D3 were ligated, cloned, and sequenced . Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins . The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97 . Phage D3 was purified by CsCl equilibrium gradient centrifugation (rho = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa . The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel . The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor . This is identical to the situation observed with coliphage HK97.

J Bacteriol, 1999 Dec, 181(23), 7176 - 84
AlgT (sigma22) controls alginate production and tolerance to environmental stress in Pseudomonas syringae; Keith LM et al.; Pseudomonas aeruginosa and the phytopathogen P . syringae produce the exopolysaccharide alginate, which is a copolymer of D-mannuronic and L-guluronic acids . One of the key regulatory genes controlling alginate biosynthesis in P . aeruginosa is algT, which encodes the alternate sigma factor, sigma(22) . In the present study, the algT gene product from P . syringae pv . syringae showed 90% amino acid identity with its P . aeruginosa counterpart, and sequence analysis of the region flanking algT in P . syringae revealed the presence of nadB, mucA, and mucB in an arrangement virtually identical to that of P . aeruginosa . An algT mutant of P . syringae was defective in alginate production but could be complemented with wild-type algT from P . syringae or P . aeruginosa when expressed in trans . The algT mutant also displayed increased sensitivity to heat, paraquat, and hydrogen peroxide (H(2)O(2)); the latter two compounds are known to generate reactive oxygen intermediates . Signals for activation of algT gene expression in P . syringae were investigated with an algT::uidA transcriptional fusion . Like that in P . aeruginosa, algT transcription in P . syringae was activated by heat shock . However, algT expression in P . syringae was also stimulated by osmotic stress and by exposure to paraquat, H(2)O(2), and copper sulfate . The latter two compounds are frequently encountered during colonization of plant tissue and may be unique signals for algT activation in P . syringae.

J Paediatr Child Health, 1999 Oct, 35(5), 505 - 6
Ecthyma gangrenosum as a very early herald of acute lymphoblastic leukaemia; Pouryousefi A et al.; Ecthyma gangrenosum is a manifestation of cutaneous infection by Pseudomonas aeruginosa . The lesion may be associated with immunocompromise, in particular neutropenia . We present a case of ecthyma gangrenosum in a previously healthy child who 3 months later developed acute lymphoblastic leukaemia; we use this to illustrate the lack of information on the growth rate of malignant and premalignant leukaemic cells in children.

Gene, 1999 Oct 1, 238(2), 417 - 25
Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa; Hassan MT et al.; Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance . An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained . A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined . Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified . The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system . The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc . The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds . DNA-DNA hybridization indicated strong conservation of czr in other environmental P . aeruginosa isolates and in the P . aeruginosa type strain PAO1, a clinical isolate . This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1 . A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs . The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Appl Microbiol Biotechnol, 1999 Oct, 52(4), 524 - 33
Recombinant protein composed of Pseudomonas exotoxin A, outer membrane proteins I and F as vaccine against P . aeruginosa infection; Chen TY et al.; We have constructed a chimeric protein composed of the receptor binding and membrane translocation domains of Pseudomonas exotoxin A (PE) with the outer membrane proteins I and F, together designated as PEIF . The potential of PEIF as a vaccine against Pseudomonas infection was evaluated in BALB/c mice and New Zealand white rabbits . We examined titers of anti-PE and anti-OprF antibodies, and the ability both to neutralize PE cytotoxicity and to increase opsonophagocytic uptake of Pseudomonas aeruginosa strain PAO1, serogroups 2 and 6 . The results showed that PEIF can induce antibodies not only to neutralize the PE cytotoxicity but also to promote the uptake of various strains of P . aeruginosa by murine peritoneal macrophages . In a burned mouse model, PEIF afforded significant protection against infection by the homologous P . aeruginosa strain PAO1, heterologous serogroup 2, and the PE hyperproducing strain PA103 . These observations thus indicate that PEIF may be used as a novel vaccine against P . aeruginosa infection.

Eur J Biochem, 1999 Dec, 266(2), 616 - 23
Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros; Ishibashi J et al.; A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli . A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE) . Analysis of the O . rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules . O . rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus . A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH2 (Ala22-Lys30-NH2), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma . This peptide showed antibacterial activity against S . aureus, methicillin-resistant S . aureus, E . coli and Pseudomonas aeruginosa . We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH2, ALLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2 . These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria . The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose . These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH2.

Science, 1999 Nov 19, 286(5444), 1561 - 5
Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa; Ernst RK et al.; Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA) . Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions . CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment . CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Am J Health Syst Pharm, 1999 Nov 1, 56(21), 2217 - 23
Influence of fluoroquinolone purchasing patterns on antimicrobial expenditures and Pseudomonas aeruginosa susceptibility; Rifenburg RP et al.; The influence of using ofloxacin in place of ciprofloxacin on hospital fluoroquinolone expenditures, total antimicrobial expenditures, and susceptibility of Pseudomonas aeruginosa to fluoroquinolones was studied . Hospitals with fluoroquinolone expenditures of at least $1 per occupied bed per year were administered annual surveys covering the years 1993 through 1996 . The two most recent consecutive years of data were compared among hospitals that used ciprofloxacin as their primary fluoroquinolone during both years (group 1), hospitals whose ofloxacin purchases increased from accounting for < or =25% of total fluoroquinolone expenditures during year 1 to accounting for >25% during year 2 (group 2), and hospitals whose ofloxacin purchases accounted for at least 25% of total fluoroquinolone expenditures for both years (group 3) . A total of 109 hospitals were included in the study . Most hospitals spent more on fluoroquinolones and total antimicrobials in year 2 than year 1 . Group 3 hospitals had a significant increase in expenditures for fluoroquinolones and non-fluoroquinolone antipseudomonal antimicrobials . Group 2 hospitals did not realize antimicrobial cost savings and had higher rates of Pseudomonas aeruginosa resistance than hospitals that used ciprofloxacin . Whether a hospital changed its pattern of ciprofloxacin and ofloxacin purchasing was not significantly associated with expenditures for fluoroquinolones, nonfluoroquinolone antimicrobial agents, or all antimicrobials . Susceptibility of P . aeruginosa to ciprofloxacin was lower in hospitals with greater proportions of ofloxacin use . Individual hospital, ciprofloxacin expenditures, and study year were found to be predictive of P . aeruginosa susceptibility to ciprofloxacin among all pooled hospitals.

Burns, 1999 Nov, 25(7), 611 - 6
Pseudomonas aeruginosa septicaemia in burns; Gang RK et al.; Out of 1415 patients treated as inpatients at Al-Babtain Center for Burns and Plastic Surgery, Ibn Sina Hospital, Kuwait spanning over a period of 6 years from June 1992 to June 1998, 102 developed clinically and microbiologically proven septicaemia . Only 15 out of them had either single or multiple episodes of septicaemia due to Pseudomonas aeruginosa and were studied during their stay in the hospital . Five of them were males and 10 females, with a mean age of 26 years (range 3-51 years) and mean total body surface area of burns (TBSA) of 66% (range 25-90%) . All of them had flame burns and resuscitation was found to be difficult in eight patients either due to delayed hospitalization or accompanied inhalation injury . Seven patients were intubated, four due to inhalation injury and three for septicaemic complications . Among the 15 patients under study, a total of 36 septicaemic episodes were detected of which 21 were due to P . aeruginosa . This organism was found in the first episodes in nine patients, in second episodes in six, in third episodes in three and fourth, fifth and sixth episodes in one patient, each at a variable postburn day . Ten patients had 38 sessions of excision and skin grafting, six of them survived . Nine of the 15 patients under study died due to septicaemia, but only six of them had P . aeruginosa as the last isolate . Except for one, all patients had > 40% TBSA burn, two had difficult resuscitation and four were intubated . The day of death varied between 3 to 52 days postburn (mean 19 days) . This study showed that females with flame burns are susceptible to P . aeruginosa septicaemia . Difficult resuscitation and intubation also proved to be important risk factors . Septicaemia could occur quite early in the postburn days and the mortality due to this organism was quite high . Early excision and grafting with other effective management may result in a better outcome.

Biochim Biophys Acta, 1999 Nov 16, 1435(1-2), 71 - 83
Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa; Uratani Y et al.; For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form . In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy . CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant . The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm . CD spectral analyses with three different methods (S.W . Provencher, J . Glockner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y . Yang, C.-S.C . Wu, H.Z . Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol . 130 (1986) 208-269; N . Sreerama, R.W . Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal . Biochem . 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet . Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG . These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.

J Immunol, 1999 Nov 15, 163(10), 5505 - 11
Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages; St-Denis A et al.; The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection . In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections . To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha . While phagocytosis of L . donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection . Intracellular survival of a L . donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection . However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression . Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila . In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L . pneumophila . Permissiveness to L . pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones . Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.

Mol Microbiol, 1999 Nov, 34(3), 399 - 413
The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence; Vasil ML et al.; During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes . This review will largely recount what we have learned in the past few years about how P . aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen . More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator - Fur) and a Fur-regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed . These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions . These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g . Krebs cycle), (iii) genes required to survive oxidative stress (e.g . superoxide dismutase), (iv) genes necessary for scavenging iron (e.g . siderophores and their cognate receptors) or genes that contribute to the virulence (e.g . exotoxin A) of this opportunistic pathogen . Despite this recent expansion of knowledge about the response of P . aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed . Virtually nothing is known about which of the distinct iron acquisition mechanisms P . aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.

Mol Microbiol, 1999 Oct, 34(2), 317 - 26
LipC, a second lipase of Pseudomonas aeruginosa, is LipB and Xcp dependent and is transcriptionally regulated by pilus biogenesis components; Martinez A et al.; We have isolated cosmids that complement a Pseudomonas aeruginosa export-impaired mutant by increasing growth on lipid agar, a medium that requires lipase expression and export . These cosmids encode a previously unidentified lipase, LipC, which has high homology to the P . aeruginosa lipA gene product . Like LipA, LipC activity requires the chaperone activity of the lipB gene product and a functional xcp gene cluster for export . However, expression of LipC is barely detectable in a wild-type background . Transposon insertions that increase lipC promoter activity have been obtained that inactivate two pilus biogenesis genes, pilX and pilY1 . This suggests that these proteins either directly or indirectly repress the expression of LipC and may be involved in transducing an extracellular signal that regulates this lipase.

J Bacteriol, 1999 Nov, 181(22), 6865 - 75
Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy; Yao X et al.; Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 . Air-dried sacculi from E . coli had a thickness of 3.0 nm, whereas those from P . aeruginosa were 1.5 nm thick . When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness . Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results . When E . coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 10(7) N/m(2) was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed . Dried sacculi were more rigid with a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) and at times could be broken by the atomic force microscope tip . Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod . Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi . The elasticity of intact E . coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure . Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R . E . Burge, A . G . Fowler, and D . A . Reaveley, J . Mol . Biol . 117:927-953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A . L . Koch, Adv . Microbial Physiol . 24:301-366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm . Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.

Br J Pharmacol, 1999 Oct, 128(4), 845 - 8
The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction; Lawrence RN et al.; The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages . We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels . OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery . Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant . Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active . Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig . The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 311 - 6
In vitro transcription analysis of rpoD in Pseudomonas aeruginosa PAO1; Aramaki H et al.; The rpoD gene encoding the principal sigma factor (sigma(70)) of Pseudomonas aeruginosa is transcribed from two promoters, P(C) and P(HS) . The sequence of P(C) is similar to the Escherichia coli sigma(70) consensus promoter sequence and that of P(HS) is similar to the E . coli sigma(H) consensus promoter sequence . Synthesis of rpoD mRNA from P(C) is constitutive under both steady-state and heat-shock growth conditions, while that of P(HS) is transiently induced upon heat-shock . To gain a better understanding of the regulation of rpoD expression, we examined in vitro transcription of rpoD using two RNA polymerases (Esigma(70) and Esigma(H), containing sigma(70) and sigma(H), respectively) purified from P . aeruginosa . DNase I footprinting analysis showed specific bindings of Esigma(70) and Esigma(H) to P(C) and P(HS) promoter regions, respectively . In the in vitro runoff transcription assay, Esigma(H) transcribed the template from P(HS) both at 30 degrees C and 42 degrees C but not from P(C) . However, Esigma(70) transcribed rpoD not only from P(C) both at 30 degrees C and 42 degrees C but also from P(HS) at 42 degrees C.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 305 - 10
Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157; Duncan SH et al.; Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa . The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity . Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid . Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Am J Respir Crit Care Med, 1999 Nov, 160(5 Pt 1), 1711 - 6
Dry powder versus intravenous and nebulized gentamicin in cystic fibrosis and bronchiectasis . A pilot study; Crowther Labiris NR et al.; Aminoglycosides are a mainstay of therapy for patients with cystic fibrosis (CF) or non-CF bronchiectasis who are infected with Pseudomonas aeruginosa (Psa) . Traditionally, aerosolized antibiotics are delivered by liquid nebulization . The objective of this study was to determine whether a gentamicin dry powder inhaler (DPI) is as microbiologically active and potentially safe as gentamicin inhaled via a small-volume nebulizer (SVN) or given intravenously . The study was done according to a randomized, single-dose, and triple crossover protocol . Ten patients with CF or non-CF bronchiectasis and chronically infected with Psa were recruited . Patients received a single dose of either gentamicin 160 mg via DPI or SVN, or gentamicin at 5 mg/kg by intravenous infusion . In seven of the 10 patients, the minimum inhibitory concentration (MIC) was achieved in sputum after DPI and SVN, with mean (95% confidence interval) gentamicin concentrations at 2 h after administration of 13.1 microgram/g sputum (range: 2.2 to 23.9 microgram/g) and 97.2 microgram/g sputum (range: 0.3 to 194.2 microgram/g), respectively, whereas gentamicin levels in the sputum after intravenous administration failed to reach the MIC . Gentamicin given by DPI and SVN significantly decreased the sputum Psa density (p < 0.05), by almost one order of magnitude . No significant decline in bacterial counts was observed after intravenous gentamicin . When gentamicin was inhaled, blood concentrations were minimal, and were below concentrations known to cause systemic toxicity . For treatment of Psa infections susceptible to gentamicin, gentamicin administration by DPI appeared to be as efficient as by SVN, despite the delivery of a 7-fold lower dose to the airways.

Biochemistry, 1999 Nov 9, 38(45), 14941 - 54
Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro reconstitution of aryl-4, 2-bisthiazoline synthetase activity from PchD, PchE, and PchF; Quadri LE et al.; Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores . The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming with phosphopantetheine to enable covalent tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates . Two cyclization domains (Cy1 in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings . The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product . A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.

Biol Pharm Bull, 1999 Oct, 22(10), 1110 - 2
In vitro transcriptional analysis of the cytochrome P-450cam hydroxylase operon; Aramaki H et al.; To characterize the promoters of cytochrome P-450cam hydroxylase operon (camDCAB) and the repressor gene (camR), in vitro run-off transcription assays were performed using RNA polymerase (RNAP) holoenzyme reconstituted with the core enzyme and the sigma70 protein of Pseudomonas aeruginosa . Both the mRNAs of camDCAB and camR were accurately transcribed from the respective promoter by the reconstituted RNAP holoenzyme . Both the transcriptions were repressed by CamR protein and the repressions were suppressed by D-camphor, consistent with the regulation in P . putida . These results suggest that the RNA polymerase containing sigma70 recognizes the promoter of camDCAB as well as that of camR.

Int Ophthalmol, 1998-99, 22(3), 163 - 7
Prophylaxis of experimental Pseudomonas aeruginosa endophthalmitis after vitrectomy using ceftazidime in the irrigating solution; Liang C et al.; PURPOSE: To test the efficacy of ceftazidime in irrigating solution during vitrectomy to prevent experimental Pseudomonas aeruginosa endophthalmitis . METHODS: Thirty-two rabbit eyes were divided into 6 groups . Vitrectomy using one of two different irrigating solutions was followed by intravitreal injection of P . aeruginosa: Group 1, balanced salt solution (BSS) followed by 100 colony-forming units (CFU) P . aeruginosa; Group 2, BSS fortified with ceftazidime 175 microg/mL (CBSS) followed by intravitreal injection of 100 CFU P . aeruginosa; Group 3, BSS followed by 500 CFU P . aeruginosa; Group 4, CBSS followed by 500 CFU P . aeruginosa; Group 5, BSS followed by 5000 CFU P . aeruginosa; and Group 6, CBSS followed by 5000 CFU P . aeruginosa . The eyes were examined clinically . Vitreous samples were cultured and histology was performed . RESULTS: Group 1: Three of 5 eyes showed mild to moderate vitreous opacities . Group 2: No vitreous opacities developed . Group 3: All eyes demonstrated endophthalmitis . Group 4: All 6 eyes had clear vitreous with visible fundus . Group 5: Severe endophthalmitis occurred in all 6 eyes . Group 6: Four eyes had clearly visible fundus, 2 eyes had hazy vitreous with red reflex of the fundus . Bacterial growth in groups 3, 4, 5, and 6 was seen in 4/4, 1/6, 6/6, and 0/6 eyes, respectively . CONCLUSION: When 100-5000 CFU P . aeruginosa were injected after vitrectomy, ceftazidime in the irrigating solution inhibited the signs of intraocular inflammation, and the rate of positive bacterial culture.

Vaccine, 1999 Nov 12, 18(7-8), 665 - 74
Protection of mice against P . aeruginosa infections by large-scale affinity-purified human IgG specific to P . aeruginosa outer membrane proteins; Lee NG et al.; In order to develop an effective means to treat Pseudomonas aeruginosa infections, we designed a large-scale process for purification of human IgG specific to P . aeruginosa outer membrane proteins (Oprs) from normal human sera . The process we developed includes affinity column chromatography using P . aeruginosa Oprs as ligands, protein A column chromatography and ultrafiltration, which enriched P . aeruginosa Oprs-specific IgG antibody by 500-fold . The purified anti-Oprs IgG was specific to the Oprs as confirmed by an ELISA competition assay and retained opsonophagocytic-killing capacity . In vivo protective efficacy of anti-Oprs IgG was evaluated by passive protection assays in mice where the 50% protective dose of anti-Oprs IgG against P . aeruginosa infections was 41 microg/kg, which was 20 times lower than that of normal serum IgG . When administered to mice 3 h after bacterial challenge, only anti-Oprs IgG afforded protection . These data demonstrate the feasibility of use of the purification process in producing functionally active target-specific human antibodies for clinical use and provide a rationale for use of anti-Oprs IgG as a valuable adjunct to treat P . aeruginosa infections.

Antimicrob Agents Chemother, 1999 Nov, 43(11), 2624 - 8
Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides; Aires JR et al.; A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1 . Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB . This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon . Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host . Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides . Furthermore, this system appeared to function with an outer membrane protein, OprM . While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P . aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob . Agents Chemother . 43:415-417, 1999) . Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P . aeruginosa to aminoglycosides . Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P . aeruginosa had no detectable effect on susceptibility to these agents.

Technol Health Care, 1999, 7(5), 359 - 70
Reduction of the bacterial load by the silver-coated endotracheal tube (SCET), a laboratory investigation; Hartmann M et al.; Microaspiration enabled by high-volume-low-pressure cuffed endotracheal tubes is the most likely explanation for ventilator-associated pneumonia . To decontaminate the secretion at the proximal end of the cuff we developed a silver-coated endotracheal tube (SCET) . In an in vitro model we investigated the efficacy of SCET to lower the bacterial load of secretion and aspirate . We developed a continuously contaminated and mechanically ventilated oropharynx-larynx-lung model to investigate the reduction of the bacterial count by SCET compared to controls . The model was continuously contaminated via the oropharynx-larynx with Pseudomonas aeruginosa ATCC 27853 . During the investigation period of 50 hours the bacterial count of oropharynx-larynx and lung was measured as colony-forming-units/ml . In addition, the characteristic curve of silver ion release of SCET was determined . SCET significantly reduced the bacterial count in oropharynx-larynx at all timepoints (p < 0.05) . In lung the bacterial count was significantly lower beginning with the 36th hour of recording (p < 0.05) . A reduction of greater than 2 log was found from 28 hours on in oropharynx-larynx and from 50 hours on in lung . The release of silver ions was very rapid and was described by a mono-exponential function with a time-constant tau of about 60 minutes and a saturation concentration of 200 +/- 80 microg/l . SCET showed a significant inhibition of growth of P . aeruginosa in the continuously contaminated and mechanically ventilated oropharynx-larynx-lung model . SCET by thus might be helpful in reducing ventilator-associated pneumonia.

Eur Respir J, 1999 Sep, 14(3), 545 - 52
The potential of various lipopolysaccharides to release monocyte chemotactic activity from lung epithelial cells and fibroblasts; Koyama S et al.; Although the cytotoxicity of lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa, i.e . Limulus amoebocyte lysate activity, is less potent than that from Escherichia coli 0127:B8, P . aeruginosa induces prominent sustained lung inflammation, as in cystic fibrosis . The present study was designed to examine the potential for several LPSs obtained from E . coli and P . aeruginosa to release monocyte chemotactic activity (MCA) from lung cells . LPSs differentially stimulated A549 cells, BEAS-2B cells and lung fibroblasts to release MCA (P . aeruginosa >E . coli 0127:B8 from Difco >055:B5 from Sigma >026:B6 (Sigma)) . E . coli 0127:B8 (Sigma) and 0111:B4 (Sigma) did not stimulate these cells . MCA was determined by means of checkerboard analysis . Molecular sieve column chromatography revealed four chemotactic peaks . The release of MCA was inhibited by cycloheximide and lipoxygenase inhibitors . Experiments with blocking antibodies suggested that much of the MCA was secondary to monocyte chemoattractant protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) . Thus, the concentrations of these chemoattractants were examined and it was found that the potency of the various LPSs to stimulate MCA closely paralleled their potency in releasing MCP-1 and GM-GSF . Serum augmented the release of MCP-1 and GM-CSF . However, the differences among LPSs from E . coli and P . aeruginosa in stimulating A549 cells were observed . These data suggest that Pseudomonas aeruginosa lipopolysaccharide may stimulate lung cells to release more monocyte chemotactic activity than lipopolysaccharides derived from Escherichia coli, leading to sustained prominent lung inflammation.

J Appl Microbiol, 1999 Sep, 87(3), 323 - 31
Comparative responses of Pseudomonas stutzeri and Pseudomonas aeruginosa to antibacterial agents; Tattawasart U et al.; The sensitivity of six strains of Pseudomonas stutzeri (NCIMB 568, 10783, 11358, 11359, JM 302, JM 375) to cationic antiseptics, mercury compounds, the parabens, phenolics, EDTA and various antibiotics was compared with Pseudomonas aeruginosa NCIMB 8626 . All Ps . stutzeri strains were highly sensitive to chlorhexidine diacetate, organomercurials and triclosan, but rather less so to quarternary ammonium compounds (QACs) . They were also sensitive to other biocidal agents and more sensitive to many antibiotics than the strain of Ps . aeruginosa . There was little correlation between uptake of chlorhexidine diacetate or cetylpyridinium chloride by dense suspensions of organisms, leakage of intracellular constituents and loss of cell viability.

