Pediatr Infect Dis J, 2003 Jul, 22(7), 609 - 12 Cluster of Trichosporon mucoides in children associated with a faulty bronchoscope; Singh N et al.; BACKGROUND: Several outbreaks of Pseudomonas aeruginosa infection associated with a specific model of fiberoptic bronchoscope have been reported . In a 3-week period in September 2000, we noticed an increased number of Trichosporon mucoides isolates recovered from bronchoalveolar lavage (BAL) specimens collected at our hospital . We investigated the circumstances surrounding these isolates . METHODS: Outbreak investigation was conducted, and the medical records of the affected patients were reviewed retrospectively for evidence of positive cultures for T . mucoides from BAL specimens . Specimens collected during the investigation were inoculated onto fungal culture medium and yeasts were identified with API-20C (BioMerieux-Vitek) . RESULTS: During the 3-week period BAL specimens from six patients yielded growth of T . mucoides . These six high risk patients had emergency bronchoscopy performed as a workup for pneumonia and/or respiratory distress . A Model BF XP-40 bronchoscope (Olympus) had been used in all six patients . Cultures of the bronchoscope (external body and the lumen), bronchoscope disinfector, 2% glutaraldehyde disinfecting solution and water filters/supply were performed . Only fluid from the bronchoscope lumen yielded growth of T . mucoides . Air sample cultures of the bronchoscopy suites were negative . Medical records review disclosed that affected patients were not readmitted with infection with T . mucoides and had no adverse outcomes . The bronchoscope was removed from service and returned to the manufacturer . CONCLUSION: Routine surveillance and aggressive investigation identified persistent T . mucoides contamination of one bronchoscope . The bronchoscope manufacturer later recalled the BF XP-40 model for corrective revision. J Mol Microbiol Biotechnol, 2003, 5(4), 230 - 9 PseudoCyc, a pathway-genome database for Pseudomonas aeruginosa; Romero P et al.; A pathway-genome database (PGDB) describes the entire genome of an organism, as well as its biochemical pathways, reactions, and enzymes . Our PathoLogic software can generate a PGDB from an annotated genome of an organism, predicting the metabolic reactions and pathways corresponding to the enzymes present in the annotation . We have used PathoLogic to generate a PGDB for PSEUDOMONAS AERUGINOSA, strain PAO1, called 'PseudoCyc', which includes 139 predicted pathways and 800 predicted reactions involving 623 chemical species and 718 enzymes . Analysis of the PathoLogic predictions of arginine metabolism and the beta-ketoadipate pathway, which are landmark pathways in P . AERUGINOSA, showed that they were for the most part correctly predicted . These studies also provided possible locations for two genes involved in the beta-ketoadipate pathway, PCAI and PCAJ, which are missing from the PAO1 annotation . PseudoCyc adds an extended dimension to the genome of P . AERUGINOSA, providing researchers with a helpful tool for the analysis of the genomic, proteomic, and metabolic information of the organism . The finding of the probable location of the PCAI and PCAJ genes is but one example of the discoveries facilitated by such a PGDB . PseudoCyc, along with PGDBs for 12 other organisms, is available at Chemistry, 2003 Jun 16, 9(12), 2812 - 8 Laser spectroscopic studies of interactions of UVI with bacterial phosphate species; Knopp R et al.; We have investigated the interactions of UVI with two bacterial phosphate-containing species: Gram-positive Bacillus sphaericus and Gram-negative Pseudomonas aeruginosa . The Gram-positive B . sphaericus was investigated by using Raman spectroscopy and time-resolved laser-induced fluorescence spectroscopy (TRLFS) . We found that living cells, spores, and intact heat-killed cells complexed UVI (pH 4.5) through phosphate groups bound to their surfaces, while decomposed cells released H2PO4- and precipitated UVI as UO2(H2PO4)2 . TRLFS of UVI showed that Gram-negative P . aeruginosa--genetically engineered to accumulate polyphosphate, subsequently degrade it, and secrete phosphate--precipitated UVI quantitatively at pH 4.5 . The same bacterial strain, not induced to secrete phosphate, sorbed only a small amount of UVI. J Postgrad Med, 2003 Jan-Mar, 49(1), 11 - 6; discussion 16 Lipid peroxidation by Pseudomonas aeruginosa in the pathogenesis of nosocomial sepsis; Giamarellos-Bourboulis EJ et al.; BACKGROUND: To study whether Pseudomonas aeruginosa may directly trigger peroxidation of polyunsaturated fatty acids, since lipid peroxidation is a mechanism involved in the pathogenesis of sepsis . METHODS: Gamma-linolenic acid (GLA) was administered intravenously at a dose of 25mg/kg in an infusion time of 10 minutes to seven male rabbits . Blood samples were collected from the hepatic veins and from the carotid artery at regular time intervals . One clinical isolate was ex vivo incubated with the serum derived from the latter samples and concentrations of malondialdehyde (MDA) were determined during incubation in the growth medium by the thiobarbiturate assay . RESULTS: Elevated concentrations of MDA compared to their basal levels were found over the first three hours of incubation in the presence of samples collected 30 to 60 minutes after the end of the infusion of GLA . After infusion of GLA concentrations of arachidonic acid in the serum increased to concentrations comparable to those detected in sepsis . CONCLUSION: Direct triggering of lipid peroxidation by nosocomial isolates might be proposed as a pathogenetic mechanism of sepsis. J Clin Invest, 2003 Jul, 112(2), 275 - 85 Application of a mathematical model to prevent in vivo amplification of antibiotic-resistant bacterial populations during therapy; Jumbe N et al.; The worldwide increase in the prevalence of multi-antibiotic-resistant bacteria has threatened the physician's ability to provide appropriate therapy for infections . The relationship between antimicrobial drug concentration and infecting pathogen population reduction is of primary interest . Using data derived from mice infected with the bacterium Pseudomonas aeruginosa and treated with a fluoroquinolone antibiotic, a mathematical model was developed that described relationships between antimicrobial drug exposures and