Microbiology, 1999 Oct, 145 ( Pt 10), 2863 - 73
A re-examination of twitching motility in Pseudomonas aeruginosa; Semmler AB et al.; Twitching motility is a form of solid surface translocation which occurs in a wide range of bacteria and which is dependent on the presence of functional type IV fimbriae or pili . A detailed examination of twitching motility in Pseudomonas aeruginosa under optimal conditions in vitro was carried out . Under these conditions (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) twitching motility is extremely rapid, leading to an overall radial rate of colony expansion of 0.6 mm h(-1) or greater . The zones of colony expansion due to twitching motility are very thin and are best visualized by staining . These zones exhibit concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and consolidation/cell division . Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells which move away from the colony edge . Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells . The bacteria in the lattice network then appear to settle and divide to fill out the colonized space . Our observations redefine twitching motility as a rapid, highly organized mechanism of bacterial translocation by which P . aeruginosa can disperse itself over large areas to colonize new territories . It is also now clear, both morphologically and genetically, that twitching motility and social gliding motility, such as occurs in Myxococcus xanthus, are essentially the same process.

Microbiology, 1999 Oct, 145 ( Pt 10), 2857 - 62
Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa; Alonso A et al.; Pseudomonas aeruginosa is one of the most important opportunistic pathogens involved in nosocomial infections, cystic fibrosis patients included . Hospital isolates frequently present multidrug-resistance (MDR) phenotypes as the consequence of constant antibiotic selective pressure . The kinetics of emergence of P . aeruginosa MDR mutants under antibiotic selective pressure indicated that long-term incubation in the presence of the bacteriostatic antibiotic tetracycline increases the mutation rate per cell per day of P . aeruginosa PAO1 by several orders of magnitude . The tetracycline-resistant mutants obtained were stable, showed decreased susceptibility to antibiotics belonging to different structural families, and contained an outer-membrane protein not present in the wild-type P . aeruginosa strain PAO1 . These data are consistent with the hypothesis that incubation in the presence of tetracycline favours the emergence of MDR mutants in P . aeruginosa . The results are relevant for understanding the rapid emergence of antibiotic-resistant mutants among bacterial populations during infections . Their relationship to other models of increased mutagenesis under stress is discussed with respect to the adaptive mutation phenomenon.

J Am Acad Dermatol, 1999 Nov, 41(5 Pt 2), 840 - 1
Ecthyma gangrenosum in an AIDS patient with normal neutrophil count; Kim EJ et al.; Ecthyma gangrenosum is the cutaneous manifestation of Pseudomonas aeruginosa septicemia, typically affecting immunosuppressed patients, particularly those with neutropenia . Association with HIV disease has been rarely reported . We describe an unusual presentation of solitary ecthyma gangrenosum on the face of a non-neutropenic patient with AIDS.

Infect Immun, 1999 Nov, 67(11), 6164 - 7
Cell death of human polymorphonuclear neutrophils induced by a Pseudomonas aeruginosa cystic fibrosis isolate requires a functional type III secretion system; Dacheux D et al.; With a coincubation model incorporating Pseudomonas aeruginosa and human polymorphonuclear neutrophils (PMNs), a cystic fibrosis (CF) P . aeruginosa isolate has been shown to resist the bactericidal action of PMNs and to induce their cellular death . An isogenic mutant of this CF isolate in which the type III secretion system was rendered nonfunctional was unable to induce cellular death of PMNs.

Infect Immun, 1999 Nov, 67(11), 6084 - 9
Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide; Bals R et al.; Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense . The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms . The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection . C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide . Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1 . Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9 . In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.

Infect Immun, 1999 Nov, 67(11), 5854 - 62
Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections; Rumbaugh KP et al.; The Pseudomonas aeruginosa quorum-sensing systems, las and rhl, control the production of numerous virulence factors . In this study, we have used the burned-mouse model to examine the contribution of quorum-sensing systems to the pathogenesis of P . aeruginosa infections in burn wounds . Different quorum-sensing mutants of P . aeruginosa PAO1 that were defective in the lasR, lasI, or rhlI gene or both the lasI and rhlI genes were utilized . The following parameters of the P . aeruginosa infection were examined: (i) lethality to the burned mouse, (ii) dissemination of the P . aeruginosa strain within the body of the infected mouse (by determining the numbers of CFU of P . aeruginosa within the liver and spleen), and (iii) spread of the P . aeruginosa strain within the burned skin (by determining the numbers of CFU of P . aeruginosa at the inoculation site and at a site about 15 mm from the inoculation site {distant site}) . In comparison with that of PAO1, the in vivo virulence of lasI, lasR, and rhlI mutants was significantly reduced . However, the most significant reduction in in vivo virulence was seen with the lasI rhlI mutant . The numbers of CFU that were recovered from the livers, spleens, and skin of mice infected with different mutants were significantly lower than those of PAO1 . At 8 and 16 h post burn infection, comparable numbers of CFU of PAO1 and lasI and rhlI mutants were obtained from both the inoculation and distant sites of the burned skin of infected mice . In contrast, CFU of the lasR mutant and the lasI rhlI double mutant were recovered only from the inoculation site of infected mice at 8 and 16 h post burn infection . The ability of a plasmid carrying either the lasI or rhlI gene or the lasI and rhlI genes to complement the defect of the lasI rhlI double mutant was also examined . The presence of any of these plasmids within the lasI rhlI double mutant significantly enhanced its in vivo virulence, as well as its ability to spread within the burned skin . These results suggest that the quorum-sensing systems play an important role in the horizontal spread of P . aeruginosa within burned skin and in the dissemination of P . aeruginosa within the bodies of burned-and-infected mice and contributed to the overall virulence of P . aeruginosa in this animal model.

Infect Immun, 1999 Nov, 67(11), 5827 - 33
Enhanced macrophage resistance to Pseudomonas exotoxin A is correlated with decreased expression of the low-density lipoprotein receptor-related protein; Laithwaite JE et al.; Cellular intoxification by exotoxin A of Pseudomonas aeruginosa (PEA) begins when PEA binds to its cellular receptor, the low-density lipoprotein receptor-related protein (LRP) . This receptor is particularly abundant on macrophages . We hypothesize here that inducible changes in cellular expression levels of the LRP represent an important mechanism by which macrophage susceptibility to PEA is regulated by the host . We have examined the effect of lipopolysaccharide (LPS) on LRP expression and PEA sensitivity in the macrophage-like cell line HS-P . Using a {(3)H}leucine incorporation assay to measure inhibition of protein synthesis, we have demonstrated that HS-P macrophages are highly sensitive to PEA and that PEA toxicity is decreased by the LRP antagonist receptor-associated protein . LPS pretreatment decreases HS-P PEA sensitivity in a time- and dose-dependent manner . The dose of toxin required to inhibit protein synthesis by 50% increased from 11.3 +/- 1.2 ng/ml in untreated cells to 25.7 +/- 2.0 ng/ml in cells treated with LPS . In pulse experiments, involving brief exposure to saturating concentrations of PEA, {(3)H}leucine incorporation was more than threefold higher in cells pretreated with LPS than in untreated macrophages . These changes in HS-P PEA sensitivity following LPS treatment were consistently associated with a fivefold decrease in HS-P LRP mRNA expression as measured by Northern blot analysis and a three-and-a-half-fold decrease in HS-P LRP-specific ligand internalization as determined by activated alpha(2)-macroglobulin internalization studies . These data demonstrate for the first time that modulation of LRP levels by extracellular signaling molecules can alter cellular PEA sensitivity.

Clin Infect Dis, 1999 Sep, 29(3), 621 - 5
Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis; Haussler S et al.; In the context of chronic lung infection due to Pseudomonas aeruginosa in cystic fibrosis (CF), attention has been focused on the presence of the most common mucoid phenotype . In this study, the presence of small-colony variants (SCVs) of P . aeruginosa in respiratory tract specimens from patients with CF was investigated, and the clinical conditions predisposing to SCVs were analyzed . P . aeruginosa SCVs were isolated from 33 of 86 P . aeruginosa-positive CF patients over a 2-year period . Fast-growing revertants with larger surface colonies could be isolated from SCV populations . Electron microscopy revealed no significant difference in cell size or morphology . MICs of a broad range of antipseudomonas agents for SCVs were two- to eightfold higher than values for revertants . Recovery of SCVs was correlated with parameters revealing poor lung function and was significantly associated with daily inhalation of tobramycin or colistin.

Clin Infect Dis, 1999 Sep, 29(3), 508 - 14
Is monotherapy for febrile neutropenia still a viable alternative?
Ramphal R.
Monotherapy for empirical treatment of febrile neutropenia is effective and often less costly than combination therapy but remains controversial . The controversy results from observations that combination therapy for Pseudomonas aeruginosa improved outcomes, and this approach became a standard . Many subsequent publications, including the Infectious Diseases Society of America guidelines for febrile neutropenia, now support monotherapy . However, changes in the pathogens involved in febrile neutropenia and in their resistance prompt a reevaluation . In the evaluation of new antibiotics, recent trials comparing either cefepime or meropenem with combination therapy or with ceftazidime confirm that monotherapy remains a viable therapeutic approach, with infectious mortality in the 5% range in all arms . The choice of monotherapy should, however, be made on the basis of resistance patterns seen in an institution . The agent selected should be very active against the organisms that are likely to cause rapidly fatal infections, and clinicians must be prepared to modify monotherapy as appropriate.

Lett Appl Microbiol, 1999 Sep, 29(3), 181 - 6
Molecular characterization of Pseudomonas aeruginosa 2NR degrading naphthalene; Civilini M et al.; Three naphthalene-degrading strains were isolated from compost, characterized by morphological and physiological properties and differentiated by 16S rDNA RFLP . During growth on naphthalene, Pseudomonas aeruginosa 2NR produced ortho catechol pathway intermediates and gentisic acid . The ability to accumulate and degrade gentisic acid shows that Ps . aeruginosa 2NR has a different salicylate pathway to that of the intensely studied Ps . putida NCIB 9816 . Molecular analysis showed the presence both of genes of the upper naphthalene pathway and genes of the ortho and meta catechol pathways . The insertion of nagH and nagG, coding for salicylate 5-hydroxylase in Pseudomonas sp . U2, was absent in Ps . aeruginosa 2NR, as in Ps . putida NCIMB 9816.

Diagn Microbiol Infect Dis, 1999 Sep, 35(1), 81 - 8
Comparative activity of clinafloxacin and nine other compounds tested against 2000 contemporary clinical isolates from patients in United States hospitals; Deshpande LM et al.; The in vitro activity of clinafloxacin (formerly CI-960, AM-1091, PD-127391) was compared with other fluoroquinolones, cephalosporins, gentamicin, vancomycin, imipenem, piperacillin/tazobactam, clindamycin, and metronidazole against 2000 recent clinical strains from a large number of hospitals in the United States . Overall, clinafloxacin was the most active compound tested . Against Pseudomonas aeruginosa, clinafloxacin and ciprofloxacin demonstrated comparable activity (88% and 80% susceptible, respectively), and were four- to 16-fold more potent than levofloxacin (MIC90, 16 micrograms/ml) or trovafloxacin (MIC90, 32 micrograms/ml) . Among anaerobic bacteria, clinafloxacin (MIC50s, 0.25-0.5 microgram/ml) and trovafloxacin (MIC50s, 0.5-2.0 micrograms/ml) were the most active quinolones, whereas metronidazole, imipenem and piperacillin/tazobactam were the most potent comparators . Clinafloxacin demonstrated sustained activity when compared to several available peer drugs against contemporary clinical isolates . The clinafloxacin spectrum against the 15 important pathogens monitored ranged from nil or 4.0% (vancomycin-resistant enterococci) to 100.0% (four different species) susceptible with an average percent susceptibility of 94.0% . This degree of potency and spectrum for clinafloxacin provides a wide potential for use against many species with established resistance to other anti-microbial classes.

Biochemistry, 1999 Oct 19, 38(42), 13976 - 82
Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions; Frankenberg N et al.; Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis . The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C . In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product . This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions . These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+) . The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site . No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B . P . aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using {3,5-(13)C}porphobilinogen as an NMR probe . The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation . The combined data suggest that P . aeruginosa PBGS represents a new type V enzyme . Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion . The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.

Biochemistry, 1999 Oct 19, 38(42), 13968 - 75
Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa; Frankenberg N et al.; During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen . Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST) . After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme . Mg2+ stabilized the oligomeric state but was not essential for octamer formation . Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity . EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+ . At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme . Pb2+, Cd2+, and Hg2+ did not restore activity . A stimulatory effect of monovalent ions was observed . A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found . pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2) . These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis . Some PBGS inhibitors were characterized . Finally, we succeeded in obtaining well-ordered crystals of P . aeruginosa PBGS complexed with the substrate analogue levulinic acid.

Immunol Lett, 1999 Sep 1, 69(3), 359 - 66
Effect of Pseudomonas aeruginosa exotoxin A on IFN-gamma synthesis: expression of costimulatory molecules on monocytes and activity of NK cells; Michalkiewicz J et al.; The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells . The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis . Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis . Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma . P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells . Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells . From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

Intern Med, 1999 Oct, 38(10), 813 - 6
Bronchiectasis with myeloperoxidase antineutrophil cytoplasmic antibody and bactericidal/permeability-increasing protein antineutrophil cytoplasmic antibody; Matsuyama W et al.; A 56-year-old woman was hospitalized for recurrent hemoptysis . She had been suffering from bronchiectasis for 4 years . Pseudomonas aeruginosa was persistently detected in her sputum . Serum was positive for Myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA) and bactericidal/permeability-increasing protein antineutrophil cytoplasmic antibody (BPI-ANCA) . She underwent lung resection . Histopathologically, the resected lung showed bronchiectasis with pulmonary fibrosis but did not show vasculitis . Her serum became negative for the ANCAs after the operation . To date, she has no recurrence of hemoptysis . We discuss this case of bronchiectasis with MPO-ANCA and BPI-ANCA and suggest a possible role for ANCAs in chronic airway infection.

Can J Microbiol, 1999 Sep, 45(9), 791 - 6
Transfer RNA genes and their significance to codon usage in the Pseudomonas aeruginosa lamboid bacteriophage D3; Kropinski AM et al.; Using tRNAscan-SE and FAStRNA we have identified four tRNA genes in the delayed early region of the bacteriophage D3 genome (GenBank accession No . AF077308) . These are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA) . The D3 Thr- and Gly-tRNAs recognize codons, which are rarely used in Pseudomonas aeruginosa and presumably, influence the rate of translation of phage proteins . BLASTN searches revealed that the D3 tRNA genes have homology to tRNA genes from Gram-positive bacteria . Analysis of codon usage in the 91 ORFs discovered in D3 indicates patterns of codon usage reminiscent of Escherichia coli or P . aeruginosa.

J Clin Microbiol, 1999 Nov, 37(11), 3654 - 61
Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit; Speijer H et al.; Colonization with Pseudomonas aeruginosa was studied by taking serial swab specimens from the oropharynges and anuses and tracheal and gastric aspirates from patients in an intensive care unit during a 10-month period in a setting of endemicity . Nineteen (10%) of the 192 patients included in the study were colonized on admission, while another 30 (16%) patients acquired P . aeruginosa while in the hospital . Typing of 353 isolates was performed by random amplified polymorphic DNA (RAPD) analysis, and 56 strains were selected for further typing by RAPD analysis, pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analysis . By these methods, 42, 44, and 44 genotypes were found, respectively . Computer-aided cluster analysis indicated that similar groups of related isolates were obtained by each method . By taking admission periods into account, analysis of the typing results suggested cross-acquisition of P . aeruginosa for five patient pairs . The small number of transfers and the large number of genotypes found indicate that most P . aeruginosa strains were derived from the patients themselves . The numbers of observed typing patterns and band differences between related isolates were counted for each typing method . AFLP analysis with primers without a selective base proved to be the most discriminatory method, followed by PFGE, AFLP analysis (with one selective base), and RAPD analysis . On the basis of a comparison with established strain differentiation criteria for PFGE, the criteria for differentiation of P . aeruginosa by AFLP analysis are presented.

Biochim Biophys Acta, 1999 Sep 22, 1440(2-3), 244 - 52
Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene; Deziel E et al.; Liquid chromatography/mass spectrometry using electrospray ionisation was used to analyse rhamnolipids produced by a Pseudomonas aeruginosa strain with mannitol or naphthalene as carbon source . Identification and quantification of 28 different rhamnolipid congeners was accomplished using a reverse-phase C(18) column and a 30 min chromatographic run . Isomeric rhamnolipids that were not chromatographically resolved could be identified by interpretation of their mass spectra and their relative proportions estimated . The most abundant rhamnolipid produced on mannitol contained two rhamnoses and two 3-hydroxydecanoic acid groups . The most abundant rhamnolipid produced from naphthalene contained two rhamnoses and one 3-hydroxydecanoic acid group.

Gene, 1999 Sep 17, 237(2), 361 - 71
Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of mycobacterium tuberculosis RmlD and pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI; Hoang TT et al.; Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors . This is probably due to the relatively high copy number of the ColE1-based expression vectors . To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed . These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively . Since pViet and pNam do not replicate in E . coli B strains, an E . coli K-12 host strain {SA1503(DE3)} was constructed . This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter . The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity . The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P . aeruginosa FabI (enoyl-ACP reductase).

Genetika, 1999 Jul, 35(7), 886 - 90
{Comparison of the frequency of imi-r resistant Pseudomonas aeruginosa mutants generated by infection of the bacteria with various bacteriophage-transposons}; Pleteneva EN et al.; We compared the frequencies of imipenem-resistant (imi-r) mutants of a Pseudomonas aeruginosa laboratory strain PAO1 infected by the phages-transposons (PT) specific for this pseudomonad species . The frequency of imi-r mutants among the lysogenic bacteria that appeared after infection reflects the frequency of integrative (conserved) PT transposition into ompD2 gene responsible for synthesis of porin, the protein required for the passage of antibiotic through the cell membrane . After infection by either PT the proportion of imi-r mutants among the lysogenic bacteria was higher than that of spontaneous mutants . The imi-r mutants induced by PT infection form colonies that differ in morphology when grown on different media . The frequencies of imi-r mutants induced by all PT are similar, except for HW12, PM57, and PM62 assigned to a species of the group B3 . The phages of this species induce imi-r mutants at a high frequency . Variations in frequencies and colony morphology of imi-r mutants are discussed.

FEBS Lett, 1999 Sep 10, 458(1), 32 - 6
Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa; Gotoh N et al.; The membrane topology of a resistance-nodulation-division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined . Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments . To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (beta-lactamase) fusion plasmids were constructed and analyzed . The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8 . It is therefore proposed that the N- and C-termini are located in the cytoplasm and the predicted orientation is consistent with the 'positive-inside rule' . This topological model can be applied to other RND proteins.

Microbiology, 1999 Sep, 145 ( Pt 9), 2587 - 94
Enzyme polymorphism in Pseudomonas aeruginosa strains recovered from cystic fibrosis patients in France; Martin C et al.; Each of 314 strains of Pseudomonas aeruginosa recovered from 87 French cystic fibrosis (CF) patients was typed by multilocus enzyme electrophoresis to investigate the genetic diversity, the relatedness and the molecular epidemiology of strains isolated from cases of chronic pulmonary colonization . Comparison of allele profiles at 18 enzyme loci identified 17 electrophoretic types (ETs) . Of the 314 isolates, 290 (92%) were either ET1 (n = 127) or ET2 (n = 163), which differed only at the shikimate dehydrogenase (SKD) locus . The mean genetic diversity (H) was 0.138 . These results suggest that there is cross-colonization between patients and/or that two predominant groups of strains are able to colonize French CF patients . Sequential isolates collected from 18 patients during a period of 12-28 months were analysed to assess genomic variability and its relationship to clinical outcome . Six patients were colonized by a stable strain . For the others, double infections or changes in colonization over time were observed . No relationships were detected between the clinical outcome and the persistence of stable isolates, the emergence of transient superinfecting variants, the presence of multiple ETs or the shift of ET during the monitoring.

J Chromatogr B Biomed Sci Appl, 1999 Sep 24, 732(2), 271 - 6
Use of liquid chromatography-mass spectrometry coupling for monitoring the serralysin-catalyzed hydrolysis of a peptide library; Louis D et al.; The use of a peptide library of limited size, is considered to be more appropriate for studying a protease with a complex specificity, but very sensitive and efficient analytical techniques must be used . We have designed and synthesized a 49-peptide library of the type Z-AlaXXAla(amide) (X=Ala, Leu, Val, Phe, Ser, Arg, Glu) for studying the Pseudomonas aeruginosa serralysin specificity . All compounds of the peptide library could be identified by a LC-MS procedure . After hydrolysis of the library by pseudomonal serralysin, the LC-MS procedure also allowed the identification of the hydrolysis products and the different cleavage sites of the substrates.

J Chromatogr B Biomed Sci Appl, 1999 Sep 10, 732(1), 39 - 46
Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography; Muck A et al.; A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed . The procedure can be used for the purification of cell wall bound LPSs after hot phenol-water extraction and for the isolation of extracellular LPSs from the supernatant, respectively . The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1 . Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling . The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.

Jpn J Antibiot, 1999 Jul, 52(7), 491 - 6
{Study on antibiotics susceptibility and mechanism of carbapenem-resistance in clinical isolates of Pseudomonas aeruginosa}; Arita K et al.; The susceptibility of 260 strains of Pseudomonas aeruginosa to several antibiotics and the mechanism of resistance to carbapenems were investigated . The number of strains of P . aeruginosa moderately resistant or resistant to ofloxacin, ceftazidime and imipenem (IPM) were 76 (29.2%), 31 (11.9%) and 30 (11.5%), respectively . There was no clear relationship between the drug resistance of P . aeruginosa and serum type . Fourteen strains (46.6%) out of 30 IPM-resistant strains were susceptible to meropenem (MEPM) . Twenty seven (90.0%) IPM-resistant strains showed cross resistant to panipenem (PAPM), and 12 strains (44.4%) out of the 27 strains showed high susceptibility to MEPM . P . aeruginosa becomes resistant to IPM and PAPM only by the decrease in the outer membrane permeability of these carbapenems . In contrast, P . aeruginosa becomes equally resistant to MEPM by concurrent occurrence of the increase in the efflux of the antibiotics and the decrease in the outer membrane permeability of the antibiotics . The possibility that both mechanisms are taken place concurrently in P . aeruginosa is considered to be low, and it was also supported by the results of the present study.

Ophthalmologica, 1999, 213(5), 305 - 10
Inflammatory response in experimental Staphylococcus and Pseudomonas endophthalmitis; Kim IT et al.; Staphylococcus epidermidis {2.0 x 10(4) colony-forming units (CFU)/0 . 1 ml} and Pseudomonas aeruginosa (2.0 x 10(3) CFU/ml) were inoculated in the vitreous humor of rabbits . In S . epidermidis endophthalmitis, the numbers of microorganisms reached a maximum (4 . 1 x 10(7) CFU/ml) at day 2 after inoculation and then declined spontaneously . However, clinical scores were observed to be worst at day 5 . In P . aeruginosa endophthalmitis, the numbers of microorganisms reached a maximum (9.3 x 10(6) CFU/ml) 36 h after inoculation . However, culture results were persistently positive until day 15 . Electroretinogram (ERG) b-wave amplitudes in S . epidermidis endophthalmitis continued to decrease from day 3 (>24%) until day 5, and then recovered to the preinoculative level of amplitudes at day 7 . ERG b-wave amplitudes in P . aeruginosa endophthalmitis continued to decrease after 24 h (>24%) . ERG b-wave amplitudes from day 7 to day 15 were flat . The inflammatory response continued under the absence of microorganisms in S . epidermidis endophthalmitis . The time in which a maximum in the number of microorganisms was reached was earlier than that in the clinical examination scores in both S . epidermidis and P . aeruginosa endophthalmitis.

Am J Physiol, 1999 Oct, 277(4 Pt 1), L777 - 86
Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P . aeruginosa; Mariencheck WI et al.; In this study, we investigate the interaction between surfactant protein A (SP-A) and a live, mucoid strain of Pseudomonas aeruginosa and identify a mechanism of clearance of this organism by alveolar macrophages . (125)I-labeled SP-A bound live, but not heat-killed, P . aeruginosa organisms in a concentration-dependent manner . Unlabeled SP-A bound live bacteria, protein isolated from whole organisms, and specific proteins of the P . aeruginosa outer membrane . The binding of SP-A to P . aeruginosa and outer membrane components was inhibited by either EDTA or mannose . Phagocytosis assays with fluorescent microscopy demonstrated that the percentage of macrophages with internalized FITC-labeled P . aeruginosa was increased 1.8-fold (19 vs . 35%) by pretreating the live bacteria with SP-A . This finding was confirmed by direct visualization of ingested bacteria by electron microscopy . Adhering macrophages to SP-A-coated surfaces attenuated the increased uptake of P . aeruginosa pretreated with SP-A, suggesting that SP-A acts as an opsonin to stimulate macrophage phagocytosis of this strain of P . aeruginosa.

Am J Physiol, 1999 Oct, 277(4 Pt 1), L749 - 59
Lung matrix incorporation of plasma fibronectin reduces vascular permeability in postsurgical bacteremia; Resnikoff M et al.; Plasma fibronectin (pFN) can incorporate into the lung extracellular matrix (ECM) as well as enhance hepatic cell phagocytic removal of bloodborne microparticulate debris that can contribute to lung vascular injury . Treatment of human pFN (hFN) with N-ethylmaleimide (NEM) blocks its ECM incorporation but not its ability to augment phagocytosis . Using hFN purified from fresh human plasma cryoprecipitate, we compared the effect of NEM-treated hFN versus normal hFN on lung transvascular protein clearance (TVPC) in postoperative bacteremic sheep to determine whether the ability of hFN to attenuate the increase in lung endothelial permeability required its ECM incorporation . Sheep with lung lymph fistulas were infused with a sublethal dose of Pseudomonas aeruginosa (5 x 10(8)) 48 h after surgery . In the first study, sheep received either FN-rich human cryoprecipitate, FN-deficient cryoprecipitate, FN purified from cryoprecipitate (hFN), FN-deficient cryoprecipitate reconstituted with purified hFN, or the sterile saline diluent . In the second study, sheep received either 200 mg of purified hFN (group I), 200 mg of NEM-treated hFN (group II), or the saline diluent (group III) . In the first study, the increase in TVPC after bacterial challenge was attenuated by FN-rich cryoprecipitate, hFN, or reconstituted FN-deficient cryoprecipitate (P < 0.05) but not by saline and FN-deficient cryoprecipitate . In the second study, TVPC increased by 2 h (P < 0.05) and peaked over 4-8 h (P < 0.05) at 380-420% above baseline in postoperative bacteremic sheep given the diluent (group III) . In contrast, intravenous infusion of hFN, but not of NEM-treated hFN, significantly (P < 0.05) attenuated this increase of lung protein clearance . Thus the ability for the intravenously infused purified pFN to attenuate the increase in lung endothelial protein permeability in sheep during postsurgical bacteremia appears to require its ECM incorporation into the interstitial ECM of the lung.

J Bacteriol, 1999 Oct, 181(20), 6300 - 5
Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa; Kohler T et al.; We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed in nfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim . Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT . By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene . Expression of a mexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged . Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termed qrh (quinone oxidoreductase homologue) was identified . Expression of a qrh::lacZ fusion was also found to be activated by MexT . Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.

J Bacteriol, 1999 Oct, 181(20), 6264 - 70
A second operator is involved in Pseudomonas aeruginosa elastase (lasB) activation; Anderson RM et al.; Pseudomonas aeruginosa LasB elastase gene (lasB) transcription is controlled by the two-component quorum-sensing system of LasR, and the autoinducer, 3OC(12)-HSL (N-3-{oxododecanoyl}homoserine lactone) . LasR and 3OC(12)-HSL-mediated lasB activation requires a functional operator sequence (OP1) in the lasB promoter region . Optimal activation of lasB, however, requires a second sequence of 70% identity to OP1, named OP2, located 43 bp upstream of OP1 . In this study, we used sequence substitutions and insertion mutations in lasBp-lacZ fusion plasmids to explore the role of OP2 in lasB activation . Our results demonstrate that (i) OP1 and OP2 synergistically mediate lasB activation; (ii) OP2, like OP1, responds to LasR and 3OC(12)-HSL; and (iii) the putative autoinducer-binding domain of LasR is not required for synergistic activation from OP1 and OP2.

Cytokines Cell Mol Ther, 1999 Jun, 5(2), 69 - 78
CD30L-ETA': a new recombinant immunotoxin based on the CD30 ligand for possible use against human lymphoma; Barth S et al.; Recombinant DNA technology makes it possible to genetically fuse V genes or cytokines to toxin domains, resulting in immunotherapeutics for selective destruction of tumor cells . Since recombinant immunotoxins can be easily manipulated in terms of affinity or cytotoxic potency and produced in large quantities, we have developed a new CD30 ligand-based fusion toxin (CD30L-ETA') . Human CD30L cDNA was ligated into a pET-based expression plasmid and thereby fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I . After IPTG-indiced expression in E . coli strain BL21(DE3), the 60 kDa His-tagged fusion protein (CD30L-ETA') was isolated from inclusion bodies . Denatured protein was renatured in the presence of 0.4 M arginine and a glutathione redox system . Refolded protein was purified and concentrated by ion-exchange chromatography on a HiTrap Q column . The binding properties of CD30L-ETA' were evaluated by competitive ELISA, immunohistochemical staining, and FACS analysis on CD30-expressing cells . The in vitro toxicity of the fusion protein was then tested on the CD30+ Hodgkin-derived cell line L540cy and the Burkitt's lymphoma cell line BL38 . CD30L-ETA' exhibited specific cytotoxicity against L540cy cells (IC50 = 24 ng/ml) as determined by {3H}leucine uptake assays . This is the first report on the specificity and cytotoxic potency of a chimeric CD30L fusion toxin against Hodgkin's disease-derived cells.

Eur Respir J, 1999 Aug, 14(2), 363 - 9
Effect of fluticasone propionate and salmeterol on Pseudomonas aeruginosa infection of the respiratory mucosa in vitro; Dowling RB et al.; The purpose of this study was to investigate the effect of the corticosteroid, fluticasone propionate (FP), on Pseudomonas aeruginosa infection of the respiratory mucosa of an organ culture model in vitro . Organ cultures infected with P . aeruginosa had significantly (p< or =0.05) elevated levels of mucosal damage and significantly (p< or =0.05) less ciliated cells compared to controls . Preincubation of tissue with FP (10(-6) or 10(-5) but not 10(-7) M) prior to P . aeruginosa infection significantly (p< or =0.05) reduced the bacterially induced mucosal damage in a concentration-dependent manner . FP (10(-5) M) also significantly (p< or =0.05) prevented loss of ciliated cells . FP did not alter the density of bacteria adherent to the different mucosal features of the organ cultures, but did reduce total bacterial numbers due to the reduced amount of damaged tissue, which is a preferred site of P . aeruginosa adherence . It has previously been shown that the long-acting beta2-agonist salmeterol (4 x 10(-7)M) also reduces the mucosal damage caused by P . aeruginosa infection, probably via elevation of intracellular cyclic adenosine monophosphate concentrations . Preincubation of tissue with both 10(-7)M FP and 10(-7)M salmeterol, concentrations at which they did not by themselves influence the effect of P . aeruginosa infection, significantly (p< or =0.05) reduced P . aeruginosa-induced loss of cilia . However, there was no additional benefit from adding 4 x 10(-7)M salmeterol to 10(-6)M FP . In conclusion fluticasone propionate reduced mucosal damage caused by P . aeruginosa infection in vitro and preserved ciliated cells . There was a synergistic action with salmeterol in the preservation of ciliated cells.

J Nat Prod, 1999 Sep, 62(9), 1222 - 4
Antimicrobial and antitumor activities of mycosporulone; Guiraud P et al.; The conditions for optimal production of mycosporulone (1) are given . Its cytotoxic, antimicrobial, and antitumor activities are described . The biological activities of 1 were compared with those of known antibacterial, antifungal, and antitumor agents . The compound was particularly active against Pseudomonas aeruginosa and Staphylococcus aureus (resistant to penicillin) . Compound 1 was not toxic to normal human cells (MRC(5)), although it exhibited cytotoxic activity against the human tumor cell lines MDA-MB 231 and PC(3) and the murine L-1210 leukemia cell line.

J Antimicrob Chemother, 1999 Sep, 44(3), 389 - 92
Ability of azlocillin and tobramycin in combination to delay or prevent resistance development in Pseudomonas aeruginosa; Wu YL et al.; The ability of combinations of azlocillin and tobramycin to prevent or delay resistance development in eight Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients was studied using chequerboard titration and in-vitro serial subculture . No isolate had developed resistance to tobramycin after 12 treatments with the antibiotic combination . Azlocillin resistance had not developed in four isolates after 16 exposures, and was delayed in the other four isolates for at least eight exposures . Beta-lactamase production was responsible for azlocillin resistance in two isolates and occurred to a lesser extent in a third.

J Med Microbiol, 1999 Apr, 48(4), 357 - 61
PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients; da Silva Filho LV et al.; This report describes a PCR primer pair that targets the algD GDP mannose gene of Pseudomonas aeruginosa and produces a specific 520-bp PCR product useful for P . aeruginosa identification . This PCR assay was tested with 182 isolates of P . aeruginosa and 20 isolates of other bacterial species, and demonstrated 100% specificity and sensitivity . The test was also able to detect P . aeruginosa directly in clinical samples such as sputum or throat swabs obtained from cystic fibrosis patients . The combination of this primer with a universal bacterial primer, acting as a control to assess DNA quality in the sample, resulted in a robust PCR method that can be used for rapid P . aeruginosa detection.

Neth J Med, 1999 Sep, 55(3), 106 - 9
Conjunctival and corneal colonization by Pseudomonas aeruginosa in mechanically ventilated patients . A prospective study; Smulders C et al.; In patients on mechanical ventilation the eyes may be colonized with P . aeruginosa . This study was designed to establish an association between endotracheal suctioning and this colonization . During the study period from January to August 1996, ten out of twenty-eight (36%) patients who were mechanically ventilated for than 3 days developed colonization of the respiratory tract with P . aeruginosa . In seven out of these ten patients (70%) conjunctival colonization with P . aeruginosa could be established . Subsequently three patients (11%) developed a clinical eye infection . In all patients the eye on the side corresponding to the position of the ventilator, the suction device and the location of the nurse during suctioning procedures, was colonized first . Contamination of the conjunctiva probably occurs by aerosol exposure during disconnection of the intubation tube from the ventilator for tracheal suctioning . Patients on mechanical ventilation may have an increased risk for eye infections.

Am J Respir Crit Care Med, 1999 Oct, 160(4), 1212 - 9
Characteristics of polyclonal endemicity of Pseudomonas aeruginosa colonization in intensive care units . Implications for infection control; Bonten MJ et al.; We investigated the endemicity of Pseudomonas aeruginosa in intensive care units (ICUs) through analyses of surveillance cultures (from the rectum, stomach, oropharynx, and trachea; n = 1,089), and clinical cultures (n = 2,393) from 297 consecutive patients . Multiple isolates of P . aeruginosa (n = 353) were genotyped . Variables associated with acquisition of respiratory tract colonization (RTC) were tested in a risk factor analysis . The mean daily prevalence of colonization was 34% . On admission, 22 patients had intestinal colonization and 13 had RTC . Twenty patients acquired colonization in the intestinal and 24 in the respiratory tract . Forty-four different genotypes were found; 38 (86%) were isolated from individual patients only . In all, 37 patients had RTC with a total of 38 genotypes: 13 (34%) were colonized on admission, 9 (24%) acquired RTC with a novel genotype during a stay in the ICU, five (13%) acquired colonization from their intestinal tract and three (8%) were colonized via cross-acquisition . In eight patients (21%), no route could be demonstrated for colonization . Antibiotics providing P . aeruginosa with a selective growth advantage were associated with acquired RTC . Endemicity of colonization with P . aeruginosa is characterized by polyclonality, and seems to be maintained by continuous admittance of colonized patients and selection pressure from antibiotics rather than by cross-acquisition.

Am J Respir Crit Care Med, 1999 Oct, 160(4), 1130 - 5
Aerosolized prolastin suppresses bacterial proliferation in a model of chronic Pseudomonas aeruginosa lung infection; Cantin AM et al.; High levels of active neutrophil elastase (HNE) are present in the respiratory secretions of patients with cystic fibrosis (CF) . We hypothesized that aerosolized Prolastin (alpha(1)-protease inhibitor or alpha(1)PI, purified from human blood) could suppress airway neutrophil inflammation and accelerate bacterial clearance from the lung in a model of chronic Pseudomonas aeruginosa lung infection . Because human alpha(1)PI effectively inhibits rat as well as human neutrophil elastase (NE) activity in vitro, we choose to test this hypothesis using a rat agar bead model of chronic P . aeruginosa lung infection . In this model, aerosolized Prolastin significantly decreased elastase activity (p < 0.01), lung neutrophil counts (p < 0.01), and bacterial colony counts (p < 0.01) . Prolastin had no direct bactericidal effect on P . aeruginosa in vitro . Lung tissue histopathology revealed a marked decrease in lung inflammation in animals treated with Prolastin . These studies indicate that Prolastin can significantly decrease the elastase burden in the chronically infected lung . In addition, not only does Prolastin suppress lung inflammation, but it also markedly decreases P . aeruginosa density in a rat model of chronic P . aeruginosa lung infection . These data suggest that aerosolized alpha(1)PI may represent a useful nonantibiotic adjunct in the treatment and control of infection and inflammation associated with CF lung disease.

Biochemistry, 1999 Sep 14, 38(37), 12159 - 64
Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa; Zhang L et al.; Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa . ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity . Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined . Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency . This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS . One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction . The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues . Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3 . Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.

EMBO J, 1999 Oct 1, 18(19), 5175 - 86
Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex; O'Hara BP et al.; Inducible expression of the aliphatic amidase operon in Pseudomonas aeruginosa is controlled by an antitermination mechanism which allows production of the full-length transcript only in the presence of small-molecule inducers, such as acetamide . Ligand-regulated antitermination is provided by AmiC, the ligand-sensitive negative regulator, and AmiR, the RNA-binding positive regulator . Under non-inducing or repressing growth conditions, AmiC and AmiR form a complex in which the activity of AmiR is silenced . The crystal structure of the AmiC-AmiR complex identifies AmiR as a new and highly unusual member of the response-regulator family of bacterial signal transduction proteins, regulated by sequestration rather than phosphorylation . Comparison with the structure of free AmiC reveals the subtle mechanism of ligand-induced release of AmiR.

Antimicrob Agents Chemother, 1999 Oct, 43(10), 2574 - 5
Postantibiotic effects of gatifloxacin against gram-positive and -negative organisms; Pankuch GA et al.; Gatifloxacin pneumococcal, staphylococcal and enterococcal postantibiotic effects (PAEs) were 0.5 to 4.0 h, respectively . For Escherichia coli and Pseudomonas aeruginosa, PAEs were 2.2 to 4.8 h . Pneumococcal, staphylococcal, and enterococcal postantibiotic sub-MIC effects (PA-SMEs) (four times the MICs) were 3.7 to 8.6, 2.3 to 3.8, and 1.6 h, respectively, and E . coli and P . aeruginosa PA-SMEs were >/=9.6 and 4.4 h, respectively.

Antimicrob Agents Chemother, 1999 Oct, 43(10), 2559 - 61
Low plasma cefepime levels in critically ill septic patients: pharmacokinetic modeling indicates improved troughs with revised dosing; Lipman J et al.; The pharmacokinetics of a 2-g bolus of cefepime were measured in critically ill patients with normal renal function . Variable and low trough plasma drug concentrations were found, and 8 of 10 patients had levels below the MIC at which 50% of the isolates are inhibited for Pseudomonas aeruginosa . Computer simulations predicted that continuous infusion and shorter dosing intervals would increase trough levels.

Antimicrob Agents Chemother, 1999 Oct, 43(10), 2473 - 8
Use of pharmacodynamic indices to predict efficacy of combination therapy in vivo; Mouton JW et al.; Although combination therapy with antimicrobial agents is often used, no available method explains or predicts the efficacies of these combinations satisfactorily . Since the efficacies of antimicrobial agents can be described by pharmacodynamic indices (PDIs), such as area under the concentration-time curve (AUC), peak level, and the time that the concentration is above the MIC (time>MIC), it was hypothesized that the same PDIs would be valid in explaining efficacy during combination therapy . Twenty-four-hour efficacy data (numbers of CFU) for Pseudomonas aeruginosa in a neutropenic mouse thigh model were determined for various combination regimens: ticarcillin-tobramycin (n = 41 different regimens), ceftazidime-netilmicin (n = 60), ciprofloxacin-ceftazidime (n = 59), netilmicin-ciprofloxacin (n = 38) and for each of these agents given singly . Multiple regression analysis was used to determine the importance of various PDIs (time>MIC, time>0.25 x the MIC, time>4 x the MIC, peak level, AUC, AUC/MIC, and their logarithmically transformed values) during monotherapy and combination therapy . The PDIs that best explained the efficacies of single-agent regimens were time>0.25 x the MIC for beta-lactams and log AUC/MIC for ciprofloxacin and the aminoglycosides . For the combination regimens, regression analysis showed that efficacy could best be explained by the combination of the two PDIs that each best explained the response for the respective agents given singly . A regression model for the efficacy of combination therapy was developed by use of a linear combination of the regression models of the PDI with the highest R(2) for each agent given singly . The model values for the single-agent therapies were then used in that equation, and the predicted values that were obtained were compared with the experimental values . The responses of the combination regimens could best be predicted by the sum of the responses of the single-agent regimens as functions of their respective PDIs (e.g., time>0.25 x the MIC for ticarcillin and log AUC/MIC for tobramycin) . The relationship between the predicted response and the observed response for the combination regimens may be useful for determination of the presence of synergism . We conclude that the PDIs for the individual drugs used in this study are class dependent and predictive of outcome not only when the drugs are given as single agents but also when they are given in combination . When given in combination, there appears to be a degree of synergism independent of the dosing regimen applied.

Antimicrob Agents Chemother, 1999 Oct, 43(10), 2389 - 94
Effects of antibiotic therapy on Pseudomonas aeruginosa-induced lung injury in a rat model; Ernst EJ et al.; The effect of antibiotics on the acute lung injury induced by virulent Pseudomonas aeruginosa PA103 was quantitatively analyzed in a rat model . Lung injury was induced by the instillation of PA103 directly into the right lower lobes of the lungs of anesthetized rats . The alveolar epithelial injury, extravascular lung water, and total plasma equivalents were measured as separate, independent parameters of acute lung injury . Four hours after the instillation of PA103, all the parameters were increased linearly depending on the dose of P . aeruginosa . Next, we examined the effects of intravenously administered antibiotics on the parameters of acute lung injury in D-galactosamine-sensitized rats . One hour after the rats received 10(7) CFU of PA103, an intravenous bolus injection of aztreonam (60 mg/kg) or imipenem-cilastatin (30 mg/kg) was administered . Despite an MIC indicating resistance, imipenem-cilastatin improved all the measurements of lung injury; in contrast, aztreonam, which had an MIC indicating sensitivity, did not improve any of the lung injury parameters . The antibiotics did not generate different quantities of plasma endotoxin; therefore, endotoxin did not appear to explain the differences in lung injury . This in vivo model is useful to quantitatively compare the efficacies of parenteral antibiotic administration on Pseudomonas airspace infections.

Eur J Biochem, 1999 Oct, 265(2), 619 - 26
The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core; Mei G et al.; The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism . The denaturation transition may be fitted by a simple two-state model . The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role . Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar . Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin . In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine . The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein . The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements . The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser . These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability . This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.

Biochemistry, 1999 Sep 28, 38(39), 12690 - 7
Backbone dynamics of azurin in solution: slow conformational change associated with deprotonation of histidine 35; Kalverda AP et al.; 15N relaxation measurements have been performed on the type Iota blue copper protein azurin from Pseudomonas aeruginosa . The relaxation times show that one loop (residues 103-108) and one turn (residues 74-77) display fast internal motions . The rest of the protein is rigid with an average order parameter S(2) of 0.85 +/- 0 . 05 . The copper binding site shows the same degree of rigidity even though is it composed of several loops and lies outside the beta-sheet sandwich . Substantial exchange broadening was found for a number of residues surrounding the side chain of His-35 . The average exchange rate has been determined from NMR exchange spectroscopy experiments and is 45 +/- 6 s(-)(1) at 41 degrees C . The exchange broadening is caused by the protonation/deprotonation equilibrium of His-35 . The NMR results indicate that the two structures of azurin observed by X-ray diffraction of crystals at pH 5.5 and 9.0 {Nar, H., Messerschmidt, A., Huber, R., Van de Kamp, M., Canters, G . W . (1991) J . Mol . Biol . 221, 765-772} are present in solution and that they interconvert slowly.

Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11229 - 34
Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa; Pesci EC et al.; Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner . In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain . Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone . The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P . aeruginosa virulence genes . We report here that P . aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal . It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase . We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P . aeruginosa las and rhl quorum sensing systems, respectively . The demonstration that 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P . aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones.

Infect Control Hosp Epidemiol, 1999 Sep, 20(9), 620 - 3
Nosocomial infections caused by multiresistant Pseudomonas aeruginosa; Arruda EA et al.; A case-control study was done to evaluate factors associated with nosocomial infections by multiresistant Pseudomonas aeruginosa (MRPA) . Results showed that MRPA was associated with the use of immunosuppressive and antimicrobial drugs . Five typing methods indicated that the MRPA infections were due to multiple strains rather than a single strain.