changes in drug-susceptible and -resistant bacterial subpopulations at an infection site . Dosing regimens and consequent drug exposures that amplify or suppress the emergence of resistant bacterial subpopulations were identified and prospectively validated . Resistant clones selected in vivo by suboptimal regimens were characterized . No mutations were identified in the quinolone resistance-determining regions of gyrA/B or parC/E . However, all resistant clones demonstrated efflux pump overexpression . At base line, MexAB-OprM, MexCD-OprJ, and MexEF-OprN were represented in the drug-resistant population . After 28 hours of therapy, MexCD-OprJ became the predominant pump expressed in the resistant clones . The likelihood of achieving resistance-suppression exposure in humans with a clinically prescribed antibiotic dose was determined . The methods developed in this study provide insight regarding how mathematical models can be used to identify rational dosing regimens that suppress the amplification of the resistant mutant population. J Am Chem Soc, 2003 Jul 23, 125(29), 8760 - 8 Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids; Berry SM et al.; Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids . The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM) . The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT . All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings . The Se EXAFS of the M121SeM variant is also reported . In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin . The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center . A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains . Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands. Swiss Med Wkly, 2003 May 31, 133(21-22), 297 - 301 Macrolide antibiotic therapy in patients with cystic fibrosis; Schoni MH; This summary of the current knowledge of macrolide therapy serves as an example of recent progress in the therapeutic approach to treating patients with cystic fibrosis (CF) . The benefit of macrolides in the treatment of patients with diffuse panbronchiolitis and Pseudomonas aeruginosa infections, as seen in Japan, was the rational behind trials in patients with CF . Thus far, the majority of reports of positive trends in the therapeutic potential of macrolides have studied azithromycin . The data presented in peer reviewed journals are, however, too sparse to already justify firm recommendations for the general use of azithromycin, erythromycin or clarithromycin on a long-term basis for the treatment of chronic lung disease in CF. Eye Contact Lens, 2003 Jul, 29(3), 190 - 2 Unusual morphology in orthokeratology contact lens-related cornea ulcer; Wang JC et al.; PURPOSE: To report a case of unusual ulcer morphology in orthokeratology-related corneal ulcer . METHODS: A single observational case report of a 14-year-old Chinese female myope with a 1.5-month experience wearing overnight B.E . orthokeratology (Capricornia) lenses and presenting with a right stellate-shaped central cornea abscess . Cornea scrapings for Gram stains, culture, and antibiotic sensitivity were performed . The patient was prescribed hourly fortified cefazolin and gentamicin drops . RESULTS: Pseudomonas aeruginosa grew on blood and chocolate agar cultures . The ulcer was successfully treated with antibiotics and reepithelialized over 5 days . There was a residual central corneal scar . The refraction changed from -4.25 sphere OD and -1.75 -1.75 x160 OS to -3.50 -1.50 x160 OD and -1.50 -1.75 x165, giving a visual acuity of 20/ 25 OD and 20/20 OS . CONCLUSIONS: A flatter fit of orthokeratology lenses may be associated with unusual cornea ulcer morphology. Eye Contact Lens, 2003 Jul, 29(3), 185 - 6 Infectious keratitis associated with daily disposable contact lenses; Su DH et al.; PURPOSE: To report two cases of infectious keratitis associated with the use of daily disposable soft contact lenses . METHOD: Two case reports of individuals who developed infectious keratitis while wearing daily disposable soft contact lenses are presented . RESULTS: The first case is that of a 34-year-old woman who had been using daily disposable soft contact lenses for 18 months before she developed a corneal ulcer in her left eye . The cultures grew Pseudomonas aeruginosa, and she was treated successfully with fortified topical antibiotics . The second case describes a 30-year-old woman who had been using conventional soft contact lenses for 5 years before switching to daily disposable soft contact lenses 3 months before presentation . She was found to have a corneal ulcer in her left eye that grew Staphylococcus aureus on cultures, and she responded to topical antibiotic treatment . CONCLUSIONS: Although daily disposable soft contact lenses theoretically have a lower risk of infectious keratitis compared with other lens wear regimens, reports have shown that at least some risk remains . These lenses should be prescribed and used with great care to minimize contact lens-related infectious keratitis. J Mol Biol, 2003 Jul 25, 330(5), 1145 - 52 Parallel pathways in cytochrome c(551) folding; Gianni S et al.; The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism, consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species . However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways . Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates . Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH . Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale . A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling . These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways. J Interferon Cytokine Res, 2003 Jun, 23(6), 307 - 18 Metalloproteases from Pseudomonas aeruginosa degrade human RANTES, MCP-1, and ENA-78; Leidal KG et al.; The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen associated with both an acute lung disease in patients with hospital-acquired pneumonia and a chronic, progressive lung disease in individuals with cystic fibrosis . A unique characteristic of this bacterium in its natural environment is the secretion of a wide variety of factors designed to ensure its growth and survival . Evidence suggests, however, that when present in the human host, these same factors may contribute to disease . In the course of studying the effect of P . aeruginosa secretory factors on airway epithelial cells, we observed that metalloproteases in bacterial-conditioned medium, as well as purified alkaline protease and elastase, degraded human RANTES, monocyte chemotactic protein-1 (MCP-1), and epithelial neutrophil-activating protein-78 (ENA-78) . Under identical conditions, interleukin-8 (IL-8) was significantly more resistant to proteolysis . Degradation was accompanied by a loss of chemotactic activity . These data suggest that metalloproteases from P . aeruginosa could alter the relative amounts of critical immunomodulatory cytokines in the airway and, thus, could contribute to the pathophysiology observed in P . aeruginosa-associated lung disease. J Appl Microbiol, 2003, 95(2), 250 - 5 The effects of some formulation factors used in ophthalmic preparations on thiomersal activity against Pseudomonas aeruginosa and Staphylococcus aureus; Abuqaddom AI et al.; AIMS: To investigate the effects of formulation ingredients used in ophthalmic preparations on thiomersal activity against Staphylococcus aureus and Pseudomonas aeruginosa . METHODS AND RESULTS: Minimum inhibition concentrations (MICs) of the tested ingredients and their combinations were studied using partial broth dilution checkerboard method . Complex formation was determined using differential scanning calorimetry (DSC) and u.v . scan . Isotonic agents showed insignificant difference in thiomersal activity . Low concentrations of propylene glycol and glycerol (2 to about 6.5%) significantly reduced the activity of thiomersal against P . aeruginosa . Higher concentrations up to about 40%, of the two cosolvents did not affect the antibacterial activity . Viscosity increasing agents significantly reduced the antibacterial activity of thiomersal . Low concentrations of 0.05% and 0.05-0.1% of ethylenediaminetetraacetic acid resulted in a significant decrease in thiomersal activity against S . aureus and P . aeruginosa, respectively . However, concentrations above 0.25 and 0.5 up to 4% caused significant increase on the antibacterial activity against the two later micro-organisms, respectively . CONCLUSIONS: Results showed that thiomersal formed complexes with ingredients containing polyhydroxy groups and chelating agents, thus thiomersal is not recommended to be used with such ingredients . SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the importance of proper selection of ingredients and preservatives for ophthalmic preparations. Acta Paediatr, 2003 Jun, 92(6), 684 - 7 Impact of inhaled corticosteroids on the risk of early Pseudomonas aeruginosa acquisition in cystic fibrosis; Minicucci L et al.; AIM: To investigate the role of inhaled corticosteroids (IC) on the risk of Pseudomonas aeruginosa acquisition before the age of 10 y in cystic fibrosis (CF) patients . METHODS: For each subject the cumulative dose kg(-1) of IC received for each year of age was calculated until the end of follow-up . The age at CF diagnosis, the nutritional status (NS) and the number of respiratory exacerbations (RE) were used as surrogate measures for the severity of CF . RESULTS: A total of 83 patients (40 M, 43 F) entered the study . Their median length of follow-up was 4.4 y, for a total of 386 person-years at risk . Twenty-three patients acquired P . aeruginosa at a median age of 4.6 y (range 0.4-9.9 y) . The estimated survival without P . aeruginosa acquisition was 65% at 10 y of age . The effect of different risk factors (IC, NS, RE and age at CF diagnosis) on the probability of P . aeruginosa acquisition was evaluated: none of them was significantly associated with the risk of P . aeruginosa acquisition . In particular, patients receiving very high cumulative doses of IC (4th quartile) had a non-significantly increased risk of P . aeruginosa acquisition compared with those receiving low doses of IC (1st quartile) (hazard ratio = 1.73, 95% confidence limits 0.40-7.38) . CONCLUSION: This retrospective study was not able to demonstrate any role of IC in increasing the risk of P . aeruginosa acquisition . This complication seems to occur at a constant pace that is independent of CF severity and age . Prospective multi-institutional randomized studies are needed to investigate the effects of high-dose IC in CF patients. Clin Cancer Res, 2003 Jul, 9(7), 2837 - 48 A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity; Di Paolo C et al.; PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) is a tumor-associated antigen overexpressed in many solid tumors but shows limited expression in normal epithelial tissues . To exploit this favorable expression pattern for targeted cancer therapy, an Ep-CAM-specific recombinant immunotoxin was developed and its antitumor activity investigated . EXPERIMENTAL DESIGN:The immunotoxin 4D5MOCB-ETA was developed by genetically fusing a truncated form of Pseudomonas aeruginosa exotoxin A (ETA) (ETA(252-608)KDEL) to the highly stable humanized single-chain antibody fragment (scFv) 4D5MOCB . Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro . Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts . RESULTS: Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen-binding affinity . In vitro, 4D5MOCB-ETA potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC(50)s ranging from 0.005 to 0.2 pM . Upon systemic administration in mice, 4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumor:blood ratio of 5.4 . The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon, or squamous cell carcinomas . CONCLUSIONS: We describe for the first time the development of a fully recombinant Ep-CAM-specific immunotoxin and demonstrate its potent activity against solid tumors of various histological origins . 