Cent Eur J Public Health, 1999 Aug, 7(3), 140 - 4
Epidemiological analysis of Pseudomonas aeruginosa strains isolated from a selected patient population in Brno, Czech Republic; Sekaninova G et al.; In the 1996/97 period, 1,413 Pseudomonas aeruginosa (PA) strains were isolated from 843 patients of the Brno teaching hospitals of St . Anne and Bohunice together with small groups from other hospitals . In the same period, 203 PA strains, used as controls, were isolated from 187 patients treated outside hospitals . Statistical evaluation was based on 1,023 hospital isolates and 189 control strains . A total of 16 isolates were recovered from the hospital environments and two from therapeutic swimming pools . The epidemiological analysis of these PA strains was based on pyocin typing, serological typing and phage typing . The most frequently occurring pyocin types amongst our strains fell into 8 pyocin-type groups . The prevailing groups differed significantly between the hospital patient and control groups . Similarly, serological typing identified differences in the predominant serotypes between hospital and control patients . The phage typing method revealed that the control PA strains were significantly more sensitive to 21 polyvalent bacteriophages used than the hospital isolates . In relation to pyocin and serological typing, strains isolated from the hospital environment showed characteristics similar to those of the PA strains isolated from hospital patients . Our results indicate that the majority of strain isolated from hospitalised patients had their origin from human or inanimate contacts in the hospitals.

J Biol Inorg Chem, 1999 Feb, 4(1), 111 - 21
Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin; Sokerina EV et al.; We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction 3Zncyt + az(II)-->Zncyt+ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu . Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c . Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrostatic models . The two transition-state theories, Bronsted-Debye-Huckel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used . Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces . The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved . Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.

FEMS Immunol Med Microbiol, 1999 Sep, 25(4), 365 - 9
The hemagglutinating activities of Pseudomonas aeruginosa lectins PA-IL and PA-IIL exhibit opposite temperature profiles due to different receptor types; Gilboa-Garber N et al.; The two Pseudomonas aeruginosa lectins PA-IL and PA-IIL, which are very similar in subunit size, composition and properties, but differ in carbohydrate specificity, were shown to exhibit opposite temperature profiles in hemagglutination tests . The galactophilic PA-IL, which interacts with the erythrocyte I antigen (together with B or P system antigens), resembles Ii system-specific 'cold hemagglutinins' (including antibodies and lectins of animals and plants) in low (4 degrees C) temperature optimum, while the hemagglutination by the fucose- and mannose-binding PA-IIL (like that of antibodies and lectins which do not bind to these antigens) increases on raising the temperature from 4 to 37 degrees C and even to 42 degrees C . The preferential production of both P . aeruginosa lectins at 28 degrees C and their much stronger interaction with enzyme (protease or sialidase)-damaged cells, as well as the lower temperature optimum (4 degrees C) of PA-IL-binding to the host cells, may be associated with the saprophytic rather than parasitic designation of this bacterium.

FEMS Immunol Med Microbiol, 1999 Sep, 25(4), 339 - 47
Human anti-Pseudomonas aeruginosa outer membrane proteins IgG cross-protective against infection with heterologous immunotype strains of P . aeruginosa; Lee NG et al.; In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P . aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column . In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P . aeruginosa . The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P . aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells . Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P . aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P . aeruginosa strains as well as homologous strains . In contrast, despite high titers against P . (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P . aeruginosa . These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P . aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P . aeruginosa infections in humans.

Can J Microbiol, 1999 Jul, 45(7), 607 - 11
Recombinant Pseudomonas exoenzyme S and exoenzyme S from Pseudomonas aeruginosa DG1 share the ability to stimulate T lymphocyte proliferation; Bruno TF et al.; Exoenzyme S from P . aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences . We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P . aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E . coli BL21(DE3), could induce T lymphocyte activation and proliferation . Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively . Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S . These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.

Infect Immun, 1999 Oct, 67(10), 5530 - 7
Pseudomonas aeruginosa induces type-III-secretion-mediated apoptosis of macrophages and epithelial cells; Hauser AR et al.; Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is cytotoxic towards a variety of eukaryotic cells . To investigate the effect of this bacterium on macrophages, we infected J774A.1 cells and primary bone-marrow-derived murine macrophages with the P . aeruginosa strain PA103 in vitro . PA103 caused type-III-secretion-dependent killing of macrophages within 2 h of infection . Only a portion of the killing required the putative cytotoxin ExoU . By three criteria, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assays, cytoplasmic nucleosome assays, and Hoechst staining, the ExoU-independent but type-III-secretion-dependent killing exhibited features of apoptosis . Extracellular bacteria were capable of inducing apoptosis, and some laboratory and clinical isolates of P . aeruginosa induced significantly higher levels of this form of cell death than others . Interestingly, HeLa cells but not Madin-Darby canine kidney cells were susceptible to type-III-secretion-mediated apoptosis under the conditions of these assays . These findings are consistent with a model in which the P . aeruginosa type III secretion system transports at least two factors that kill macrophages: ExoU, which causes necrosis, and a second, as yet unidentified, effector protein, which induces apoptosis . Such killing may contribute to the ability of this organism to persist and disseminate within infected patients.

Infect Immun, 1999 Oct, 67(10), 5386 - 94
Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa; Motley ST et al.; Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells . Using in vivo expression technology, we have identified several promoters in Pseudomonas aeruginosa which are preferentially activated during infection of neutropenic mice . One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells . The full characterization of this protein, termed PpkA, is presented in this communication . The ppkA gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues . The catalytic domain is linked to the rest of the protein by a short proline-rich segment . The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity . The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates . Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA . Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity . Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in P . aeruginosa, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.

Infect Immun, 1999 Oct, 67(10), 5324 - 31
Expression of the soxR gene of Pseudomonas aeruginosa is inducible during infection of burn wounds in mice and is required to cause efficient bacteremia; Ha U et al.; Burn wounds are prone to infection by Pseudomonas aeruginosa, which is an opportunistic pathogen causing various human diseases . During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival . A purine-auxotrophic mutant of P . aeruginosa was unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments . An in vivo expression technology based on purEK gene expression was applied to the burned mouse infection model to isolate P . aeruginosa genes that are specifically induced during infection . Four such in vivo-inducible (ivi) genetic loci were identified, including the gene for a superoxide response regulator (soxR), the gene for a malate synthase G homologue (glcG), an antisense transcript of a putative regulator responding to copper (copR), and an uncharacterized genetic locus . SoxR of Escherichia coli is known to regulate genes involved in protecting the bacterium against oxidative stress . The expression of soxR was proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide . The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished . Furthermore, a soxR null mutant was shown to be much more sensitive than wild-type P . aeruginosa to macrophage-mediated killing . In support of this observation, a soxR null mutant exhibited a significant delay in causing systemic infections in the burned mice . Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the soxR gene in the infection of burn wounds.

Infect Immun, 1999 Oct, 67(10), 5231 - 42
P2Z-Independent and P2Z receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated from cystic fibrosis patients; Zaborina O et al.; We demonstrate that a mucoid, alginate-producing strain of Pseudomonas aeruginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5'-nucleotidase, and ATP-modifying enzymatic activities . The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+ or Mg2+ . Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1 . The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties . The N-terminal sequence of the purified ATPase isolated from an ndk knockout mutant shows its identity with that of the heat shock chaperonin Hsp60 . During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5'-nucleotidase, adenylate kinase, and a putative ATP reductase activity . These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP) . The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane . Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death . Thus mucoid P . aeruginosa cells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation . Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.

Infect Immun, 1999 Oct, 67(10), 5157 - 62
Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release; Ramafi G et al.; The effects of pathologically relevant concentrations (0.38 to 12.5 microM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro . Ca2+ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively . Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+ . These effects of 1-hp on the clearance of Ca2+ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment . The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+ exchanger appeared to be the primary target of 1-hp . These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+ from these cells.

Infect Immun, 1999 Oct, 67(10), 5076 - 82
Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells; Saleh A et al.; ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa . As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated . Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF . Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors . All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells . The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively . Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors . Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this . These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

J Microbiol Immunol Infect, 1998 Sep, 31(3), 151 - 64
Microbiologically induced corrosion of aluminum alloys in fuel-oil/aqueous system; Yang SS et al.; To investigate the microbiologically induced corrosion of aluminum alloys in fuel-oil/aqueous system, aluminum alloys A356, AA 5052, AA 5083 and AA 6061 were chosen as the test alloys and Cladosporium and several fuel-oil contaminated microbes isolated in Taiwan were used as test organisms . Aluminum alloy AA 5083 in fuel-oil/aqueous system was the most susceptible material for microbial corrosion, then followed by aluminum alloys AA 5052 and A356, and AA 6061 was more resistant to microbial aggression . Mixed culture had high capability of corrosion, then followed by Penicillium sp . AM-F5, Fusarium sp . AM-F1, Pseudomonas aeruginosa AM-B5, Ps . fluorescens AM-B9, C . resinae ATCC 22712, Penicillium sp . AM-F2, Candida sp . AM-Y1 and Ps . aeruginosa AM-B11 . From energy dispersive spectrometer analysis, aluminum and magnesium contents decreased in the corrosion area, while chlorine and sulfur contents increased . The major organic acid produced in fuel-oil/aqueous system was acetic acid, and the total organic acids content had a positive correlation with the degree of microbial corrosion.

Mikrobiologiia, 1999 May-Jun, 68(3), 299 - 303
{Relationship between oxidoreductase activity and cytochrome b5 and P-450 levels in Pseudomonas bacteria depending upon the structure of C6-substrate}; Sokol'chik TI et al.; The influence of the structure of C6 growth substrate on the activity of oxidoreductases and content of cytochromes b5 and P-450 in cells of Pseudomonas aeruginosa and P . fluorescens was studied . Among the hydrocarbons tested (hexane, 1-hexene, cyclohexene), 1-hexene was the most efficient inducer of the synthesis of cytochromes b5 and P-450 in log-phase cells of the pseudomonads . Substitution of the hydrocarbon substrate for a carbohydrate one (glucose) also resulted in a considerable increase in the activity of enzymes of the NADH-dependent electron-transport chain.

Pediatr Pulmonol, 1999 Sep, 28(3), 159 - 66
Changing epidemiology of Pseudomonas aeruginosa infection in Danish cystic fibrosis patients (1974-1995); Frederiksen B et al.; Recurrent and chronic lower airway infection with Pseudomonas aeruginosa (PA) is an important component of cystic fibrosis (CF) pulmonary disease . Different modes of treatment and control of CF patients have been introduced at the Copenhagen CF Centre over the past 20 years and have been associated with improved survival . Treatment consisted of: 1) elective antibiotics for 14 days every 3 months to patients with chronic PA infection (started in 1976), 2) cohort isolation to prevent cross-infection (patients with PA were separated from patients without PA, starting in 1981); and 3) early intensive treatment with inhaled colistin and oral ciprofloxacin from time of initial PA colonization (started in 1989) . The aim of the present study was to evaluate the impact of each of these interventions on the changes in the epidemiology of PA . Based on monthly cultures of lower airway secretions in each CF patient seen during 1974-1995, significant changes in the incidence and prevalence of the PA infection were found . The monthly prevalence of chronic PA increased significantly (P < 0.0001) from below 40% before 1976 to above 60% in 1980, which was found to be due to cross-infection among the CF patients after introduction of elective antibiotic courses in 1976 . To deal with this problem, cohort isolation was introduced in 1981, and since then the monthly point prevalence of chronic PA decreased slowly until 1989 (P < 0.0001), when early intensive treatment from initial PA colonization was introduced; this was associated with a further decrease in point prevalence to 45% in 1995 (P < 0.005) . The annual incidence of chronic PA infection also decreased significantly (P < 0.01) from 16% to below 2% after introduction of cohort isolation and early intensive treatment from initial PA isolation . Furthermore, the time from acquisition of first PA to development of chronic PA infection increased significantly, from approximately 1 year to almost 4 years after introduction of cohort isolation (P < 0.0001) . After introduction of early intensive treatment, the probability of still not having developed chronic PA infection 7 years after the first isolation of PA was above 80% (P < 0.0001) . In conclusion, the introduction of cohort isolation and early intensive treatment following the initial isolation of PA resulted in a reduced incidence and prevalence of chronic PA infection . We are not aware of other studies showing a decreasing prevalence of chronic PA infection, as survival of CF patients has increased.

Antibiot Khimioter, 1999, 44(7), 25 - 31
{Clinico-laboratory effectiveness of modern ointments with a polyethylene glycol base in the treatment of purulent wounds}; Blatun LA et al.; The 25-year experience with the underbandage treatment of soft tissue purulent wounds of various location and genesis with modern ointments with polyethylene glycol as the basis was analyzed . Levocin, levomecole, dioxycole, 5-percent dioxydinic, 1-percent iodopyronic, 0.5-percent quinifuryl and furagel ointments proved to preserve their high activity against aerobic grampositive and gramnegative flora . 10-percent mafenide acetate ointment had a high selective effect on Pseudomonas aeruginosa . The new ointments nitacid and streptonitole containing nitazole and white streptocide were highly active against both aerobic and anaerobic infections . The use of the ointments with the polyethylene glycol as the basis made it possible to decrease 2 times the period of the patient hospitalization in surgical units and to shorten the terms of the systemic antibacterial therapy . The marked therapeutic effect of such ointments due to their high dehydrating capacity and broad antibacterial spectrum enabled to consider them as the drugs of choice in the local treatment of purulent wounds during the 1st phase of the wound process, trophic and decubic ulcers, infected burns, diabetic and atherosclerotic gangrene, furuncles, carbuncles, mastitis, etc . The ointments in the water soluble vehicle can be as well used with success for the prophylactic treatment of infected wounds after the suture . The multitarget effect of the ointments in the water soluble vehicle and their ability to prevent severe purulent complications permitted to consider them as the 1st order drugs in cases of emergency.

J Biomed Sci, 1999 Sep-Oct, 6(5), 357 - 63
A nontoxic Pseudomonas exotoxin A induces active immunity and passive protective antibody against Pseudomonas exotoxin A intoxication; Chen TY et al.; Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced by Pseudomonas aeruginosa . In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(Delta576-613), for active immunization against PE-mediated disease . We first examined the toxic effects of PE and PE(Delta576-613) on 5- and 9-week-old ICR mice . The results show that the subcutaneous administration of PE(Delta576-613) at a dose of 250 microg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 microg, respectively . PE(Delta576-613) was then used to immunize ICR mice . The minimum dose of PE(Delta576-613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng . However, immunization with 250 ng PE(Delta576-613) failed to protect the immunized mice from a lethal dose of PE . The effective immunization dose of PE(Delta576-613) that could protect mice against a 2 microg PE challenge was found to be 15 microg . In addition, sera obtained from PE(Delta576-613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE . Thus, PE(Delta576-613) may be used as an alternative route to new PE vaccine development.

Indian J Exp Biol, 1999 May, 37(5), 481 - 6
Bioreactor operated production of lipase: castor oil hydrolysis using partially-purified lipase; Sharon C et al.; A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L-bioreactor . The bioreactor was run at different airflow rates . Low airflow rates (1 and 3 L/min), did not lead to effective growth and lipase production . Growth increased by about one order and lipase production increased by about 6 times, at an airflow rate of 5 L/min . Lipase production occurred during decelerated cell growth . A highly stable lipase was produced which retained its activity in the running bioreactor, even after a period of one month . This stable lipase was partially-purified using ammonium sulphate precipitation technique . Castor oil was hydrolyzed using 300U crude and partially-purified lipase, each . Approximately 21-fold, partially-purified lipase could hydrolyze 81% castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when crude lipase was used.

J Clin Invest, 1999 Sep, 104(6), 743 - 50
Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia; Kurahashi K et al.; The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model . The airspace instillation of the cytotoxic P . aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock . The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung . The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic . The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103 . Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury . We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock . Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P . aeruginosa pneumonia.

Eur J Biochem, 1999 Oct 1, 265(1), 221 - 30
Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin; Sliwinski-Korell A et al.; Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin . Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges . SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism . Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers . Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization . Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization . The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure . Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy . Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm . These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin.

Acta Crystallogr D Biol Crystallogr, 1999 Sep, 55 ( Pt 9), 1591 - 3
Purification, crystallization and preliminary X-ray analysis of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa; Sainz G et al.; The catabolic ornithine carbamoyltransferase (OTCase) from Pseudomonas aeruginosa exhibits allosteric behaviour, with two conformational states of the molecule: an active R form and an inactive T form . The enzyme is a dodecamer with a molecular mass of 455700 Da . Three crystal forms have been obtained . Crystals of allosteric state T are rhombohedral, belonging to the R3 space group, with hexagonal unit-cell parameters a = b = 180.6, c = 122.0 A . They diffract to a resolution of 4.5 A . Two crystal forms for allosteric state R have been obtained, with hexagonal and cubic symmetries . Hexagonal crystals, which diffract to a resolution of 3 . 4 A, belong to the space group P6(3) with unit-cell parameters a = b = 140.8, c = 145.6 A . The cubic crystals belong to space group I23, with unit-cell parameter a = 134.32 A and diffract to a resolution better than 2.5 A . In all crystal forms, the dodecamer exhibits a 23 point-group symmetry.

Folia Microbiol (Praha), 1999, 44(1), 93 - 7
Pseudomonas aeruginosa phage lysate as an immunobiological agent . 1 . Selection of Pseudomonas aeruginosa clinical strains for phage lysate preparation; Sekaninova G et al.; A total of 2087 Pseudomonas aeruginosa isolates collected during the period 1994-1997 were used as starting material . Out of 1704 in-patient isolates, 299 strains were selected for the preparation of phage lysates but only five strains provided stable lysates, i.e., maintained the ability to be repeatedly and completely lysed by the appropriate phage in the course of several years . A set of 193 out-patients (189) and water sources (4) isolates failed to yield strains suitable for phage lysate preparation; 190 strains isolated abroad from patients with cystic fibrosis or respiratory infections included three isolates which, despite having a high degree of mucus production, were suitable for lysate preparation . The antigenic pattern of the phage lysates was ascertained by SDS-polyacrylamide gel electrophoresis.

Int J Dermatol, 1999 Aug, 38(8), 573 - 8
The microbiology of infected and noninfected leg ulcers; Bowler PG et al.; BACKGROUND: A clinical study was undertaken to investigate and compare specifically the aerobic and anaerobic microbiology of infected and noninfected leg ulcers . METHODS: Leg ulcers, defined as being infected on the basis of clinical signs, were swab sampled and investigated for aerobic and anaerobic microorganisms using stringent isolation and identification techniques . RESULTS: Two hundred and twenty isolates were cultured from 44 infected leg ulcers, in comparison with 110 isolates from 30 noninfected leg ulcers . Statistical analysis indicated a significantly greater mean number of anaerobic bacteria per infected ulcer (particularly Peptostreptococcus spp . and Prevotella spp.) in comparison with the noninfected ulcer group (2.5 vs . 1.3, respectively) (P < 0.05) . Also, anaerobes represented 49% of the total microbial composition in infected leg ulcers compared with 36% in noninfected leg ulcers . The mean numbers of aerobes per wound in the two ulcer groups were not statistically different (P > 0.05) . The study failed to demonstrate a clear correlation between commonly implicated facultative pathogens and wound infection . The isolation rate of Pseudomonas aeruginosa was generally low and, although Staphylococcus aureus was a frequent isolate in both wound types, it was more prevalent in noninfected leg ulcers . CONCLUSIONS: This study has demonstrated the complex aerobic-anaerobic microflora which exists in leg ulcers, the prevalence of anaerobes in infected wounds, and a poor correlation between the presence of specific aerobic pathogens and wound infection . In view of these findings, the role of microbial synergistic interactions in the pathogenesis of chronic wound infection may be of greater clinical importance than the isolated involvement of any specific potential pathogen.

Cornea, 1999 Sep, 18(5), 595 - 8
Decontamination of human sclera: an in vitro study; Lucci LM et al.; PURPOSE: The human sclera is frequently used in ophthalmic surgeries and must be preserved in disinfectants that prevent its contamination . In this study the efficiency of glycerin, absolute alcohol (ethanol), and benzalkonium chloride (1:5,000) as human sclera disinfectants were compared . METHODS: Fresh human scleras were trephined, the scleral disks divided into three groups and contaminated with Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), or Bacillus cereus (ATCC 11778) for 24 h . Thereafter they were transferred to preservation vials each containing glycerin, absolute alcohol, benzalkonium chloride diluted in 70% alcohol (1:5,000) or Trypticase Soy Broth (control), respectively, and stored at room temperature . From each vial, two scleral disks were removed after 1, 2, 3, 4, 7, 10, and 14 days of immersion . Both were plated on blood agar, one being macerated, and both incubated at 37 degrees C for 48 h . RESULTS: Pseudomonas aeruginosa, S . aureus, and B . cereus were recovered from the glycerin-immersed scleral disks until the second, fourth, and fourteenth days, respectively . Bacillus cereus was recovered from those immersed in absolute alcohol until the fourteenth day, whereas disks infected with the other microorganisms and immersed in absolute alcohol presented no growth since the very first day of immersion . Bacillus cereus was recovered from scleral disks immersed in benzalkonium chloride diluted in 70% alcohol (1:5,000) only on the first day . CONCLUSION: Resistant microorganisms can survive in scleral tissue preserved in glycerin and absolute alcohol . We conclude that benzalkonium chloride diluted in 70% alcohol (1:5,000) in vitro is the best disinfectant for human sclera after 24 h.

Cornea, 1999 Sep, 18(5), 565 - 9
Risk factors for pediatric presumed microbial keratitis: a case-control study; Vajpayee RB et al.; PURPOSE: To evaluate risk factors for pediatric presumed microbial keratitis and to describe the clinical picture, microbiologic spectrum, treatment modalities, posttreatment sequelae, and visual outcome in cases of pediatric presumed microbial keratitis . METHODS: A case-control study design was used to identify the risk factors associated with pediatric presumed microbial keratitis . Fifty cases of fresh corneal ulceration aged 12 years or younger were compared with 50 controls . The study variables included were age, gender, immunization status, nutritional status (weight for height), and socioeconomic status . The clinical presentation of the cases with corneal ulceration, microbiologic spectrum, and treatment modalities also were evaluated . All the cases were followed up for a minimum of 3 months, and the posttreatment sequelae and visual outcome were analyzed . RESULTS: The mean (+/- standard deviation) age of children with corneal ulceration and controls was 4.8 (+/-3.8) years and 5.1 (+/-2.8) years, respectively . Incomplete immunization status (AOR {95% confidence interval (CI)}, 1.34 {0.62-2.9}) and poor nutritional status {AOR (95% CI) 1.06 (0.68-1.6)} were not found to be the predictors of corneal ulceration . Lower socioeconomic status was significantly associated with the occurrence of corneal ulceration {AOR (95% CI) 1.52 (1.1-2.3)} . Corneal trauma (38%) and systemic illness (24%) were the most often associated predisposing factors . Seventy percent of the cases were culture positive . Staphylococcus (70%) species was the most frequently isolated, followed by Pseudomonas aeruginosa (10%) . Fungi were isolated in five eyes . Postresolution visual acuity at 3 months could be recorded only in 31 eyes and a visual acuity of 6/18 or better was achieved in 22% of these cases . CONCLUSION: Corneal ulceration in pediatric age group in India is associated with poverty.