4D5MOCB-ETA is currently being evaluated in a Phase I study in patients with refractory squamous cell carcinoma of the head and neck. Clin Microbiol Infect, 2003 May, 9(5), 388 - 96 Different trends in antibiotic resistance rates at a university teaching hospital; Sorberg M et al.; OBJECTIVES: To investigate long-term trends in antibiotic resistance of common bacterial species isolated at a university hospital and in its intensive care units (ICUs) . METHODS: Levels of antibiotic resistance of common bacterial pathogens were investigated at the Karolinska Hospital during the 12-year period 1988-99 . Resistance rates were analyzed for the entire hospital, as well as for ICUs combined . RESULTS: At the Karolinska Hospital, we found increased ciprofloxacin resistance among Escherichia coli isolates, from 0% in 1991 to 11% in 1999 . In the ICUs, the corresponding increase was from 0% to 4.8% during the same period . Co-trimoxazole resistance levels increased from 7.5% to 14%, with lower levels for the ICUs . For ampicillin, cefuroxime, and gentamicin, the levels of resistance were similar in the whole hospital and in the ICUs . Among Pseudomonas aeruginosa isolates, imipenem resistance was higher in the ICUs . For ciprofloxacin, resistance increased from 2.5% in 1991 to 13% in 1999 in the whole hospital, with similar figures for the ICUs . CONCLUSION: The resistance rates at the Karolinska Hospital were still generally low, but were increasing for some antibiotic-microbe combinations . The results emphasize the importance of including all sectors of a hospital in resistance surveillance studies, and also the value of long surveillance periods. Clin Microbiol Infect, 2003 May, 9(5), 370 - 9 Development of resistance in Pseudomonas aeruginosa obtained from patients with cystic fibrosis at different times; Spencker FB et al.; OBJECTIVE: Determination of the extent of changes in quantitative resistance in Pseudomonas aeruginosa isolates from patients with cystic fibrosis over a period of approximately 2 years . METHODS: Three hundred and ninety nine isolates of P . aeruginosa collected from 34 pediatric patients in the period between April 1994 and April 1996 were investigated . During the 2 years the children were treated with a combination of a betalactam and an aminoglycoside, approximately every 3 months . In between they received ciprofloxacin orally, when required . The minimal inhibitory concentrations (MICs) of 38 clones of P . aeruginosa defined by different patterns in macrorestriction analysis (pulse field gel electrophoresis, PFGE) were established for 12 antibiotics: gentamicin, amikacin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin, imipenem, meropenem, ceftazidime, cefepime, and piperacillin by means of broth microdilution tests according to DIN 58940 . RESULTS: Twenty-four of the 38 clones developed increased MIC values during the time of observation especially for aminoglycosides and quinolones . Comparatively less affected were ceftazidime, imipenem and meropenem . An association between the number of the intravenous treatment courses and the increase of the MIC values could not be verified . CONCLUSIONS: A trend towards an increase of the MICs against antipseudomonal agents was observed over a limited period of time . It is necessary to prevent this development possibly by employing suitable combinations of antibiotics and the introduction of new substances. Genetika, 2003 May, 39(5), 595 - 620 {Role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria}; Krylov VN; The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer . Examples of phage participation in this process proved to a different extent are described . Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent phi KZ-like phages) of conditional pathogen Pseudomonas aeruginosa . Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution . An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches. J Antimicrob Chemother, 2003 Aug, 52(2), 247 - 52 Epub 2003 Jul 01. Liposomal tobramycin against pulmonary infections of Pseudomonas aeruginosa: a pharmacokinetic and efficacy study following single and multiple intratracheal administrations in rats; Marier JF et al.; OBJECTIVE: To determine the pharmacokinetics and efficacy of tobramycin against pulmonary infections of Pseudomonas aeruginosa in rats after intratracheal administration of conventional and liposomal formulations . METHODS: Male Sprague-Dawley rats were inoculated with 10(6) cfu of a mucoid variant of P . aeruginosa (MIC of tobramycin for PA 508 = 1 mg/l) and tobramycin (conventional or liposomal formulations) was administered in single (490 microg) and multiple dose (490 microg during 4 days) experiments . Rats were killed at multiple time points to determine the residual cfu of P . aeruginosa and tobramycin amounts in lungs . Pharmacokinetic parameters were calculated using a two-compartment model with NONMEM . RESULTS: Mean (+/-S.D.) elimination half-life (t(1/2beta)) and pulmonary exposure (AUC) of the conventional formulation were 14.0 +/-4.0 h and 663 +/-89 microg x h/lungs, respectively . The pharmacokinetic profile of liposomal tobramycin was markedly different, with a longer t(1/2beta) (34.4 +/-5 h, P < 0.05), resulting in an increased AUC (3890 +/-560 microg x h/lungs, P < 0.05) . chi(2) analyses were carried out on cfu data distributed in the following categories: below 10(3), 10(3)-10(5), and above 10(5) cfu . In the single dose experiments, approximately 90% of the observations were above 10(5) cfu for both formulations . Significant differences in cfu distribution were observed after multiple treatments, with approximately 10% of the observations falling below 10(3) cfu of P . aeruginosa for the conventional formulation versus 30% for the liposomal formulation . CONCLUSION: The liposomal formulation of tobramycin promoted drug retention in lungs and improved its efficacy after multiple treatments. J Am Soc Mass Spectrom, 2003 Jul, 14(7), 742 - 51 Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation; Guina T et al.; In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors . For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS) . To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts . The experiments confirmed expression of 1331 P . aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium . A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium . Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P . aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes . This result was confirmed by 2-D PAGE analysis of P . aeruginosa outer membrane proteins . This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes. Chem Biol, 2003 Jun, 10(6), 563 - 71 Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer; Smith KM et al.; The autoinducer (AI) that initiates the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa is an acyl-homoserine lactone (acyl-HSL) . We initiated a study of the requirements for binding of the AI to its protein effector LasR by synthesizing a library of analogs with the HSL moiety replaced with different amines and alcohols . We tested each compound for both agonist and antagonist activity using a QS-controlled reporter gene assay and found several new agonists and antagonists . A representative antagonist was further tested for its ability to inhibit virulence factors . This data progresses our understanding of the LasR-AI interaction toward the rational design of therapeutic inhibitors of QS. HNO, 2003 Jun, 51(6), 480 - 5 Epub 2003 Apr 08. {Primary nasal epithelial cells and fibroblasts have inflammation-inducing functions}; Rudack C et al.; BACKGROUND: The appearance of neutrophils in rhinitis and sinusitis led to the working hypothesis that neutrophil-specific attractants commonly called chemokines are generated by stimulation with proinflammatory cytokines and bacteria . The receptor mechanism of chemokine synthesis by bacterial products is under discussion and still has to be elucidated . PATIENTS AND METHODS: The primary nasal cultures of epithelial cells and fibroblasts ( n=4) were incubated with TNF-alpha (tumor necrosis factor) for 24 and 72 h . Bacterial stimulation of the cell cultures was performed by adding supernatants from the mucoid phenotype of Pseudomonas aeruginosa (PA01) in dilution 1:5 for 24 and 72 h . Supernatants were collected and the concentration of the chemokines interleukin-8 (IL-8), GRO-alpha (growth-related oncogene-alpha), and ENA-78 (epithelial neutrophil-activating peptide) were determined by ELISA technique . Our results revealed that the protein concentration of the chemokines GRO-alpha and IL-8 was upregulated by TNF-alpha as well as by bacterial supernatants in epithelial cells . CONCLUSION: We conclude that GRO-alpha and IL-8 were inducible by bacterial supernatants in nasal epithelial cells and fibroblasts. Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1310 - 2 Epub 2003 Jun 27. Crystallization and X-ray analysis of a bacterial non-haem iron-containing phenylalanine hydroxylase from the Gram-negative opportunistic pathogen Pseudomonas aeruginosa; Ekstrom F et al.; Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment . A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated . The single-domain monomeric protein harbours a non-haem iron at the active site . The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone . Here, the cloning, purification and crystallization of native and SeMet-labelled P . aeruginosa phenylalanine hydroxylase are reported . Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A . Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge. Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1241 - 2 Epub 2003 Jun 27. Crystallization and preliminary X-ray diffraction analysis of lectin-1 from Pseudomonas aeruginosa; Karaveg K et al.; Carbohydrate recognition plays a role in the pathogenesis of Pseudomonas aeruginosa, a common cause of opportunistic infection in humans . Crystals of a carbohydrate-binding protein from P . aeruginosa, lectin PA-1, have now been obtained . The crystals belong to space group I222, with unit-cell parameters a = 40.25, b = 72.30, c = 133.82 A, and diffract to beyond 1.9 A resolution on a rotating-anode X-ray source . Details of crystal-growth conditions, diffraction data collection and processing are reported. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 167 - 71 Mucosal vaccination with a recombinant OprF-I vaccine of Pseudomonas aeruginosa in healthy volunteers: comparison of a systemic vs . a mucosal booster schedule; Gocke K et al.; We compared the immunogenicity of two vaccination schedules with either a systemic or a mucosal booster, both following a mucosal primary vaccination with a recombinant outer membrane fusion protein of Pseudomonas aeruginosa (OprF-I) in 12 healthy volunteers . The systemic booster induced higher levels of OprF-I-specific serum antibodies of IgG isotype, with a mean+/-S.E.M . of 32.6+/-7.8x10(7) enzyme-linked immunosorbent assay (ELISA) units (EU) as compared to the nasal booster with 14.6+/-2.1x10(7) EU (P=0.05) . Specific serum IgA antibodies and antibodies in saliva did not differ between the two vaccination groups . We conclude that a combined mucosal/systemic vaccination with the OprF-I vaccine may offer an enhanced systemic immunogenicity . Further studies on the long-term immunogenicity and induction of antibodies on the respiratory airway surface are warranted. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 161 - 6 Clinical study to assess the immunogenicity and safety of a recombinant Pseudomonas aeruginosa OprF-OprI vaccine in burn patients; Mansouri E et al.; In a recent clinical trial we evaluated the safety and immunogenicity of a recombinant OprF-OprI vaccine consisting of the mature outer membrane protein I (OprI) and amino acids 190-342 of OprF of Pseudomonas aeruginosa in burn patients and compared the elicited antibodies with antibodies against tetanus as response to a simultaneous immunization given on the day of admission . Safety and immunogenicity of the vaccine had been tested before in healthy human volunteers as published in 1999 . In this first clinical trial we immunized eight burn patients suffering from second or third degree burns involving between 35% and 55% of the body surface three times with 100 microg of the OprF-OprI vaccine . The vaccine was found to be very well tolerated . The patients did not show any serious side effects - and in particular no activation of the mediator cascade was observed . None of the subjects showed systemic P . aeruginosa infections during or after the treatment of their burns . The serological tests (ELISA) for detection of antibodies against P . aeruginosa and tetanus toxoid showed seroconversion for seven patients after inoculation . The data indicate that OprF-OprI can be a useful vaccine in the therapeutic management of burn injuries. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 155 - 60 Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa; Thomas LD et al.; Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals . Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group . The bacterium produces various immunomodulatory products that enable it to survive in the lung . Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine . This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P . aeruginosa . N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic . A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P . aeruginosa challenge . Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue . The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase . Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form . When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate . This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine. FEMS Immunol Med Microbiol, 2003 Jul 15, 37(2-3), 147 - 53 DNA vaccines against chronic lung infections by Pseudomonas aeruginosa; Staczek J et al.; Vaccines containing outer membrane protein F (OprF) of Pseudomonas aeruginosa are effective in reducing lesion severity in a mouse pulmonary chronic infection model . One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector . When delivered three times biolistically by gene gun, the F/I vaccine induces protection that is antibody-mediated in outbred mice . To demonstrate the role of F/I-induced antibody-mediated immunity, B-cell-deficient {B(-)} and B-cell-intact {B(+)} mice were immunized with F/I, challenged with Pseudomonas, and examined for lesion severity . As expected, F/I-immunized B(+) mice had fewer and less severe lesions than vector-immunized B(+) mice . However, surprisingly, F/I- and vector-immunized B(-) mice were equally protected to levels similar to F/I-immunized B(+) mice . Examination of immune cell populations and cytokine levels indicated a relative increase in the quantity of CD3+ T-lymphocytes in vector- or F/I-immunized and challenged B(-) mice compared to B(+) mice . These data indicate the protective role played by cell-mediated immunity in B(-) mice, which supports our hypothesis that cell-mediated immunity can play an important role in protection against P . aeruginosa. Am J Respir Cell Mol Biol, 2003 Dec, 29(6), 661 - 8 Epub 2003 Jun 26. CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury; Vanderbilt JN et al.; The proinflammatory CXC chemokines GRO, CINC-2alpha, and macrophage inflammatory protein (MIP)-2 are a closely related family of neutrophil chemoattractants . Here, we report that freshly isolated alveolar Type II (TII) cells express these chemokine mRNAs at much higher levels than do freshly isolated Type I cells or alveolar macrophages (AM) . TII cells also express CXCR2, the receptor for these chemokines . Lung injury caused by acid or Pseudomonas aeruginosa (Pa) caused an increase in TII cell expression of chemokine mRNAs and GRO protein . We compared the time courses of chemokine mRNA expression in cultured TII cells and AM . In TII cells, GRO mRNA levels were stable over 4 h, but decreased to undetectable levels by 24 h . CINC-2alpha and MIP-2 mRNA levels were low in freshly isolated cells, increased over 2-4 h in culture, and by 24 h dropped to undetectable levels . In contrast, none of these chemokine mRNAs were detected in freshly isolated AM, but expression was induced by tissue culture . In summary, we have shown that TII alveolar epithelial cells produce three of the major proinflammatory CXC chemokines (GRO, CINC-2alpha, and MIP-2) and their cognate receptor CXCR2 . Chemokine expression is upregulated in response to lung injury . These observations support a central role for the TII cell as an immunologic effector cell in the alveolus and raise intriguing questions about how CXC chemokines and receptors modulate diverse normal and pathologic cellular responses in the alveoli. Vet Microbiol, 2003 Jul 30, 94(4), 295 - 301 Mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from canine infections; Teresa Tejedor M et al.; Chronic otitis externa in dogs is often associated with Pseudomonas aeruginosa infection . Fluoroquinolones are often used for treating such infections . Fluoroquinolone resistance mechanisms were characterized in 10 strains of P . aeruginosa isolated from chronic canine otitis externa . Nine out of ten strains harbored a mutation in the gyrA gene and presented an overexpression of efflux pump(s) . There was a good correlation between the lipophilicity of the fluoroquinolone being tested and the effect of the efflux pump inhibitor in the final MIC . Therefore, both mechanisms, mutation in the gyrA gene and increased efflux pump(s), seem to play an important role in the acquisition of fluoroquinolone resistance in veterinary clinical isolates of P . aeruginosa . Levels of resistance to fluoroquinolones suggest that they could not be a good choice for systemic therapy of Pseudomonas otitis. Biochim Biophys Acta, 2003 Jun 20, 1622(1), 36 - 41 A stable isotope dilution assay for the quantification of the Pseudomonas quinolone signal in Pseudomonas aeruginosa cultures; Lepine F et al.; A stable isotope dilution method was developed to analyse 2-heptyl-3,4-dihydroxyquinoline, also called the Pseudomonas quinolone signal (PQS), directly in Pseudomonas aeruginosa cultures by liquid chromatography coupled to mass spectrometry (LC/MS) . PQS, along with the isobaric 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), were quantified in various Pseudomonas liquid cultures using a deuterated PQS analog as internal standard . The kinetic of production of these quinolines in a growing culture of P . aeruginosa PA14 showed that their production starts at the end of the logarithmic growth phase and is maximal at the onset of the stationary growth phase . The concentration of PQS reached a maximum at 13 mg/l and then decreased, while the HQNO concentration reached 18 mg/l and then remained stable . Culture supernatants of P . aeruginosa strains PAO1 and PA14 produced similar concentrations of PQS whereas no PQS or HQNO could be detected in culture supernatants of the P . aeruginosa strain PAK or in the other Pseudomonas species tested, including phytopathogenic pseudomonads. Jpn J Antibiot, 2003 Apr, 56(2), 138 - 41 {Antibacterial activity of biapenem against recent clinical isolates}; Hara T et al.; Antibacterial activity of biapenem (BIPM) against clinical isolates of 8 species between 2000 and 2002 was compared with those of imipenem/cilastatin (IPM/CS), meropenem (MEPM), panipenem/betamipron (PAPM/BP) and ceftazidime (CAZ) . The MICs of biapenem for Gram-positive bacteria were higher than those of IPM/CS and PAPM/BP, equal to those of MEPM and lower than those of CAZ . The MICs of BIPM for Gram-negative bacteria were higher than those of MEPM, equal to those of IPM/CS and PAPM/BP, and