J Biomater Sci Polym Ed, 1999, 10(8), 827 - 44
Surface roughness enhances upward migration of bacteria on polymer fibers above liquid cultures; Coughlin RW et al.; Monofilament polypropylene (PP) fibers, very similar to fibers that have been used as monofilament tailstrings of interuterine contraceptive devices, were suspended vertically in bacterial liquid monocultures so that a portion of a fiber extended above the liquid surface . In some cases these highly oriented, cold drawn fibers were abraded prior to insertion in the cultures in order to produce surface roughness characterized by axial channels and protruding microfibrils that partially peeled from the fiber surface thereby forming the channels . Extent of migration on a fiber was assessed by aseptically cutting it into small segments, followed by culturing each segment on agar containing growth medium . Such assessment of the PP fibers after 48 h of incubation in the cultures revealed upward migration of Eschericia coli, Pseudomonas aeruginosa, and Staphylococcus aureus over significantly longer distances on the pre-roughened fibers than on those not so pre-treated . Mean measured distances of migration during 48 h were: for E . coli 2.7+/-0.6 mm on roughened fibers (n = 16) and 0.4+/-0.7 mm on fibers not roughened (n = 17); for S . aureus 9.0+/-4.3 mm on roughened fibers (n = 13) and 0.2+/-0.3 mm on fibers not roughened (n = 14); for P . aeruginosa 8.5+/-3.7 mm on roughened fibers (n = 26) and 0.2+/-0.5 mm on fibers not roughened (n = 5) . Although no statistically significant (95% confidence level) difference could be discerned between the migration distances of S . aureus and P . aeruginosa, each of these species migrated a greater distance on the PP than did E . coli . The migrations observed are attributed predominantly to wicking of the liquid cultures upward in the axial grooves developed on the surface of the PP by the eruption and peeling of microfibrils from the surface . Surface tension of the growth medium was significantly lower than that of water and its contact angle on PP was less than 90 deg, thereby indicating a tendency to wet the PP . Bacterial growth in the medium further reduced its contact angle on PP, thereby indicating an even greater tendency to wet PP after such growth.

Am J Physiol, 1999 Sep, 277(3 Pt 1), L616 - 27
Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs; Cadogan E et al.; We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression . Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later . Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs . In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01) . Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity . Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors . Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection . Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not . Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

Aust N Z J Ophthalmol, 1999 Jun-Aug, 27(3-4), 218 - 20
Effect of tear-specific immunoglobulin A on the adhesion of Pseudomonas aeruginosa I to contact lenses; Lan J et al.; PURPOSE: The aims of the study were (i) to determine the immunodominant antigens of Pseudomonos aeruginoso 1 (Paer1) to tear secretory immunoglobulin A (sIgA); (ii) to study the role of sIgA in inhibiting bacterial adhesion to contact lenses . METHODS: SDS-PAGE and Western blotting were used to study the interaction of tear sIgA with Paer1; an adhesion assay was used to study the effect of sIgA on the adhesion of Paer1 to the contact lenses . RESULTS: The results of our study showed that the immunodominant molecules of Paer1 which reacted with tear sIgA were of molecular masses of 105, 50, 45 kDa; the binding of sIgA to Paer1 resulted in a reduction of the adhesion of Paer1 to worn Etafilcon A contact lenses . CONCLUSIONS: In conclusion, Paer1-specific sIgA are present in tears; the immunodominant molecules of Paer1 may be adhesins and tear-specific sIgA might play an important role in protecting the eye from contact-lens-induced corneal infection by preventing bacterial attachment to contact lenses and the ocular surface.

Surgery, 1970 Jul, 68(1), 248 - 53
Treatment of experimental burn wound sepsis by postburn immunization with polyvalent Pseudomonas antigen; Nance FC et al.; 1 . Vaccination with a new heptavalent Pseudomonas aeruginosa antigen was found to reduce mortality rates from experimental pseudomonal burn wound sepsis . 2 . Adjunctive use of the heptavalent vaccine improved survival of animals with pseudomonal sepsis which were treated with topical antibiotics . 3 . Elevated antibody titers following vaccination could be correlated with survival in this model.

Mikrobiol Z, 1999 May-Jun, 61(3), 30 - 6
{An experimental validation of the use of levomycetin preparations for the treatment of burns infected with Pseudomonas aeruginosa}; Tkachenko VL et al.; Application of 1% of chloramphenicol (gel and cream) for local treatment of Pseudomonas aeruginosa burn infection has been studied in experiment . In vivo, both medical forms show pronounced therapeutic effect, they promote elimination of P . aeruginosa from wounds and decrease inflammation . In noninfected thermal trauma in laboratory animals application of gel and cream of chloramphenicol reduces transition from the phase of inflammation to the phase of reparation by 3-8 days and prevents infection of the burn wound by conditionally pathogenic microflora.

Jpn J Ophthalmol, 1999 Jul-Aug, 43(4), 300 - 2
Extensive destruction of the eyeball by invasion of basal cell carcinoma of the eyelid; Jeong S et al.; BACKGROUND: Eyeball destruction caused by invasion of basal cell carcinoma of the eyelid . CASE: A 100-year-old woman showed extensive eyeball destruction caused by the invasion of basal cell carcinoma of the eyelid . Complete ophthalmologic examinations, including computed tomographic (CT) scans of the orbit, were performed . The patient underwent incisional biopsy and bacteriological examination of the exudate from the lesion . OBSERVATIONS: Orbital CT scan showed a mass in the extraconal space of the right orbit, with extension to the adjacent sinus cavity without brain involvement . The remnant of the eyeball was posteriorly displaced . Pseudomonas aeruginosa was identified by culture examination of the exudate . Histological study of the biopsy specimen showed basal cell carcinoma of the noduloulcerative type . CONCLUSIONS: Basal cell carcinoma of the eyelid had caused severe periorbital and eyeball destruction.

Eur J Clin Microbiol Infect Dis, 1999 Jul, 18(7), 473 - 7
Pseudomonas aeruginosa bacteremia in patients infected with human immunodeficiency virus type 1; Vidal F et al.; A prospective analysis of 43 episodes of Pseudomonas aeruginosa bacteremia in HIV-1-infected subjects was performed and the results compared with the incidence and outcome of Pseudomonas aeruginosa bacteremia in other high-risk patients, such as transplant recipients, leukemia patients, or patients hospitalized in the intensive care unit . The incidence of bacteremia/fungemia as a whole and of gram-negative and Pseudomonas aeruginosa bacteremia in particular was greater in HIV-1-infected subjects than in the unselected general population admitted . In contrast, the incidence of Pseudomonas aeruginosa bacteremia in HIV-1-infected patients did not differ from that in patients with other high-risk conditions . In patients with HIV-1 infection, independent risk factors for presenting Pseudomonas aeruginosa bacteremia were nosocomial origin (OR, 2.7; 95% CI, 1.3-5.7), neutropenia (OR, 2.7; 95% CI, 1.07-6.8), previous treatment with cephalosporins (OR, 3.6; 95% CI, 1.1-11.6), and a CD4+ cell count lower than 50 cells/mm3 (OR, 3.1; 95% CI, 1.7-8.6) . Primary bacteremia and pneumonia were the most common forms of presentation . Fourteen (33%) patients died as a consequence of the bacteremia . The presence of severe sepsis (OR, 17.5; 95% CI, 3.2-68) and the institution of inappropriate definitive antibiotic therapy (OR, 2.7; 95% CI, 1.1-13) were independently associated with a poor outcome . One year after the development of bacteremia, only eight (19%) patients remained alive.

Jpn J Antibiot, 1999 Jun, 52(6), 458 - 68
{Estimation of antibacterial activity of various antibiotics against Pseudomonas aeruginosa by score method}; Kouda M et al.; Antibacterial activity of various antibiotics against Pseudomonas aeruginosa isolated from each hospitals depends on the variety or amount of antibiotics used in each hospital . The antibiotic, which is effective to P . aeruginosa in a certain hospital is not always effective to that in other hospital . The excellent antibiotics in antibacterial activity have low MIC and hard to progress in resistance, and such antibiotics may be effective against P . aeruginosa isolated from any hospitals . Therefore we thought that the antibiotic, which was progress to resistance, would show a great difference in MIC among hospitals, and we investigated MIC and difference of MIC of various antibiotics against P . aeruginosa isolated from six hospitals . Furthermore, we converted the data of MICs and difference of MIC among six hospitals into the score, and tried to estimate antibacterial activity of various antibiotics by using those scores . From the results of analysis in this report, we think the antibiotics actually surpass in antibacterial activity may be imipenem, cefozopran, cefsulodin and amikacin . New analytical method proposed in this report will become one of potential methods to estimate antibacterial activity of antibiotics against bacteria isolated from inpatient with bacterial infections.

Jpn J Antibiot, 1999 Jun, 52(6), 449 - 57
{In vitro antimicrobial activity of carbapenem antibiotics against Pseudomonas aeruginosa, measured using a low-concentration Mueller-Hinton Agar culture medium}; Igari J; Ohya et al . noted that the antibacterial activity of carbapenem-family antibiotics against Pseudomonas aeruginosa was significantly enhanced through lowering the basic amino acid concentration in the culture medium . They reported that there was a marked difference in antimicrobial activity of panipenem (PAPM) against Pseudomonas aeruginosa between the culture medium with Mueller-Hinton Agar (MHA) diluted with distilled water and the non-diluted culture medium . We used 2,312 strains of fresh Pseudomonas aeruginosa, isolated from clinical materials, to examine the antibacterial activity of PAPM in non-diluted and diluted culture media . For testing the susceptibility, we employed the Showa disc method and agar plate dilution method . In the Showa disc method, the inhibition diameters of the tested microbial strains showed a larger distribution for both 16-fold and 40-fold diluted MHA, compared to the non-diluted MHA . The MIC values in the agar plate dilution method were also smaller in distribution for the 16-fold as well as the 40-fold diluted MHA, compared to the non-diluted culture media . Approximately 90% of the strains showed decreased MIC values, 2-8 times in the 16-fold diluted MHA and 2-16 times in the 40-fold diluted MHA . From the above results, we confirmed that the in vitro antibacterial activity of PAPM against Pseudomonas aeruginosa was enhanced through lowering the MHA concentration.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 85 - 90
The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis; Olvera C et al.; Pseudomonas aeruginosa produces exoproducts correlated with its pathogenicity . One of these virulence-associated traits is the surfactant rhamnolipid . The production of alginate and lipopolysaccharide (LPS) are also of importance for P . aeruginosa virulence . The product of the algC gene (which is involved in alginate production through its phosphomannomutase activity and in LPS synthesis through its phosphoglucomutase activity) participates in rhamnolipid production, presumably catalyzing the first step in the deoxy-thymidine-diphospho-L-rhamnose (dTDP-L-rhamnose) pathway, the conversion of glucose-6-phosphate to glucose-1-phosphate . Other structural alg genes, encoded in the alg operon, are not involved in rhamnolipid nor LPS production . These results show that the AlgC protein plays a central role in the production of the three P . aeruginosa virulence-associated saccharides: alginate, LPS and rhamnolipid.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 67 - 72
nalB-type mutations causing the overexpression of the MexAB-OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome; Saito K et al.; Two mutations, one at the nalB and the other at the mexR locus, in the Pseudomonas aeruginosa chromosome are known to cause overexpression of the MexAB-OprM efflux pump . Based on the following results, we concluded that nalB is the mutation that has occurred within the mexR gene of the P . aeruginosa chromosome . (i) Nucleotide sequencing of the mex operon upstream region of 21 independent nalB-type mutants including the original nalB9 revealed that all the mutations were located within the mexR gene . The mutations were classified into three different groups and nine types including single base substitutions, single base deletions and base insertions . (ii) Substitution of the mutant mexR with the wild-type mexR and replacement of the wild-type mexR with a defined mexR mutation resulted in the expression of wild-type and nalB-type MexAB-OprM respectively, which was confirmed by testing the antibiotic susceptibility and beta-galactosidase activity of the mexA-lacZ translational fusion.

Phys Ther, 1999 Sep, 79(9), 839 - 46
Bactericidal effect of 0.95-mW helium-neon and 5-mW indium-gallium-aluminum-phosphate laser irradiation at exposure times of 30, 60, and 120 seconds on photosensitized Staphylococcus aureus and Pseudomonas aeruginosa in vitro; DeSimone NA et al.; BACKGROUND AND PURPOSE: Studies have demonstrated a bactericidal effect of laser irradiation when lasers with power outputs of (6 mW are directed toward pathogenic or opportunistic bacteria previously treated with a photosensitizing agent . The purpose of this study was to determine the bactericidal capabilities of irradiation from lasers with power outputs of less than 6 mW on photosensitized microorganisms . METHODS: Two bacteria that commonly infect skin lesions, Staphylococcus aureus and Pseudomonas aeruginosa, were used . The 2 lasers used, the 0.95-mW helium -neon laser and the 5-mW indium-gallium-aluminum-phosphate laser, emit light at a wavelength close to the absorption maxima of the sensitizing agent chosen, toluidine blue O . This agent was used because of its proven effectiveness in sensitizing bacteria . For each bacterial strain, toluidine blue O was added to a 108 cells/mL solution until a 0.01% weight/volume ratio was obtained . These mixtures were spread on agar-coated petri dishes, which were then exposed to 1 of the 2 lasers for 30, 60, and 120 seconds . The cultures were then grown overnight and examined for one or more visible zones of inhibition . The areas surrounding the irradiated zone provided a control for the effects of toluidine blue O alone . To determine the effects of laser irradiation without prior toluidine blue O sensitization, separate plates were established using unsensitized bacteria . RESULTS: Although inconsistencies between plates were noted, both lasers produced at least one zone of inhibition in both bacterial species at all 3 time periods . The 5-mW laser, however, produced a greater number of these zones . CONCLUSION AND DISCUSSION: Laser-induced microbial killing of photosensitized organisms could have clinical applications in the treatment of infected skin lesions, pending in vivo studies.

Chem Pharm Bull (Tokyo), 1999 Aug, 47(8), 1121 - 7
Phenolic constituents of Cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant Staphylococcus aureus; Hatano T et al.; Thirteen phenolic glycosides including six new compounds were isolated from seeds of Cassia tora (Leguminosae) . The structures of the new compounds, rubrofusarin triglucoside (7), nor-rubrofusarin gentiobioside (9), demethylflavasperone gentiobioside (10), torachrysone gentiobioside (11), torachrysone tetraglucoside (12) and torachrysone apioglucoside (13), were elucidated on the basis of spectroscopic and chemical evidence . The effects of the phenolic glycosides, their aglycones and several other compounds structurally related to them on Escherichia coli K12, Pseudomonas aeruginosa PAO1 and some strains of Staphylococcus aureus were then examined . Among them, torachrysone (15), toralactone (16), aloe-emodin (18), rhein (19) and emodin (20) showed noticeable antibacterial effects on four strains of methicillin-resistant Staphylococcus aureus with a minimum inhibitory concentration of 2-64 micrograms/ml . On the other hand, the phenolic compounds tested did not show strong antibacterial effects on E . coli and P . aeruginosa.

Microbiol Mol Biol Rev, 1999 Sep, 63(3), 523 - 53
Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa; Rocchetta HL et al.; Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host . Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties . The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism . Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band . The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P . aeruginosa . The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations . In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes . Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed . The recent identification of additional genes within the P . aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis . These studies demonstrate that P . aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.

Clin Infect Dis, 1999 Aug, 29(2), 245 - 52
Surveillance of antimicrobial use and antimicrobial resistance in United States hospitals: project ICARE phase 2 . Project Intensive Care Antimicrobial Resistance Epidemiology (ICARE) hospitals; Fridkin SK et al.; The search for the means to understand and control the emergence and spread of antimicrobial resistance has become a public health priority . Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) has established laboratory-based surveillance for antimicrobial resistance and antimicrobial use at a subset of hospitals participating in the National Nosocomial Infection Surveillance system . These data illustrate that for most antimicrobial-resistant organisms studied, rates of resistance were highest in the intensive care unit (ICU) areas and lowest in the outpatient areas . A notable exception was ciprofloxacin- or ofloxacin-resistant Pseudomonas aeruginosa, for which resistance rates were highest in the outpatient areas . For most of the antimicrobial agents associated with this resistance, the rate of use was highest in the ICU areas, in parallel to the pattern seen for resistance . These comparative data on use and resistance among similar areas (i.e., ICU or other inpatient areas) can be used as a benchmark by participating hospitals to focus their efforts at addressing antimicrobial resistance.

Mol Microbiol, 1999 Sep, 33(5), 1069 - 80
Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation; Baynham PJ et al.; The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate . Transcriptional activation of algD results in the high-level synthesis of alginate, an important P . aeruginosa virulence factor with antiphagocytic and adherence properties . Previously, we have identified a protein(s), AlgZ, expressed in mucoid P . aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter . Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription . In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays . A biochemical enrichment procedure, resulting in a 130-fold enrichment for AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained . Using the P . aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli . Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P . aeruginosa . A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins . An algZ deletion mutant was constructed in the mucoid CF isolate FRD1 . The resulting strain was non-mucoid and exhibited no detectable algD transcription . As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT . The cloning of algZ is a crucial step in determining the mechanism of algD activation.

Histochem J, 1999 Jul, 31(7), 485 - 93
Binding properties of the galactose-detecting lectin Pseudomonas aeruginosa agglutinin (PA-IL) to skeletal muscle fibres . Quantitative precipitation and precipitation inhibition assays; Kirkeby S et al.; Pseudomonas aeruginosa agglutinin (PA-IL) staining and the influence of various carbohydrates on lectin binding to muscle sections were investigated quantitatively using a scanning and integrating microspectrophotometre . A strong dose-dependent inhibition of PA-IL staining in the sections was recorded with galabiose (Galalpha1-4Gal) while lactose (Galbeta1-4Glc) had no inhibitory effect . The affinity of PA-IL to Galalpha1 carbohydrates was studied by ELISA using immobilized glycoconjugates in which Galalpha1 glycans were attached to bovine serum albumin or ceramide . PA-IL exhibited strong binding to both simple glycoconjugates having a single Galalpha moiety and to di- and trisaccharides with terminal Galalpha1 at the non-reducing end . In all cross-sectioned muscle fibres incubated with PA-IL, the staining was present as a honeycomb-shaped network through the entire cytoplasm . Further, a dense punctuate staining could be shown in most fibres . A similar staining pattern was noticed after incubation with a monoclonal antibody against ryanodine receptors and with biotinylated ryanodine suggesting that the network could represent the sarcoplasmic reticulum . Further, Western blots of a sarcoplasmic reticulum preparation showed multiple bands after incubation with PA-IL . It may therefore be proposed that glycoconjugates carrying terminal Galalpha1 show affinity for PA-IL and are located to the sarcoplasmic reticulum.

Chemotherapy, 1999 Sep-Oct, 45(5), 335 - 41
'Subinhibitory' erythromycin represses production of Pseudomonas aeruginosa lectins, autoinducer and virulence factors; Sofer D et al.; Pseudomonas aeruginosa infection is preceded by selective adhesion of the bacteria to the host target cells via diverse adhesins, including lectins . This step enables maximal damage to the target host cells by the bacterially secreted injurious toxins and enzymes . The production of both lectins and many of the virulence factors is positively controlled by transcription activators including signaling autoinducers (N-acyl-L-homoserine lactones) . We show in this communication that erythromycin at subminimal growth inhibitory concentrations simultaneously suppresses the production of P . aeruginosa hemagglutinins (including lectins), protease, hemolysin and homoserine lactone autoinducers . The antibiotic-treated bacteria also show reduced virulence to mice, endorsing clinical observations that indicate the efficiency of low-dose erythromycin treatment of persistent drug-resistant P . aeruginosa infections.

FEBS Lett, 1999 Aug 27, 457(2), 277 - 82
Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives; Borovok N et al.; A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported . The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency . TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC . Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye . For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process . Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated . Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.

Intensive Care Med, 1999 Jul, 25(7), 733 - 43
Early but not delayed continuous arteriovenous hemofiltration improves cardiovascular function in sepsis in dogs; Mink SN et al.; OBJECTIVE: Continuous arteriovenous hemofiltration (CAVH) has been advocated as treatment to remove inflammatory mediators and thereby to improve hemodynamic parameters in sepsis . However, the results obtained with CAVH have been inconsistent . In a canine model of bacteremic Pseudomonas aeruginosa pneumonia, we tested the hypothesis that the time course of the institution of CAVH may be important in obtaining a beneficial treatment effect . METHODS: Two protocols were performed in phenobarbital-anesthetized dogs . In the early hemofiltration study (EHS), CAVH for 3 h was initiated 2 h post-pneumonia before mean arterial pressure (MAP) fell . In the late hemofiltration study (LHS), CAVH for 3 h was initiated at 5 h post-pneumonia when a decrease in MAP had already occurred . Hemodynamic measurements included cardiac output (CO), stroke volume (SV), and stroke work (SW) . Myocardial depressant activity {filterable cardiodepressant substance (FCS)} found in plasma was assessed by bioassay at each measurement interval . RESULTS: In EHS, after 5 h of sepsis, SW, CO, and SV in the hemofiltered pneumonia group were higher as compared with the nonhemofiltered pneumonia group . In contrast, in LHS, no differences in hemodynamic parameters were found between the two pneumonia groups . In both EHS and LHS, plasma FCS activity was decreased to similar extents by CAVH . CONCLUSION: These results suggest the time course of institution of CAVH may be important in obtaining a beneficial treatment effect in sepsis.

Clin Exp Immunol, 1999 Sep, 117(3), 561 - 7
Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/permeability-increasing protein (BPI) and cystic fibrosis lung disease; Mahadeva R et al.; Persistent infection with Pseudomonas aeruginosa and inflammatory mechanisms play an important role in cystic fibrosis (CF) lung disease . ANCA against BPI, a potent host defence protein with anti-bacterial and anti-endotoxin properties, have been described in CF . We have assessed the relationship of anti-BPI antibodies to pulmonary disease severity in 148 CF subjects . IgA and IgG anti-BPI antibodies were found in 55.4% and 70.3% of CF patients, respectively, and higher levels were strongly associated with colonization with P . aeruginosa (P = 0.001 and 0.039 for IgA and IgG antibodies, respectively) . IgA and IgG anti-BPI antibodies were independently associated with more severe lung disease as assessed by chest radiograph score (P = 0.023) and a significantly lower forced expiratory volume in 1 s (FEV1)% (P = 0.01) . The pathophysiological relevance of the autoantibodies was investigated further by determining their epitope specificity and their effect on bacterial phagocytosis in vitro . Both isotypes of anti-BPI antibodies were specific for the C-terminus of BPI shown recently to be important for BPI-mediated opsonization, and in vitro affinity-purified anti-BPI antibodies significantly reduced BPI-induced phagocytosis of Escherichia coli compared with controls . These data indicate that anti-BPI autoantibodies are associated with colonization with P . aeruginosa and worse lung disease in CF . The inhibition of bacterial phagocytosis suggests that these autoantibodies may contribute to the persistence of P . aeruginosa in the CF lung and so play a role in perpetuating CF lung damage.

Hum Gene Ther, 1999 Aug 10, 10(12), 1923 - 30
Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer; Biffi A et al.; In vitro and in vivo studies have demonstrated that gene transfer of the CFTR (cystic fibrosis transmembrane conductance regulator) cDNA into human respiratory cells through nonviral vectors can occur safely and can be done repeatedly . Although functional evaluation of CFTR in cystic fibrosis (CF) patients enrolled in phase I clinical trials using cationic liposomes has shown a partial correction of nasal potential difference, a biological assay indicating a therapeutic relevance of CFTR gene transfer is still missing . Our aims were to study the induction of killing activity toward Pseudomonas aeruginosa (PA) in CF cells by cationic vector-mediated CFTR gene transfer and to use this assay as a therapeutic end point . Luciferase expression and GFP FACS analysis were used to evaluate the optimal vector and the efficiency of gene transfer into non-CF human respiratory cells growing from nasal polyp explants at the air-liquid interface . To prove that transgenic CFTR was expressed in CF cell cultures under the same experimental conditions, a specific RT-PCR was performed . Challenge of the outgrowths with a known amount of PA showed a bacterial clearance activity by non-CF respiratory cells, while in the case of CF cells it even resulted in bacterial growth . Cationic vector-mediated CFTR cDNA determined the recovery of bacterial clearance activity only under those conditions yielding 5% or more of GFP-positive cells . The results shown in this study might be helpful in considering cationic vectors as therapeutic nonviral vectors for transferring CFTR into human CF respiratory cells, as well as for restoring the bacterial killing activity defective in cystic fibrosis.

Res Microbiol, 1999 Jul-Aug, 150(6), 403 - 6
Beta-lactamase-mediated resistance to extended spectrum cephalosporins among clinical isolates of Pseudomonas aeruginosa; Ben-Mahrez K et al.; The role of chromosomal cephalosporinases and secondary beta-lactamases in resistance to extended spectrum cephalosporins in clinical isolates of Pseudomonas aeruginosa was investigated . Strains 687, 59, and 58 expressed an inducible chromosomal cephalosporinase, efficiently enhanced with cefoxitin and imipenem . The inducible activity in strain 802 was produced at a moderately elevated basal level and may be involved in resistance to extended spectrum cephalosporins and aztreonam . All strains produced secondary beta-lactamases inhibited by clavulanate: strains 687, 59, and 58 had carbenicillinases with pIs of 5.7 and 5.3 . Strain 802 expressed a secondary beta-lactamase of pI 7.6 which may be a novel extended spectrum beta-lactamase different from known enzymes of P . aeruginosa.

Carbohydr Res, 1999 Apr 30, 317(1-4), 39 - 52
Synthesis of Pseudomonas aeruginosa lipopolysaccharide core antigens containing 7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranosyl residues; Reiter A et al.; The monosaccharide allyl 7-O-carbamoyl-L-glycero-alpha-D-manno- heptopyranoside, the reducing disaccharide 7-O-carbamoyl-L-glycero-alpha-D- manno-heptopyranosyl-(1-->3)-L-glycero-D-manno-heptopyranose and the disaccharides allyl 7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranosyl-(1-->3)-L-glycero- beta- and alpha-D-manno-heptopyranoside were prepared in good yields . The 7-O-carbamoyl substituent was regioselectively introduced via NH3-NH4HCO3 treatment of a 6,7-O-carbonate group . Glycosylation steps were carried out using Me3SiOTf or BF3.Et2O promoted coupling of allyl alcohol with trichloroacetimidate or fluoride glycosyl donors, respectively . The deprotected allyl glycosides were reacted with cysteamine to afford spacer glycosides which were subsequently linked to bovine serum albumin . The artificial antigens which are related to the dephosphorylated heptose region of the lipopolysaccharide core region from Pseudomonas aeruginosa classified into RNA group I may be used for the characterization of monoclonal antibodies directed against inner core epitopes of human-pathogenic Pseudomonas species.

Respir Med, 1999 Jul, 93(7), 476 - 80
Inhaled antibiotic therapy in non-cystic fibrosis patients with bronchiectasis and chronic bronchial infection by Pseudomonas aeruginosa; Orriols R et al.; The aim of this study was to investigate the long-term effectiveness and safety of inhaled antibiotic treatment in non-cystic fibrosis patients with bronchiectasis and chronic infection by Pseudomonas aeruginosa, after standard endovenous and oral therapy for long-term control of the infection had failed . After completing a 2-week endovenous antibiotic treatment to stabilize respiratory status, 17 patients were randomly allocated to a 12-month treatment either with inhaled ceftazidime and tobramycin (group A) or a symptomatic treatment (group B) . One patient from group A abandoned inhaled treatment because of bronchospasm and another from group B died before the end of the study . The remaining 15 patients, seven from group A and eight from group B, completed the study . Both groups had similar previous characteristics . The number of admissions and days of admission (mean +/- SEM) of group A {0.6 (1.5) and 13.1 (34.8)} were lower than those of group B {2.5 (2.1) and 57.9 (41.8)} (P < 0.05) . Forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), PAO2 and PACO2 were similar in the two groups at the end of follow-up, showing a comparable decline in these parameters . There were no significant differences either in the use of oral antibiotics or in the frequency of emergence of antibiotic-resistant bacteria between groups . Microbiological studies suggested that several patients had different Pseudomonas aeruginosa strains . None of the patients presented impaired renal or auditory function at the end of the study . This study suggests that long-term inhaled antibiotic therapy may be safe and lessen disease severity in non-cystic fibrosis patients with bronchiectasis and chronic bronchial infection by Pseudomonas aeruginosa which do not respond satisfactorily to antibiotics administered via other routes.

J Bacteriol, 1999 Sep, 181(17), 5498 - 504
Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD); Kutchma AJ et al.; A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A {CoA}:acyl carrier protein {ACP} transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced . This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled . The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster . A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change . A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid . Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful . We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria . Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed . In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.

J Bacteriol, 1999 Sep, 181(17), 5489 - 97
Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis; Hoang TT et al.; The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced . Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function . The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively . The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts . The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan . An polyhistidine-tagged FabI protein was purified and characterized . Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan . A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified . The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant . When coupled to FabI, purified P . aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine . This reaction was NADH dependent and inhibited by triclosan . The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.

J Bacteriol, 1999 Sep, 181(17), 5426 - 32
Amino acid-mediated induction of the basic amino acid-specific outer membrane porin OprD from Pseudomonas aeruginosa; Ochs MM et al.; Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources . Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem . The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon . The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression . The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays . The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism . In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen . Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.

Microbiology, 1999 Aug, 145 ( Pt 8), 2061 - 7
A chimaeric plant virus vaccine protects mice against a bacterial infection; Brennan FR et al.; The plant virus cowpea mosaic virus (CPMV) is an efficient carrier of foreign peptides for the generation of strong humoral immune responses . Peptides derived from both viruses and bacteria are strongly immunogenic when displayed on the surface of CPMV and elicit high titres of peptide-specific antibody . However, the protective effects of antibodies generated using bacterial epitopes in this system have yet to be demonstrated . In this study the ability of chimaeric virus particles (CVPs) to afford protection against bacterial infection was assessed . Immunization of outbred mice with CPMV expressing a peptide derived from outer-membrane protein F of Pseudomonas aeruginosa (CPMV-PAE5) generated high titres of P . aeruginosa-specific IgG that opsonized the bacteria for phagocytosis by human neutrophils and afforded protection upon challenge with two different immunotypes of P . aeruginosa in a model of chronic pulmonary infection . When examined 8 d after challenge, CVP-immunized mice had fewer severe lung lesions and fewer bacteria in their lungs compared to mice immunized with wild-type virus . Different levels of protection were seen with CPMV-PAE5 when Freund's or alum adjuvants were used . These studies highlight the ability of CVPs to generate protective immunity against infectious disease agents.

Respiration, 1999, 66(4), 373 - 6
Massive pulmonary hemorrhage due to cytomegalovirus infection in a Japanese patient with alpha-1-antitrypsin-deficient emphysema; Tsushima K et al.; Although alpha(1)-antitrypsin (AAT) deficiency is one of the most common hereditary diseases and a recognized cause of emphysema in Caucasians, variants of this deficiency are extremely rare among Orientals . We present here a Japanese emphysema patient with the AAT deficiency variant originally identified as S(iiyama) . After an 8-year follow-up period, the patient suffered from repeated pulmonary Pseudomonas aeruginosa infection for 4 years . He died suddenly of massive pulmonary hemorrhage . The pathologic examination revealed a necrotic hematoma in the right S10 lobe, which exhibited pneumonia due to cytomegalovirus (CMV) infection . Pulmonary hemorrhage due to CMV can occur and be fatal in patients with emphysema and AAT deficiency.

J Antimicrob Chemother, 1999 Jul, 44(1), 91 - 7
Treatment of experimental pneumonia in rats caused by a PER-1 extended-spectrum beta-lactamase-producing strain of Pseudomonas aeruginosa; Mimoz O et al.; The antibacterial activity of imipenem, cefepime and piperacillin-tazobactam alone or in combination with amikacin against a Pseudomonas aeruginosa strain producing an extended-spectrum beta-lactamase (PER-1) were compared using an experimental model of pneumonia in non-leucopenic rats . Animals were infected intratracheally with 8.0 +/- 0.4 log10 cfu of P . aeruginosa, and therapy was initiated 3 h later, by which time animal lungs showed bilateral pneumonia containing >7 log10 P . aeruginosa cfu/g of tissue . Since rats eliminate antibiotics much more rapidly than humans, renal impairment was induced in all animals to simulate the pharmacokinetic parameters of humans . MICs determined using an inoculum of 4 log10 cfu/mL were as follows: imipenem, 1 mg/L; cefepime, 8 mg/L; piperacillin-tazobactam, 32 mg/L; and amikacin, 16 mg/L . A noticeable inoculum effect was observed with the four antimicrobial agents tested, which was greatest for cefepime and piperacillin-tazobactam . In-vitro studies indicated that imipenem was the beta-lactam with the greatest bactericidal effect and that amikacin was synergic only in combination with cefepime and imipenem . Cefepime and piperacillin-tazobactam alone failed to decrease bacterial counts in the rats' lungs 60 h after therapy onset, whereas imipenem and, to a lesser extent, amikacin significantly reduced the number of viable microorganisms . Combination of amikacin with any of the three beta-lactams tested was synergic, despite a high amikacin MIC for the infecting strain . These results paralleled our in-vitro data showing a marked inoculum effect for cefepime and piperacillin-tazobactam . Based on the results of this study, the best treatment for infections caused by this type of extended-spectrum beta-lactamase-possessing strain would be imipenem plus amikacin.

FEMS Immunol Med Microbiol, 1999 Aug 15, 25(3), 313 - 21
The role of Pseudomonas aeruginosa elastase as a potent inflammatory factor in a rat air pouch inflammation model; Kon Y et al.; Pseudomonas aeruginosa, an opportunistic pathogen, can cause life threatening infections in patients compromised by underlying respiratory disease like bronchiectasis, cystic fibrosis and diffuse panbronchiolitis . Most strains of P . aeruginosa produce some kind of protease with broad substrate specificities during the infectious state in the host . P . aeruginosa elastase, one of the strongest exotoxins, has a tissue-damaging proteolytic activity and is capable of degrading such plasma proteins as immunoglobulins, complement factor and cytokines . The present study focused on the effect of P . aeruginosa elastase and was designed to evaluate the neutrophil accumulation at the inflammation site mediated by P . aeruginosa elastase in the inflammatory response in the host . An air pouch model in rats, considered as a useful model of inflammation, was used to analyze the number of leukocytes, the volume of exudate and the concentration of interleukin-8 after the injection of P . aeruginosa elastase into the pouch cavity . The number of neutrophils and the volume of exudate in the pouch cavity increased significantly at 4 h, peaked at 8 h in a dose-dependent manner and then decreased at 24 h . The concentration of interleukin-8 in pouch fluid peaked 4 h earlier than the peak of the neutrophil number . The enzymatic activity of P . aeruginosa elastase seemed to reinforce the inflammation process . The influence of lipopolysaccharide contamination was negligible . Although these observations were made in the subcutaneous cavity, they indicate that P . aeruginosa elastase plays a role as an immunoprovocative factor in the inflammatory response in cases of infection with P . aeruginosa.

Braz J Med Biol Res, 1999 Aug, 32(8), 1021 - 8
Structure and function of the cystic fibrosis transmembrane conductance regulator; Morales MM et al.; Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) . Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel . Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients . The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP . The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain . In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Bratisl Lek Listy, 1999 Mar, 100(3), 164 - 7
{Prophylactic effects of ceftriaxone in patients after endoscopic papillosphincterotomy}; Batovsky M et al.; In thirty patients after EPS and gall extraction for cultivation ceftriaxon in preventive dose 1 g was administered . This group of patients was compared with a group of 30 patients after EPS without preventive administration of antibiotic from clinical and biochemical point of view . Most frequently occurring bacteria in the gall of patients after EPS were Pseudomonas aeruginosa and E . coli . All of the detected bacteria were sufficiently sensitive to ceftriaxon . Preventive effect of ceftriaxon was manifested in statistically significant fall of hyperbilirubinemia and hyperamylasemia 24 hours after EPS (p > 0.05) . (Tab . 5, Ref . 8.)

Anesteziol Reanimatol, 1999 May-Jun, (3), 46 - 54
{Hospital infections caused by Pseudomonas aeruginosa . Significance in intensive therapy}; Sidorenko SV et al.; The significance of P . aeruginosa as an agent of hospital infections in intensive care departments is determined by high prevalence of this microorganism, its natural and acquired resistance to antibiotics of various groups, and severity of the infection it induces . The resistance of P . aeruginosa to antibiotics is different in different regions . Among the strains isolated in Moscow in intensive care wards for newborns 9% were resistant to meropenem, 10% to amicacine, 15% to imipramine, 16% to cefepime, 37% to ceftasidime, 45% to piperacylline/tasobactam, 45% to ciprofloxacine, and 60% to gentamicin; 1.5% of these strains were resistant to all tested antibiotics . High prevalence of antibiotic resistance among P . aeruginosa impedes the choice of drugs for empirical antibiotic therapy and increases the significance of microbiological diagnosis . Even if an agent is sensitive to such antibiotics as semisynthetic penicillines and aminoglycosides, their use as monotherapy in infections caused by P . aeruginosa is ineffective . Carbapenemes, III- IV generations cefalosporines, and fluoroquinolones can be used as mono therapy.

Afr J Med Med Sci, 1996 Sep, 25(3), 221 - 4
Antimicrobial potentials of Diospyros mespiliformis (Ebenaceae); Adeniyi BA et al.; The petroleum ether, chloroform, methanol, and water extracts of the leaves, stem bark, and root of Diospyros mespiliformis were studied for their antimicrobial activities . The crude extracts showed broad spectrum antimicrobial activities against 9 Gram-positive bacteria, 8 Gram-negative bacteria, and 6 fungal strains . Of the four extracting solvents, chloroform produced extracts with the best antimicrobial activities, while the chloroform extract of the root exhibited the highest antimicrobial activity . Some tetracycline resistant strains of Staphylococcus aureus and gentamicin resistant strains of Pseudomonas aeruginosa were sensitive to some of the extracts tested . Preliminary phytochemical screening revealed the presence of the following metabolites: anthraquinones, tannins, triterpene, saponins, steroids, and sugars and the absence of alkaloids . The antimicrobial activities observed are discussed in relation to the chemical constituents reportedly isolated from several species of this plant and their traditional uses.

Infect Immun, 1999 Sep, 67(9), 4794 - 800
Porin from Pseudomonas aeruginosa induces apoptosis in an epithelial cell line derived from rat seminal vesicles; Buommino E et al.; Micromolar concentrations of porin, purified from the outer membranes of Pseudomonas aeruginosa, induced in vitro the classic morphological and biochemical signs of apoptosis in an epithelial cell line (SVC1) derived from the rat seminal vesicle secretory epithelium . The programmed cell death (PCD) was p53 independent and associated with significant decrease of bcl-2 expression, a marked increase of c-myc transcriptional activity, and an absence of the mRNA coding for tissue transglutaminase . The Ca(2+) influx, caused by the porin treatment of SVC1 cells, appears to play an important role in the triggering of apoptosis in our biological model . The possibility that the porin property of inducing PCD plays a role in the infertility of individuals chronically infected by gram-negative bacteria is discussed.

Infect Immun, 1999 Sep, 67(9), 4744 - 50
Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype; Parad RB et al.; Whether allelic variants of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) independently contribute to pulmonary outcome in CF patients has not been resolved . We used both cross-sectional and mixed-model longitudinal analyses of data from CF patients that were at least 12 years old to determine the influence on pulmonary function (percent predicted forced expiratory volume {FEV(1)}) of the CFTR gene genotype, gender, mucoid Pseudomonas aeruginosa (MPA) infection status, presence of total opsonic antibody to MPA, and, separately, the opsonic antibody activity specific to the mucoid exopolysaccharide (MEP) surface antigen . Two different factors were independently associated with the lack of MPA infection: a high level of MEP-specific opsonic activity (MSOA), implicating an immunologically based mechanism of resistance to infection, and a lack of any type of opsonic antibody to MPA, indicative of no significant exposure or infection . This latter phenotype was found in a subset of CF patients who carried at least one uncommon CFTR gene allele suggestive of a genetic basis for resistance to infection in this group of older CF patients . For CF patients in whom both CFTR gene alleles were identified by screening for the 12 most common variants (75% of alleles), cross-sectional analysis showed that MPA infection was best correlated with lower percent predicted FEV(1), while genotype (two versus one DeltaF508 CFTR gene allele) and a low level of MSOA were associated with increased risk of infection . A mixed-model analysis of longitudinal spirometric measurements that considered multiple risk factors to derive regression equations was used to determine which clinical parameters had the greatest effect on the annual rate of decline in percent predicted FEV(1) . This analysis showed that the CFTR gene genotype only modestly modified the constant (y intercept) of the derived equations, while gender and MPA infection status had the largest effects on annual rates of decline in percent predicted FEV(1) . These results indicate that the CFTR genotype is usually not a primary determinant of pulmonary function in most CF patients, but gender and MPA infection status are . Infection status is potentially influenced by both immunologic (a high level of MSOA) and genetic factors, such as carriage of a CFTR gene allele that leads to a diagnosis of CF but still confers resistance to infection that is comparable to that of the wild-type CFTR gene.

Infect Immun, 1999 Sep, 67(9), 4613 - 9
Pseudomonas aeruginosa exoenzyme S stimulates murine lymphocyte proliferation in vitro; Barclay NG et al.; The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients . Previous studies have established that an exoproduct of P . aeruginosa (exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response . An animal model would facilitate studies of the pathophysiologic consequences of this activation . As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined . When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated . The optimum response occurred after 2 to 3 days in culture, at 4 x 10(5) cells per well and 5.0 micrograms of exoenzyme S per ml . The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response . Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1 . There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S . Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S . Thus, murine lymphocytes, like human lymphocytes, respond to P . aeruginosa exoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis of P . aeruginosa infections in CF patients.

Infect Immun, 1999 Sep, 67(9), 4603 - 12
Immunization of mice with a TolA-like surface protein of Trypanosoma cruzi generates CD4(+) T-cell-dependent parasiticidal activity; Quanquin NM et al.; The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system . The gene family contains three tandemly repeated members that have 97 to 100% sequence identity . The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa . Based on these similarities, we have designated this gene family tolT . Immunization of mice with recombinant TolT generates a population of CD4(+) T lymphocytes that recognize T . cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-gamma), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells . This population of T cells also produces both IFN-gamma and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type . T cells from mice chronically infected with T . cruzi also produce significant levels of IFN-gamma when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T . cruzi.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 713 - 7
Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials; Stone JH et al.; Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials . Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-{3,4,5-3H} leucine or {methyl-3H} thymidine during growth in a minimal medium . Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates . These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP) . The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum.

Microbios, 1999, 97(387), 103 - 15
High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa; Bhatti AR et al.; Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C . Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C . Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall . In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D . Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C . Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C . Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.

Biochim Biophys Acta, 1999 Aug 30, 1454(3), 236 - 50
Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity; Fricke B et al.; A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope . The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media . It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells . The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic steps on Red-Sepharose CL-4B (yield 58.3%, purification factor 776.3) . It consisted of a single polypeptide chain with a molecular mass of 44.6 kDa, determined by mass spectrometry . ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range . The ICMP readily hydrolyzed Glu(13)-Ala(14) and Tyr(16)-Leu(17) bonds in the insulin B-chain . Phe(25)-Tyr(26) and His(10)-Leu(11) were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'(1)-position . The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase . The amino acid sequence of internal peptides showed no homologies with the known proteinases . This outer membrane proteinase was capable of specific cleavage of alpha and beta fibrinogen chains . Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P(1)-subsite were the substrates best accepted, but they were only cleaved at a very low rate.

Clin Infect Dis, 1999 May, 28(5), 1128 - 33
Epidemiology and clinical outcomes of patients with multiresistant Pseudomonas aeruginosa; Harris A et al.; We conducted a case-series study of multiresistant Pseudomonas aeruginosa in patients who did not have cystic fibrosis . Patient characteristics, antibiotic exposures, time course of emergence of resistance, and clinical outcomes were examined . Twenty-two patients were identified from whom P . aeruginosa resistant to ciprofloxacin, imipenem, ceftazidime, and piperacillin was isolated . Nineteen (86%) had clinical infection . Patients received prolonged courses of antipseudomonal antibiotics before isolation of multiresistant P . aeruginosa . Nine of 11 patients with soft-tissue infection exhibited resolution of clinical infection but usually required surgical removal of infected tissue with or without revascularization . Overall, three patients died . In two instances in which multiple isolates with different susceptibility profiles from the same patient were available, pulsed-field gel electrophoresis profiles of serial isolates were indistinguishable or closely related . This study illustrates that multiresistant P . aeruginosa emerges in a stepwise manner after exposure to antipseudomonal antibiotics and results in adverse outcomes.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1998 Nov, 14(6), 436 - 8
{Clinical characteristics of burn caused by coal mine explosion}; Li J et al.; OBJECTIVE: To investigate the characteristics of the burn injury caused by coal mine explosion so as to enhance the cure rate . METHODS: Analyse the therapeutic result after planned standard treatment of clinical patients and review historical patients . RESULTS: 1 . Coal mine explosion includes two types, i.e., gas explosion and coal dust explosion . 2 . This kind of burn is a combined injury with pathologic changes of burns as the main feature . Mechanical injury is the chief cause of early death . Blast injury mainly damages the lungs . The occurrence of carboxyhemoglobinemia is not often . 3 . The amount of fluid infusion in the first 24 h in exudation phase is 8% less of the traditional formula . Alkaline balanced salt solution is supplied as electrolyte solution, which can provide 45% of necessary HCO3- for correction of acidosis . 4 . No thorough debridement is imposed in the treatment of burn wound . Baking with electric bulb with topical SD-Ag in semi-exposure state can be used . 5 . Inhalation injury chiefly occurs in upper respiratory tract . The main bacterial species causing complicated lung infection are Pseudomonas aeruginosa and Staphylococcus aureus . 6 . When MSOF occurs, the most frequently involved organ and system are the kidney and respiratory system . 7 . The main bacteria causing systemic invasive infection are enteric bacilli and Pseudomonas aeruginosa . Enteric bacilli infection may be enterogenic . The latter infection is chiefly the result of cross infection in hospital . CONCLUSION: The burn caused by coal mine explosion is a combined injury characterized by pathologic changes of burn as the main issue . This kind of burn has two types, i.e., gas-explosion-burn and coal-dust-explosion-burn.