lower than those of CAZ . Antibacterial activity of BIPM against Pseudomonas aeruginosa was equal to those of IPM/CS and MEPM and superior to those of PAPM/BP and CAZ . In conclusion, BIPM showed broad antibacterial activity against both Gram-positive and Gram-negative clinical isolates . These results suggest that BIPM is useful for the treatment of various bacterial infections. Mol Microbiol, 2003 Jul, 49(1), 1 - 9 Control of rpoS transcription in Escherichia coli and Pseudomonas: why so different? Venturi V. In Escherichia coli, the stationary phase alternative sigma factor sigmas controls the expression of genes involved cell survival in response to cessation of growth (stationary phase) and provides cross-protection to various stresses . Levels of sigmas increase dramatically at the onset of stationary phase and are regulated at the transcriptional, post-transcriptional and post-translational level, making this one of the most complex regulatory systems in bacteria . The basic mechanisms for the control of translation and sigmas proteolysis have been understood . However, studies on the transcriptional control in E . coli lag behind and are controversial . The cAMP-CRP complex and the two component BarA/UvrY system have been implicated and, ppGpp and polyphosphate appear to have a signalling role . sigmas has also been reported to be a general stress regulator in the fluorescent pseudomonads (Pseudomonas aeruginosa, P . fluorescens and P . putida) and recent studies on sigmas regulation highlight that transcriptional regulation in these bacteria apparently plays a major role . Global regulatory systems, the GacA/GacS two component system and quorum sensing all affect rpoS expression, as does the TetR family PsrA regulator that directly binds to- and activates the rpoS promoter in stationary phase . This striking difference in regulation between E . coli and Pseudomonas can be partly attributed to the differences in the functional role of sigmas in the two bacterial species . This report will review mainly recent studies on rpoS transcriptional regulation and will try to rationalize the current knowledge into a working model. Antimicrob Agents Chemother, 2003 Jul, 47(7), 2093 - 9 Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR; Tabata A et al.; The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated . A P . aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method . It was revealed that P . aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride . Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P . aeruginosa . Moreover, the P-12-resistant strain exhibited cross-resistance to BAC . Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa . The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR . A correlation between the level of expression of OprR and the level of resistance of P . aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain . The knockout mutant recovered susceptibility not only to P-12 but also to BAC . These results suggested that OprR significantly participated in the adaptation of P . aeruginosa to QACs, such as P-12 and BAC. Zhonghua Yi Xue Za Zhi, 2003 Mar 10, 83(5), 403 - 7 {Susceptibility of 570 Pseudomonas aeruginosa strains to 11 antimicrobial agents and the mechanism of its resistance to fluoroquinolones}; Lei YC et al.; OBJECTIVE: To investigate the resistance of Pseudomonas aeruginosa (P . aeruginosa) to 11 antimicrobial agents and the mechanism of its resistance to fluoroquinolones (FQs) . METHODS: The susceptibility of 570 strains of P . aeruginosa isolated clinically to 11 antimicrobial agents were detected by Kirby-Bauer disc diffusion method . The minimal inhibitory concentration (MIC) of 111 strains thereof to two FQs was determined by agar dilution method . 80 strains resistant to ciprofloxacin (MIC >or= 4 mg/L) were studied for the presence of point mutations in the gyrA and parC gene by direct sequencing and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method . RESULTS: The resistance rates of P . aeruginosa to cefepine, imipenem, amikacin, and ceftazidine, and aztreonam were 10.9%, 11.1%, 11.8%, 12.5%, and 16.6% respectively . The resistance rates to ciprofloxacin, levofloxacin, cefoperazone/sulbactam, and piperacillin were 29.2%, 32.9%, 23.5% and 33.7% respectively . The resistance rates to gentamycin and cefotaxime were 35.7% and 41.1% respectively . More than 60% of the ciprofloxacin resistant strains and the intensive care units (ICU) isolates were multidrug resistant . Among the 80 ciprofloxacin resistant strains, 66 (75%) had a mutation in gyrA codon: Thr83 (ACC)-->Ile (ATC), 52 strains (65%) had a mutation at parC codon: Ser87 (TCG)-->Leu (TTG), and 52 strains (65%) had both the above mentioned gyrA and parC mutations; while gyrA or parC mutation was not observed in the 31 ciprofloxacin susceptible strains . The MIC of ciprofloxacin for the 52 strains with mutations in both gyrA and parC genes was 19.80 mg/L +/- 2.11 mg/L, significantly higher than those for the 14 strains with mutation only in gyrA gene (11.88 mg/L +/- 2.73 mg/L, P < 0.05) and the 143 strains without gyrA and parC gene mutants (11.89 mg/L +/- 2.12 mg/L, P < 0.05) . CONCLUSION: Resistance to antimicrobial agents of P . aeruginosa strains remains a problem . In particular, those ciprofloxacin resistant strains and ICU isolates are resistant to most of the antimicrobial agents . The resistance to fluoroquinolones of the clinical isolates of P . aeruginosa is mainly due to the mutations in gyrA and parC genes encoding the target enzyme of fluoroquinolones. Zhonghua Yi Xue Za Zhi, 2003 Mar 10, 83(5), 395 - 8 {Incidence and pathogens of nosocomial bacteremia in China}; Wu AH et al.; OBJECTIVE: To investigate the incidence and pathogens of nosocomial bacteremia (NB) in China . METHODS: The data of incidence and pathogens of NB reported from Jun 1998 to Dec 2001 by the hospitals of the Chinese nosocomial infections surveillance system (CNISS) were collected and analyzed . RESULTS: A total of 4882364 patients were surveyed and 2 371 cases of NB were reported . The incidence rate of NB was 48.6/100000, accounting for 1.3% of nosocomial infection . The incidence of bacteremia was higher in units of hematology, infectious disease, neonatology, and burns, and among the surgical patients with contaminated wound . 