J Med Microbiol, 1999 Aug, 48(8), 765 - 70
Effect of passive immunotherapy on murine gut-derived sepsis caused by Pseudomonas aeruginosa; Matsumoto T et al.; The effect of passive immunotherapy with antisera against heat-killed Pseudomonas aeruginosa and three of its exo-enzymes (elastase, alkaline protease and exotoxin A) in gut-derived P . aeruginosa sepsis was evaluated . Mice were given a suspension of P . aeruginosa strain D4 in their drinking water, together with ampicillin (200 mg/kg) to disrupt the normal bacterial flora . Cyclophosphamide was then administered to induce translocation of P . aeruginosa that had colonised the gastrointestinal tract so that gut-derived septicaemia was produced . In this model, intraperitoneal administration of antiserum against heat-killed bacteria, 100 microl/mouse, twice a day for 3 consecutive days significantly increased the survival rate over that of mice treated with normal rabbit serum . Antiserum against elastase, alkaline protease, or a combination of these two antisera, failed to provide significant protection . In contrast, antiserum against exotoxin A significantly increased the survival rate over that of mice treated with normal rabbit serum . These results indicate that passive immunisation with antiserum against heat-killed bacteria and exotoxin A, but not with antiserum against either elastase or alkaline protease, protects mice against gut-derived sepsis caused by P . aeruginosa.

Nephron, 1999, 82(4), 324 - 30
The outer membrane protein I of Pseudomonas aeruginosa PAO1, a possible pollutant of dialysate in hemodialysis, induces cytokines in mouse bone marrow cells; Ino M et al.; The finding of outer membrane protein I (OprI) of Pseudomonas aeruginosa in hemodialyzers used by patients with end-stage renal failure led us to study the possible role of OprI as cytokine inducer . However, there are few reports on the biological activity of OprI, because it is difficult to obtain highly purified OprI . In this study, we attempt to establish a procedure for the efficient purification of OprI, which does not include lipopolysaccharide, from the bacterial culture broth, not hemodialyzers, to demonstrate that OprI is a potent cytokine inducer . From bacterial culture broth (1 liter), P . aeruginosa PAO1, which was confirmed previously by the sequence coding, was separated by centrifugation, high-performance liquid chromatography, and disk electrophoresis . Mouse bone marrow cells were stimulated by purified OprI, and the supernatants of the culture were analyzed by several enzyme-linked immunosorbent assay kits . The tumor necrosis factor alpha production stimulated by purified OprI was confirmed and degraded within 24 h . Furthermore, interleukin (IL) 1alpha, IL-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor were also induced by OprI despite the absence of lipopolysaccharide . We conclude that OprI has the potential to induce tumor necrosis factor alpha production in mouse bone marrow cells and that tumor necrosis factor alpha contributes to the induction of inflammatory cytokines, namely IL-1alpha, IL-1beta, IL-6, and granulocyte/macrophage colony-stimulating factor, while lipopolysaccharide has little effect on these cells . These results suggest the presence of a pathway of inflammatory signal transduction triggered by OprI . In addition, OprI is possibly one of the harmful dialysate pollutants in hemodialysis patients besides the well-known lipopolysaccharide.

J Clin Microbiol, 1999 Sep, 37(9), 2987 - 91
Evaluation of reference dilution test methods for antimicrobial susceptibility testing of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis; Saiman L et al.; The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics . The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P . aeruginosa strains, is unknown . Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies . Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r >/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r >/= 0.87) and for ciprofloxacin (r = 0.86) . Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods . Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons) . However, CF strains were more likely to have serious discords than were non-CF strains (P < 0 . 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains . In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution . Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.

J Clin Invest, 1999 Aug, 104(4), 409 - 18
Augmentation of pulmonary host defense against Pseudomonas by FcgammaRIIA cDNA transfer to the respiratory epithelium; Worgall S et al.; Fcgamma receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule . The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector-mediated gene transfer of FcgammaRIIA cDNA to induce expression of the human FcgammaRIIA receptor . Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcgammaRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with (51)Cr-labeled SRBCs . When A549 cells were infected with an Ad vector expressing a FcgammaRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed . In vivo expression of FcgammaRIIA in the lung after intratracheal administration of the AdFcgammaRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcgamma receptor expression . Similar results were observed with a chimeric FcgammaRIIA construct containing the extracellular domain of FcgammaRIIIA . Together, these data demonstrate that Ad-mediated FcgammaRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.

Microbiol Immunol, 1999, 43(5), 409 - 17
Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells; Sar B et al.; Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P . aeruginosa) . We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P . aeruginosa, induces IL-8 generation in bronchial epithelial cells (K . Oishi et al . Infect . Immun . 65: 2648-2655, 1997) . We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta . We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR . New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells . Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells . In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion . PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM . Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture . We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro . When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM . Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8 . PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells . These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P . aeruginosa.

Scand J Infect Dis, 1999, 31(2), 163 - 8
Impact and pattern of gram-negative bacteraemia during 6 y at a large university hospital; Harbarth S et al.; In order to characterize the impact and pattern of Gram-negative bacteraemia (GNB) at a Swiss University hospital and to assess the effect of multi-resistance on mortality, we conducted a 6-y retrospective cohort study using linear regression and multivariate Cox-proportional hazard analysis . 1766 patients had 1835 episodes of GNB; 61% were community-acquired . The incidence of GNB increased linearly (r2 = 0.90, p = 0.014) from 7.07 episodes to 8.32 episodes per 1000 admissions, but this trend was no longer significant after adjustment for the number of blood cultures drawn/y . The in-hospital mortality for patients with GNB decreased from 20% in 1989 to 16% in 1994 (r2 = 0.94, p = 0.005) . The risk ratio for death remained unchanged over the study period and was 7-fold higher for patients with GNB than for patients without GNB . Factors independently associated with an increased hazard of death after GNB were: severity of illness as measured by exposure to intensive care (hazard ratio {HR}, 1.5); age = 66-79 y (HR 1.8); GNB due to Klebsiella spp . (HR 1.7) or Pseudomonas aeruginosa (HR 1.6); and polymicrobial infection (HR 1.6) . Multi-resistance was not associated with an increased risk of death (HR 1.0) . Although the crude mortality of GNB decreased, the population-attributable risk ratio for death remained significant . These data suggest the absence of a major impact of multi-resistant GNB on patient mortality.

Acta Paediatr, 1999 Jul, 88(7), 783 - 5
Aminoglycoside-associated hypomagnesaemia in children with cystic fibrosis; Akbar A et al.; Hypomagnesaemia in children with cystic fibrosis (CF) is under-recognized . We report a child with CF who developed significant hypomagnesaemia following intravenous (i.v.) treatment with aminoglycosides for exacerbations of Pseudomonas aeruginosa infection . Three additional cases have also been observed . Investigations in two patients have revealed excessive renal loss of magnesium . It is postulated that renal tubular damage secondary to the cumulative effects of repeated courses of aminoglycosides resulted in hypomagnesaemia, and we suggest screening for this problem by monitoring serum magnesium regularly in all patients with CF receiving multiple courses of aminoglycosides.

Eur Respir J, 1999 Jun, 13(6), 1301 - 9
Fibronectin and alpha5beta1 integrin mediate binding of Pseudomonas aeruginosa to repairing airway epithelium; Roger P et al.; Initial infection of the airway by Pseudomonas aeruginosa may occur through a variety of bacterial strategies including binding to epithelial receptors present at the surface of the respiratory epithelium . In order to characterize the adherence sites for P . aeruginosa in damaged and repairing bronchial tissue, an ex vivo model of airway epithelial injury and repair was developed using primary cell cultures of nasal cells from 14 subjects with polyposis . P . aeruginosa strongly adhered to flattened dedifferentiated (FD) bronchial and nasal cytokeratin 13-positive epithelial cells in the process of migration for repair . In in vitro experiments, competitive binding inhibition assays demonstrated that alpha5beta1 integrins and cellular fibronectin, in particular the RGD sequence, are receptors involved in P . aeruginosa adherence to FD nasal epithelial cells . Fluorescent cell sorting analysis and immunofluorescence techniques revealed that the alpha5beta1 integrins are overexpressed and apically exposed in FD nasal epithelial cells . One 50 kDa outer membrane protein was identified in piliated and nonpiliated strains of P . aeruginosa that was involved in binding to cellular fibronectin and alpha5beta1 epithelial integrins . These results demonstrate that Pseudomonas aeruginosa adherence is related to the dedifferentiation of airway epithelium during the repair process which unmasks and upregulates the alpha5beta1 integrin expression and induces active synthesis of cellular fibronectin . These epithelial receptors are then used by a Pseudomonas aeruginosa 50 kDa outer membrane protein as sites of bacterial adherence.

Drug Chem Toxicol, 1999 Aug, 22(3), 555 - 62
The effects of ellagic acid on arylamine N-acetyltransferase activity in the bacterium Pseudomonas aeruginosa; Lo HH et al.; Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by ellagic acid (EA), a naturally occurring dietary plant phenol . By measuring the acetylation of 2-aminofluorene (2-AF), the NAT activity was determined . In P . aeruginosa ATCC 27853, a NAT activity of 1.37 +/- 0.25 nmol/min/10(10) CFU for intact cell and a NAT activity of 5.92 +/- 0.20 nmol/min/mg protein for cytosolic preparation were measured . EA (ranging from 1 to 0.125 mM) showed a dose-dependent inhibition of NAT activities in the analysis of both intact cell and cytosolic preparations . Enzymatic kinetics were determined and found that EA was a potent non-competitive inhibitor of NAT activity in P . aeruginosa ATCC 27853 . EA inhibition of NAT activities in P . aeruginosa ATCC 27853 was time-dependent for at least 4 hrs . These data strongly indicated that EA could suppress NAT activity in P . aeruginosa.

J Photochem Photobiol B, 1999 May, 50(1), 59 - 65
Pseudomonas aeruginosa UV-A-induced lethal effect: influence of salts, nutritional stress and pyocyanine; Fernandez RO et al.; The presence of NaCl in plating media shows an important protection against the Pseudomonas aeruginosa UV-A-induced lethal effect, contrasting with the known sensitizing action of salts on UV-A-irradiated Escherichia coli cells . MgSO4 exhibits a similar protection, but lower concentrations than for NaCl are needed to achieve the same effect . NaCl protection from lethal effects involves an osmotic mechanism, while MgSO4 could act by a different process . On the other hand, when cells grown in a complete medium are then incubated for 20 min in a synthetic medium and irradiated with UV-A, a very marked protection is obtained . This protection is dependent on protein synthesis, since treatment with tetracycline, during the nutritional stress, blocks its induction . These results offer a new example of cross-protection among different stressing agents . In our experimental conditions, natural phenazines of P . aeruginosa are not present in the cells, ruling out the possibility that these pigments act as photosensitizers . Conversely, pyocyanine (the major phenazine produced by this microorganism) prevents the UV-A killing effect in a concentration-dependent way when present in the irradiation media . Finally, UV-A irradiation induces, as in E . coli, the accumulation of guanosine tetraphosphate and guanosine pentaphosphate, although the physiological meaning of this finding has yet to be determined.

J Bacteriol, 1999 Aug, 181(16), 5111 - 3
Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa; Powell B et al.; The rnc operon from Pseudomonas aeruginosa has been cloned and characterized . The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria . Multicopy plasmids carrying the rnc operon of P . aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli . In particular, the P . aeruginosa era homolog rescues the conditional lethality of era mutants in E . coli, and the presumptive protein has 60% identity with the Era of E . coli . We discuss these data and evidence suggesting that a GTPase previously purified from P . aeruginosa and designated Pra is not an Era homolog.

Burns, 1999 Aug, 25(5), 415 - 23
Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa infection in a murine burn wound model; Felts AG et al.; The leading cause of morbidity and mortality in severe burn wound patients is infection . Treatment of burn wound infection is complicated by the emergence of antibiotic resistant organisms . A potential therapeutic alternative to antibiotic drugs is the local administration of polyclonal antibodies, termed passive local immunotherapy (PLI), directly to the burned tissue . A mouse burn wound infection model to simulate full thickness burn wound infection was used to evaluate the efficacy of passive local immunotherapy as a viable prophylactic or therapeutic agent . Pooled human immunoglobulins (IgG), delivered locally to the site of infection, are shown to be more effective at preventing fatal burn wound sepsis than treatment by intravenous infusion of IgG . A single 10 mg dose of human IgG administered locally to the burned, infected tissue site, either 24 hours prior to bacterial challenge, or within 3 hours after bacterial challenge, enhanced animal survival significantly (P < 0.001 and P < 0.05 respectively) compared to control animals . In addition, reduced levels of bacteria were found in local and systemic tissues of IgG-treated mice compared to control mice (P < 0.05) . These data support the local use of polyclonal immunoglobulin preparations as an efficacious and cost effective means to prevent and treat burn wound infections.

J Bacteriol, 1999 Aug, 181(16), 4890 - 5
The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation; Swanson BL et al.; We have previously described a Pseudomonas aeruginosa gene, ptxR, which enhances exotoxin A production at the transcriptional level . We have also described another gene, ptxS, which is transcribed divergently from ptxR and interferes with the enhancement of exotoxin A synthesis by ptxR . However, the mechanisms through which ptxR and/or ptxS are regulated is not known . In this study, we attempted (by using the DNA gel shift assay) to determine if P . aeruginosa contains a potential regulatory protein that binds specifically to the ptxR or ptxS upstream region . In the initial analysis, different-sized gel shift bands were detected when a probe containing the ptxR-ptxS intergenic region was incubated with the lysate of P . aeruginosa PAO1 . The strongest binding activity was detected with a smaller fragment that represents the ptxS upstream region . Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream of ptxS . The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of the ptxS isogenic mutant PAO1::ptxS . However, the binding band was regenerated when a plasmid carrying ptxS intact was introduced into PAO1::ptxS . In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter . The effect of PtxS on ptxS expression was examined by using a ptxS-lacZ fusion plasmid . The level of beta-galactosidase activity produced by PAO1::ptxS carrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid . Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment . Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site . These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within the ptxS upstream region.

Rinsho Byori, 1999 Jun, 47(6), 494 - 500
{Diagnosis of sepsis based on the host response}; Matsumoto T; Sepsis is often a fatal condition, and excessive production of host inflammatory mediators including cytokines, are considered to be responsible for its lethality . We investigated the biology of TNF-alpha, IL-1, IL-6, and IL-10 with particular emphasis on its role in murine gut-derived sepsis due to Pseudomonas aeruginosa and found that these cytokine levels in serum showed significant increases at lethal conditions . Furthermore, we studied the cytokine levels in serum and BALF of mice after pulmonary infection with Klebsiella pneumoniae . The results showed that inflammatory cytokines in BALF demonstrated high levels at the early stage of infection in response to local inflammation of the lung . On the other hand, cytokine levels in serum abruptly increased at the late stage of infection in response to systemic inflammation . These results revealed that it is important to discriminate between systemic inflammation and local inflammation to diagnose sepsis.

Biochim Biophys Acta, 1999 Aug 5, 1428(2-3), 334 - 40
Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal-tobramycin formulation for the treatment of chronic infections in lungs; Sachetelli S et al.; In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria . One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P . aeruginosa and other related bacteria . Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated . The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t . ) immunizations in BALB/c mouse . No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera . These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.

Am J Pathol, 1999 Aug, 155(2), 473 - 81
Genistein inhibits constitutive and inducible NFkappaB activation and decreases IL-8 production by human cystic fibrosis bronchial gland cells; Tabary O et al.; The inflammatory pathogenesis in airways of patients with cystic fibrosis (CF) is still unresolved . We demonstrate here that in in situ human DeltaF508 homozygous CF bronchial tissues, submucosal gland cells exhibit an absence of inhibitor factor kappaBalpha (IkappaBalpha) and high levels of chemokine interleukin-8 (IL-8) expression . These results were confirmed by cultured human CF bronchial gland cells in which a lack of cytosolic IkappaBalpha and high levels of constitutively activated nuclear factor kappaB (NFkappaB) associated with an up-regulation of IL-8 production (13-fold increase) were found when compared to non-CF (control) disease bronchial gland cells . We also demonstrated that the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, significantly reduces the endogenous and Pseudomonas aeruginosa lipopolysaccharide-induced IL-8 production in cultured CF bronchial gland cells by increasing cytosolic IkappaBalpha protein levels . Overall, results show that genistein is a potent inhibitor of the activated NFkappaB identified in CF gland cells . This strong inhibition of constitutively activated NFkappaB and the resulting down-regulation of IL-8 production by genistein in the CF gland cells highlights the key role played by cytosolic IkappaBalpha in the regulation of inflammatory processes in CF human airway cells.

Am Surg, 1999 Aug, 65(8), 706 - 9; discussion 710
Trends in nosocomial pneumonia in surgical patients as we approach the 21st century: a prospective analysis; Crabtree TD et al.; To compare outcome and prognostic factors of pneumonia in surgical patients, we prospectively studied all episodes of nosocomial infection at all sites in 1997 on the surgical services at a single hospital . Pneumonia accounted for 74 of 287 episodes of infection . The crude mortality for pneumonia was 31.1 versus 12.2 per cent for all other infections (P < 0.001) . Pneumonia patients had a higher severity of illness compared with those with infections at other sites (18.7 +/- 0.8 vs 14.0 +/- 0.5; P < 0.001) . Crude mortality remained higher in pneumonia patients when compared with an infected control group matched for severity of illness and age (31% vs 15%; P = 0.02) . Staphylococcus aureus (15%) was the most common isolate, followed by Pseudomonas aeruginosa (9%) . Resistant Gram-positive cocci accounted for 7 per cent of all isolates but was associated with a 60 per cent mortality vs 28 per cent with other organisms (not significant; P = 0.1) . The Acute Physiology and Chronic Health Evaluation (APACHE) II score for patients with resistant Gram-positive cocci was 22 +/- 1 versus 18 +/- 1 with other organisms (P = 0.03) . Nonsurvivors of pneumonia were older (58 +/- 2 vs 51 +/- 3; P = 0.03), had a higher APACHE II score (23 +/- 1 vs 17 +/-1; P < 0.001), and were diagnosed later in their hospital course (18 +/- 4 days vs 11 +/- 1; P = 0.05) compared with survivors . Pneumonia-associated mortality in surgical patients remains high compared with other infections even when correcting for differences in severity of illness . Although resistant Gram-positive cocci appear to be increasing in frequency, they may represent markers of severe illness rather than true pathogens . Increasing age, severity of illness, and length of stay before diagnosis were all associated with a worse prognosis.

J AAPOS, 1999 Jun, 3(3), 183 - 4
Pseudomonas-induced bilateral endophthalmitis with corneal perforation in a neonate; Wasserman BN et al.; Neonatal endophthalmitis is a rare entity that may be exogenous or endogenous . Pseudomonas aeruginosa is a ubiquitous gram-negative rod that may appear as a nosocomial source of infection in the neonatal intensive care unit . A case of bilateral Pseudomonas-induced endophthalmitis is presented, and a discussion of the case and of the relevant literature follows.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 411 - 9
Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum beta-lactamase VEB-1 in Pseudomonas aeruginosa; Naas T et al.; A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram . Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1 . Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P . aeruginosa JES was amplified and subsequently sequenced . In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB . In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000 . P . aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries . The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.

Appl Environ Microbiol, 1999 Aug, 65(8), 3730 - 4
The branched-chain dodecylbenzene sulfonate degradation pathway of Pseudomonas aeruginosa W51D involves a novel route for degradation of the surfactant lateral alkyl chain; Campos-Garcia J et al.; Pseudomonas aeruginosa W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon . A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety . The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.

Biochem Biophys Res Commun, 1999 Aug 2, 261(2), 452 - 5
Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa; Germ M et al.; Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics . We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes . The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide . When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation . The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants . We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration . Among three extrusion pumps in P . aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion .

Electrophoresis, 1999 Jun, 20(7), 1578 - 85
Micellar electrokinetic chromatography for analyzing active site specificity of Pseudomonas aeruginosa elastase; Viglio S et al.; The geometry of the catalytic site of Pseudomonas aeruginosa elastase was reexamined, exploiting the specific feature of micellar electrokinetic chromatography (MEKC), i.e., its ability to detect a decrease of intact substrate and simultaneous formation of reaction products . We carried out a detailed investigation using two tri- and six tetra-peptide 4-nitroanilides (NA) differing from each other by only one or more amino acids as stable substrates . The kinetic cleavage parameters Km and k(cat) determined by MEKC and the catalytic efficiency Km/k(cat) values calculated allowed us to better define the substrate specificity of this proteinase.

J Infect, 1999 May, 38(3), 176 - 81
Pseudomonas aeruginosa infection in human immunodeficiency virus infected patients; Meynard JL et al.; OBJECTIVES: (1) To determine the incidence and outcome of Pseudomonas aeruginosa infection in HIV-infected patients . (2) To study the antimicrobial susceptibility of P . aeruginosa isolates in this particular population . (3) To identify risk factors for these infections . PATIENTS AND METHODS: A retrospective case-control study performed in a 28-bed infectious-diseases unit in a 940-bed university hospital . All cases were defined as HIV-infected patients with severe infections due to P . aeruginosa, including bacteremia, lower or upper respiratory tract infections, infections related to a central venous catheter, and cutaneous/muscular infection . Each case was matched with an HIV-seropositive control not infected by P . aeruginosa and hospitalized on the same dates as the cases . RESULTS: One thousand and thirty-five HIV-infected patients were hospitalized during the study period . A first severe P . aeruginosa infection was documented in 41 patients, giving an overall annual incidence note of 2.51 episodes per 100 admissions . Forty of the 41 case notes were available for analysis . They consisted of 17 cases of bacteraemia, four upper respiratory tract infections, 10 lower respiratory tract infections, three catheter-related infections, and six cutaneous/muscular infections . Of these 40 cases, 60% were nosocomial and the remainder were community-acquired . The overall mortality rate was 22% (47% in bacteraemic forms) . Twenty five percent of patients relapsed after an average of 37 days . The case-control comparison showed that AIDS was more frequent among the cases (92% vs . 74%, P = 0.04), who also had a lower PN count (P = 0.005), and a lower CD4 cell count (15.7 +/- 18.8/mm3 vs . 118 +/- 211/mm3; P = 0.0007) . The number of days spent in hospital in the previous 3 months (29.3 +/- 20.7 vs . 19.7 +/- 14, P = 0.04) was significantly higher among the cases . In a multivariate analysis, examining treatments received in the previous month, only co-trimoxazole {OR = 5.5 (1.1-26.9)}, penicillins {OR = 5.2 (1.1-25.3)}, steroids {OR = 5.5, (1.2-25.5)} and a CD4 cell count below 50/mm3 {OR = 13.2 (1.4-129.4)} were identified as risk factors . CONCLUSION: P . aeruginosa infection is a not frequent bacterial disease in highly immunodeficient HIV-infected patients . It is frequently fatal and must be borne in mind in the advanced stages of HIV disease, especially when patients have received co-trimoxazole (trianthoprim-sulphamethoxazole), penicillins or steroids.

Respir Physiol, 1999 May 3, 115(3), 301 - 7
Repeatability and methodology of resting energy expenditure in patients with cystic fibrosis; Bell SC et al.; In this study, intra-individual variation of resting energy expenditure (REE) in adults with cystic fibrosis (CF) and the effect of measurement duration were determined . Twelve adults with CF and chronic Pseudomonas aeruginosa (Ps . aeruginosa) infection and 12 healthy volunteers, matched for age and sex were studied whilst clinically stable on days 1.2, and 15 . Respiratory gas exchange was monitored by continuous measurement of oxygen uptake (VO2) and carbon dioxide production (VCO2) using a ventilated hood indirect calorimeter . Coefficients of variation (CVs) were 4.3% in patients and 2.4% in controls comparing days 1 and 2 . The CV for patients was 5.0% and for controls 2.9% comparing days 1 and 15 . The effect of measurement duration on REE was assessed in eight of the CF patients . REE remained stable for 40 min but tended to rise by 80 min . Plasma catecholamine concentrations were stable between study days in patients but fell with time in controls suggesting some adaptation to experimental procedure . The greater variability of REE in patients was related to change in serum CRP over 2 weeks . REE is a repeatable measurement in clinically stable patients with CF, though variability was greater in patients than healthy subjects . This has implications for the design and interpretation of longitudinal studies of REE in patients with chronic lung disease.

Kansenshogaku Zasshi, 1999 Jun, 73(6), 618 - 22
{A neutropenic acute myeloid leukemia patient complicated with chronic otitis media due to Aspergillus niger and yeast-like fungi caused by superinfection}; Itoh K et al.; There have been few reports describing otomycosis in association with compromised hosts . So we report a neutropenic acute myeloid leukemia (AML) patient complicated with otomycosis caused by superinfection . A 51-year-old male was admitted because a third relapse of AML in March 1998 . Two years ago, he was diagnosed as having chronic otitis media involving the VII cranial nerve due to Pseudomonas aeruginosa coinciding with AML . Then, he had suffered from a right-sided earache and otic discharge in accord with every myelosuppression, which improved on treatment with otic administration of ofloxacin . After 1 course of induction chemotherapy, he developed a spiking fever with severe earache and otic discharge at a nadir period of WBC . Ear swab cultures yielded Aspergillus niger and yeast-like fungi . So, he was treated with intravenous administration of amphotericin B (AMPH-B): initial dose was 5 mg/day and was gradually increased to 30 mg/day . Thereafter, the otic symptoms subsided and never recurred . Subsequently, he was given another antifungal agent, itraconazole . Although induction chemotherapies resulted in failure, he did not suffer otic symptoms until his death due to cerebral bleeding in January 1999 . For neutropenic patients without rapid hematological improvement, we recommend intensive antifungal therapy as the first-line of therapy for otomycosis rather than local therapy.

J Chromatogr A, 1999 Jun 18, 846(1-2), 125 - 34
Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography; Viglio S et al.; Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures . Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide) . When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only . Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated . MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.

Can J Microbiol, 1999 Apr, 45(4), 347 - 51
Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant; Liu YT et al.; The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively . The amino acid residues essential for function of the recombinase, protease, and ATPase in E . coli recA protein are conserved in G . oxydans . Of 24 amino acid residues believed to be the ATP binding domain of E . coli RecA, 17 are found to be identical in G . oxydans RecA . Interestingly, nucleotide sequence alignment between the SOS box of G . orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure . A G . oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette . Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization . Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G . oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation . Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G . oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

Mikrobiologiia, 1999 Mar-Apr, 68(2), 211 - 4
{Characteristics of carbohydrate metabolism of R-, S- i M-dissociants of Pseudomonas aeruginosa}; Mil'ko ES et al.; R and S dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2 differed but little in their growth in a minimal defined medium with glucose as the source of carbon and energy . At the same time, the number of cells of M dissociant in the late exponential phase was five orders of magnitude less than that of R and S dissociants . The growth of M dissociant was accompanied by the accumulation of formate in the culture liquid and a concurrent decrease in pH . All three dissociants contained the key enzymes of the Entner-Doudoroff pathway of glucose oxidation; however, the activities of these enzymes, especially 6-phosphogluconate dehydrogenase, were low in M dissociant . Conversely, the activity of formate dehydrogenase in cells of M dissociant was higher than in other dissociants . The activity of 6-phosphogluconate dehydrogenase, a key enzyme of the pentosephosphate pathway of glucose oxidation, was detected only in S dissociant . The peculiarities of the carbohydrate metabolism of M dissociant are probably responsible for its poor growth on glucose and determine the more pronounced anaerobic type of its metabolism.

Mikrobiologiia, 1999 Mar-Apr, 68(2), 206 - 10
{Effect of carbon, nitrogen, and phosphorus sources of nutrition on the growth of R-, S- and M-dissociants of Pseudomonas aeruginosa}; Maksimov VN et al.; The effect of glucose, nitrate, and phosphate on the stationary-phase growth parameters of the R, S, and M dissociants of the hydrocarbon-oxidizing bacterium Pseudomonas aeruginosa K-2 was studied . The data obtained were analyzed in terms of the Mitscherlich equation . S dissociant required less glucose than other dissociants, whereas M dissociant required less nitrogen and phosphorus . These findings were confirmed by the results of investigation of the combined action of glucose, nitrate, and phosphate in a 3 x 3 x 3 factor experiment . It is anticipated that M dissociant must prevail under conditions of nitrogen and phosphorus deficiency, and S dissociant must be dominant in the case of optimally chosen proportions between the biogenic elements studied.

J Microencapsul, 1999 Jul-Aug, 16(4), 419 - 29
Antibacterial activity of liposome-encapsulated antibiotics against Pseudomonas aeruginosa attached to the matrix of human dermis; Trafny EA et al.; The present studies were undertaken to compare the antibacterial activity of liposome vesicles containing amikacin, ciprofloxacin or polymyxin B in the removal of P . aeruginosa organisms from microcolonies growing on sections of the matrix of human dermis . Encapsulation efficiency of antimicrobials inside cationic liposomes was 30% for amikacin, 50% for ciprofloxacin, and 100% for polymyxin B . The sections of dermis were colonized for 72 h with P . aeruginosa strains isolated from burn wounds . After that time, an intense growth of microorganisms on the dermis surface was observed . The sessile organisms were treated (with mild shaking) with solutions of either liposomal or free amikacin, ciprofloxacin, and polymyxin B for 1 h, and also with a mixture of liposomal or free ciprofloxacin and polymyxin B (1:1) for 20 min . After treatment with liposomal antimicrobials, the mean per cent of viable cells attached to the dermis was 48.7% for liposomal amikacin, 17.4% for liposomal ciprofloxacin, 19.1% for liposomal polymyxin B, and 3.6% for a mixture of liposomal ciprofloxacin and liposomal polymyxin B . Removal of P . aeruginosa from microcolonies growing on the dermal matrix was more effective when liposomal formulations were used compared to the free antibiotics . Therefore, cleansing of the contaminated matrix of human dermis with liposomal ciprofloxacin, liposomal polymyxin B or with the mixture of both liposomal antibiotics seems to increase the efficacy at the removal of attached bacterial cells.

J Biol Chem, 1999 Jul 30, 274(31), 21823 - 9
Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange; Ganesan AK et al.; Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128) . Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange . Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions . While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras . ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype . Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange . Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments . Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.

MMWR Morb Mortal Wkly Rep, 1999 Jul 9, 48(26), 557 - 60
Bronchoscopy-related infections and pseudoinfections--New York, 1996 and 1998.
{Is the a connection between prolonged carriage and clonal hospital-to-hospital clonal spread of multiresistant Pseudomonas aeruginosa of the O12 serotype? Are the specific habits of the hospitals involved the cause?}
Watine J, Hacini J, Vidal I.

Hopital General, Rodez, FranceResults obtained using our expert computer program (SIR, 12A, Montpellier, France) over the last seven years (1991-1998) suggest that the increased incidence of superficial pus infection or colonization with O12 Pseudomonas aeruginosa, together with the significantly longer carriage duration for this organism as compared to non O12 . P . aeruginosa, may contribute to the current endemic clonal spread of O12 P . aeruginosa in a general hospital in Rodez, France, and in neighboring extended-care facilities . This ecological observation may reflect poor compliance with basic hygiene procedures in the Rodez hospital and in neighboring hospitals . The O12 P . aeruginosa clone in the Rodez hospital is indistinguishable from the one that has disseminated throughout Europe, suggesting that the ecological observation and hypothesis described herein may deserve to be investigated in other institutions that are also the site of endemic clonal O12 P . aeruginosa dissemination.

Infect Immun, 1999 Aug, 67(8), 3872 - 8
Essential role of transcription factor nuclear factor-kappaB in regulation of interleukin-8 gene expression by nitrite reductase from Pseudomonas aeruginosa in respiratory epithelial cells; Mori N et al.; Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases . Recently, we have reported that nitrite reductase from P . aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells . To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to nitrite reductase . Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity . This increased IL-8 gene expression was inhibited by mutating the nuclear factor-kappaB (NF-kappaB) binding element . Nitrite reductase enhanced nuclear localization of the NF-kappaB binding complex . Furthermore, nitrite reductase induced the degradation of IkappaBalpha, the major cytoplasmic inhibitor of NF-kappaB, and the expression of IkappaBalpha mRNA . These data support the critical role of the activation of NF-kappaB in nitrite reductase-induced IL-8 gene expression in airway epithelium.

Curr Eye Res, 1999 Jul, 19(1), 26 - 32
Complement defects in aged mice compromise phagocytosis of Pseudomonas aeruginosa; Hazlett LD et al.; PURPOSE: The role of complement in phagocytosis and killing of P . aeruginosa was examined using serum from aged vs young donor mice . METHODS: Phagocytosis, complement hemolytic and microbicidal assays were used . RESULTS: Serum from young donor mice contained a heat-labile factor which significantly enhanced phagocytic activity of cells from young mice compared with similarly treated aged donor serum . Use of cobra venom factor (CVF) to destroy C3 and the terminal complement components in serum from young or aged donor mice also significantly decreased the phagocytic activity of young cells . EGTA treatment of young or aged donor serum, to activate the alternative pathway and selectively inhibit activation of the classical pathway, resulted in a significant decrease in phagocytosis by young cells in the presence of donor serum from either group . Alternative pathway mediated hemolysis also was measured and was significantly reduced in aged vs young donor serum . PMN microbicidal activity was tested using cells from young mice in the presence of aged vs young donor serum, but no significant differences were noted . CONCLUSION: These data provide evidence that defects in the alternative pathway of complement in the serum of aged animals lead to decreased phagocytic activity of cells from young mice, but not impaired bacterial killing.

Anal Biochem, 1999 Aug 1, 272(2), 216 - 23
An enzyme-linked immunosorbent assay for the association of the catalytic domain of diphthamide-specific ribosyltransferases to eukaryotic elongation factor-2; Prentice GA et al.; The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2) . PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+) . The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions . It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable . The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent . The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin . This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2 . A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM . The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA .

J Biotechnol, 1999 Apr 30, 70(1-3), 27 - 32
Microbial antagonism: a neglected avenue of natural products research; Burgess JG et al.; Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces . We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates . Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds . This is a much higher proportion than free living marine isolates or soil bacteria . In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals . Thus the number of strains able to produce antibiotics appears to be much higher than previously thought . Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa . An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed.

Am J Physiol, 1999 Jul, 277(1 Pt 1), L183 - 90
Efficient killing of inhaled bacteria in DeltaF508 mice: role of airway surface liquid composition; McCray PB Jr et al.; Cystic fibrosis mice have been generated by gene targeting but show little lung disease without repeated exposure to bacteria . We asked if murine mucosal defenses and airway surface liquid (ASL) Cl(-) were altered by the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation . Naive DeltaF508 -/- and +/- mice showed no pulmonary inflammation and after inhaled Pseudomonas aeruginosa had similar inflammatory responses and bacterial clearance rates . We therefore investigated components of the innate immune system . Bronchoalveolar lavage fluid from mice killed Escherichia coli, and the microbicidal activity was inhibited by NaCl . Because beta-defensins are salt-sensitive epithelial products, we looked for pulmonary beta-defensin expression . A mouse homolog of human beta-defensin-1 (termed "MBD-1") was identified; the mRNA was expressed in the lung . Using a radiotracer technique, ASL volume and Cl(-) concentration ({Cl(-)}) were measured in cultured tracheal epithelia from normal and DeltaF508 -/- mice . The estimated ASL volume was similar for both groups . There were no differences in ASL {Cl(-)} in DeltaF508 -/- and normal mice (13.8 +/- 2.6 vs . 17.8 +/- 5.6 meq/l) . Because ASL {Cl(-)} is low in normal and mutant mice, salt-sensitive antimicrobial factors, including MBD-1, may be normally active.

Am J Physiol, 1999 Jul, 277(1 Pt 1), L204 - 17
Airway epithelial tight junctions and binding and cytotoxicity of Pseudomonas aeruginosa; Lee A et al.; The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosa to apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells . Binding of noncytotoxic P . aeruginosa strain O1 was assessed with P . aeruginosa that expressed green fluorescent protein . Binding of cytotoxic P . aeruginosa strain 6206 was assessed with FITC-labeled P . aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide . The role of direct contact of P . aeruginosa to epithelial cells was tested with filters with small (0.45-micrometer) or large (2.0-micrometer) pores . High transepithelial resistance (R(t)) Calu-3 and cultured bovine tracheal monolayers (R(t) > 1,000 Omega . cm(2)) bound P . aeruginosa very infrequently (<1 P . aeruginosa/100 cells) at the apical membrane, but P . aeruginosa bound frequently to cells near "free edges" at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes . Wounded high R(t) epithelia showed increased P . aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P . aeruginosa, and dead and living cells near the wound bound P . aeruginosa similarly . Compared with high R(t) epithelia, low R(t) CFT1 (R(t) = 100-200 Omega . cm(2)) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P . aeruginosa binding throughout the monolayer . Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs . 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P . aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low R(t) than in high R(t) airway epithelia . Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide . P . aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low R(t) CFT1 cells.

J Pept Res, 1999 Jun, 53(6), 618 - 25
Synthesis and biological evaluation of a pyoverdin-beta-lactam conjugate: a new type of arginine-specific cross-linking in aqueous solution; Kinzel O et al.; Arginine specific reagents such as phenylglyoxal and other alpha-dioxo compounds react with arginine side chains by forming adducts with a stoichiometry of 2:1 or a mixture of 2:1 and 1:1 . These adducts are labile in neutral and slightly alkaline aqueous solution . We developed a new type of cross-linking reaction with aliphatic beta-dioxo compounds . They can be used for the well-defined, irreversible covalent attachment of molecules carrying a primary amino group to arginyl residues of water soluble peptides . The reaction proceeds under mild conditions in aqueous solution, essentially without the formation of side products . A pyoverdin-cephalexin conjugate was synthesized in order to promote its cellular uptake by Pseudomonas aeruginosa . Preliminary biological investigations of the conjugate indicated that it enters the bacterial cell via the pyoverdin-mediated iron uptake pathway.

J Med Invest, 1999 Feb, 46(1-2), 79 - 85
Relationships between activity of daily living, and oral cavity care and the number of oral cavity microorganisms in patients with cerebrovascular diseases; Michishige F et al.; We examined the relationships among the activity of daily living (ADL), oral cavity care, and the number of oral cavity microorganisms in 40 patients with cerebrovascular diseases (CVD) . The CVD patients were classified into 4 groups, I, II, III and IV based on their ADL and the method used for oral cavity care . The ADL was highest in group I and lowest in group III . Only the patients of only group III could not eat by themselves and were receiving naso-esophageal feeding . Oral cavity care was performed by the patients themselves in groups I and IV, but was performed by caregivers in groups II and III . The group IV patients had no teeth, but could eat by themselves using full dentures . The numbers of microorganisms in the pharyngeal swabs from the 4 groups were measured and expressed as colony-forming units (cfu) . The numbers of both Staphylococci spp . and Candida spp . were significantly higher in group III than in the other groups . Moreover, Pseudomonas aeruginosa was isolated only from patients of group III (in about 66%) . The oral cavity care by caregivers was almost the same in groups II and III, but the numbers of oral cavity microorganisms were significantly higher in group III than in group II . These results indicated that microorganisms grow more easily in the oral cavities of CVD patients with low ADL compared with CVD patients with higher ADL, and that eating is thought to be important for the prevention of an increase of microorganisms in the oral cavity.

Eur J Biochem, 1999 Jul, 263(2), 478 - 85
Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa; Walther-Rasmussen J et al.; AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI) . In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P . aeruginosa strain and biochemically characterized . The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6 . When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous . The specific activities of the variants were very similar . MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa) . MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms . The N-terminal sequences of three of the beta-lactamases were identical to the published sequence {Lodge, J.M . , Minchin, S.D., Piddock, L.J.V . & Busby, J.W . (1990) Biochem . J . 272, 627-631}, while two variants were truncated by two amino-acid residues, one of which was acidic . The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found . Our results indicate that the P . aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene . The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.

Glycobiology, 1999 Aug, 9(8), 757 - 64
Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates; Scharfman A et al.; Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis . It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process . In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed . The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Le(a), Le(y), Le(x), sialyl- and 3'-sulfo-Le(x), and blood group A determinants) . We used also neoglycoconjugates containing Gal(alpha1-2)Galbeta and sialyl- N -acetyllactosamine determinants . The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl- N -acetyllactosamine epitopes . The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Le(x)determinant than for all the other glycoconjugates studied . The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides . In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Le(x)neoglycoconjugate, its sialylated and its sulfated derivatives . Individual variations in the binding of these strains to the three glycoconjugates were observed . However, three strains out of four had a higher affinity for the sialyl-Le(x)than for the 3'-sulfo-Le(x)derivative.

J Orthop Trauma, 1999 Jun-Jul, 13(5), 332 - 7
Comparison of castile soap, benzalkonium chloride, and bacitracin as irrigation solutions for complex contaminated orthopaedic wounds; Conroy BP et al.; OBJECTIVE: The purpose of the present study was to determine the effects of cleaning a contaminated orthopaedic wound with different classes of wound irrigation solutions . STUDY DESIGN: Rats with a contaminated orthopaedic wound were randomized into treatment groups: normal saline (NS), castile soap (CS), benzalkonium chloride (BzC), bacitracin (Abx), or sequential irrigation with BzC, CS, and NS . INTERVENTION: Pseudomonas aeruginosa {P . aeruginosa; 1 x 10(6) colony-forming units (CFU)}, or Staphylococcus aureus (S . aureus; 1 x 10(6) CFU) were placed into a paravertebral wound (containing a wire implant placed through a spinous process) and allowed to incubate for fifteen minutes . The wound was then irrigated with three liters of either NS, 0.05 percent CS, 0.03 percent BzC, Abx (33,000 units per liter) or underwent a sequential irrigation treatment (one liter each of BzC, CS, NS) . MAIN OUTCOME MEASUREMENTS: The animals were observed daily for wound complications for fourteen days and then killed, and cultures of the wound were obtained . RESULTS: Pseudomonas aeruginosa: Both CS and the sequential irrigation treatment significantly lowered the rate of positive wound cultures when compared with NS (p < 0.05) . Irrigation with BzC resulted in a higher rate of positive wound cultures and complications . The sequential irrigation treatment prevented the wound complications associated with irrigation with BzC alone . Staphylococcus aureus: Only BzC irrigation significantly lowered the rate of positive wound cultures when compared with NS (p < 0.05) . CONCLUSION: The rate of positive wound cultures due to P . aeruginosa is effectively reduced by irrigation with CS alone or by the sequential irrigation treatment . When used alone, the antiseptic BzC results in a higher rate of positive wound cultures and wound complications . The wound complications seen with irrigation with BzC alone are prevented by the sequential irrigation treatment (BzC followed by CS and NS) . The rate of positive wound cultures in this model due to S . aureus is not decreased by irrigation with CS; however, the rate of positive wound cultures is safely and effectively decreased with the use of BzC.

Mayo Clin Proc, 1999 Jul, 74(7), 698 - 701
Bronchial mucormycosis with progressive air trapping; Collins DM et al.; A previously healthy 70-year-old woman developed fever, cough, and exertional dyspnea . Her symptoms progressed over a 2-month period despite treatment by her primary care physician with 2 courses of oral antibiotics and the addition of prednisone . Hypoxemia and the finding of hyperglycemia with mild ketoacidosis led to hospital admission . Serial chest radiographs demonstrated diffuse heterogeneous pulmonary opacities and progressive air trapping in the right lower lobe . Fiberoptic bronchoscopy revealed a deep penetrating ulcer with exposed bronchial cartilage of the bronchus intermedius and dynamic airway obstruction with complete closure during expiration . Biopsy of the ulcer revealed Rhizopus arrhizus . Respiratory failure stabilized with the patient on conventional mechanical ventilation and receiving amphotericin B . Before surgery could be performed, Pseudomonas aeruginosa pneumonia and septic shock developed, and the patient died.

J Clin Microbiol, 1999 Aug, 37(8), 2687 - 93
Fluorescent oligonucleotide probes for clinical and environmental detection of Acanthamoeba and the T4 18S rRNA gene sequence type; Stothard DR et al.; The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoeba are described . Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA) . The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific for Acanthamoeba as shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba 18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformis and Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosa and Escherichia coli) . The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba 18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types . T4 is the subgenus group associated most closely with Acanthamoeba keratitis (AK) . GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls . GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed . Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoeba in 21 of 24 specimens from the 22 patients . The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genus Acanthamoeba in both environmental and clinical samples . In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.

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