1757 strains of pathogens had been isolated, among which 585 (33.3%) were gram-positive bacteria, 962 (54.8%) gram-negative bacteria, 186 (10.6%) fungi, and 24 (1.4%) other pathogens . The most common pathogens were Escherichia coli (17.2%), Coagulase negative staphylococcus (CoNS 13.4%), fungi (10.6%), Klebsiella (9.1%), Staphylococcus aureus (8.1%), Pseudomonas aeruginosa (6.8%), and Enterobactor (6.3%) spp . CONCLUSION: Patients in unit of hematology, infectious diseases, burns and neonatology and surgical patients with class III incision have higher incidence of nosocomial bacteremia . Gram-negative bacteria were the prominent pathogens, the most common pathogens were E . coli, CoNS, fungi, Klebsiella, S . aureus, P . aeruginosa, and Enterobactor spp. Microb Drug Resist, 2003 Summer, 9(2), 161 - 5 Molecular epidemiology of extended-spectrum beta-lactamases produced by clinical isolates in a university hospital in Greece: detection of SHV-5 in Pseudomonas aeruginosa and prevalence of SHV-12; Neonakis IK et al.; To assess the nature and diversity of various types of SHV and TEM derivatives in our hospital a survey was conducted . Sixty-seven extended-spectrum beta-lactamases (ESBL)-producing nosocomial pathogens, isolated over a 12-month period, were analyzed by means of PCR and direct sequencing . SHV-5 was the predominant ESBL found in our region (38 strains) . Other less frequent variants included SHV-2 and SHV-12 with two and three isolates, respectively . For the first time, an outbreak of 11 Pseudomonas aeruginosa producing SHV-5 was encountered . All blaTEM-positive strains carried the non-ESBL TEM-1 . The incidence of non-SHV non-TEM ESBLs was remarkably high as almost one out of three isolates harbored such an ESBL . The epidemiological and clinical impact of these findings must be carefully investigated and interpreted. Infect Immun, 2003 Jul, 71(7), 4144 - 50 Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae; Rabin SD et al.; ExoU, a protein transported by the type III secretion system of Pseudomonas aeruginosa, is an important cytotoxin, though its mechanism of action is unclear . Here we show that the intracellular expression of ExoU is cytotoxic to Saccharomyces cerevisiae . Furthermore, internal amino- and carboxyl-terminal deletions confirmed that regions of ExoU previously shown to be essential for killing mammalian cells were also required for killing yeast cells . These findings indicate that S . cerevisiae is a useful model organism for the study of ExoU. Infect Immun, 2003 Jul, 71(7), 4059 - 66 The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa; Lau GW et al.; Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients . The molecular basis of the host-P . aeruginosa interaction and the effect of specific P . aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown . We examine interactions between P . aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P . aeruginosa human isolate PA14 . Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-kappa B-like transcription factors, allow increased P . aeruginosa infectivity within fly tissues . In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an I kappa B-like factor that inhibits the Toll signaling, reduce infectivity . Our finding that Dorsal activity is required to restrict P . aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity . Additionally, our results provide the basis for future studies into interactions between P . aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans. Infect Immun, 2003 Jul, 71(7), 3875 - 84 Construction and characterization of a Pseudomonas aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine; Theilacker C et al.; Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa . The mucoid exopolysaccharide (MEP) from P . aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection . MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P . aeruginosa infection . When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies . To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker . In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit . Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity . Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P . aeruginosa . Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies . Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate . However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate . Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P . aeruginosa in vitro . These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer. Infect Immun, 2003 Jul, 71(7), 3866 - 74 Modification of Pseudomonas aeruginosa interactions with corneal epithelial cells by human tear fluid; Fleiszig SM et al.; Both cytotoxic and invasive strains of Pseudomonas aeruginosa can damage corneal epithelial cells in vitro, but neither can infect healthy corneas in vivo . We tested the hypothesis that whole human tear fluid can protect corneal epithelia against P . aeruginosa virulence mechanisms . Cultured corneal epithelial cells were inoculated with 10(6) CFU of one of 10 strains of P . aeruginosa (five cytotoxic, five invasive)/ml with or without reflex tear fluid collected from the conjunctival sacs of human volunteers . Cytotoxicity was assessed by observation of trypan blue staining and measurement of lactate dehydrogenase release; invasion was quantified by using gentamicin survival assays . Tear fluid retarded growth of only 50% of the P . aeruginosa strains (three of five invasive strains, two of five cytotoxic strains) yet protected corneal cells against invasion by or cytotoxicity of 9 of 10 strains . The only strain resistant to the tear cytoprotective effects was susceptible to tear bacteriostatic activity . Dilution of tear fluid threefold significantly reduced cytoprotection, while bacteriostatic activity prevailed with dilutions beyond 100-fold . Sulfacetamide (1 mg/ml) with bacteriostatic activity matching that of tear fluid was less cytoprotective than tear fluid (80% protection with tear fluid, 48% with sulfacetamide) . Video microscopy revealed bacterial chain formation in both tear fluid and sulfacetamide, but tear fluid also blocked bacterial swimming motility . After prolonged tear contact, bacteria regained normal growth rates, swimming motility, and cytotoxic activity, suggesting a breakdown of protective tear factors . Boiled tear fluid lost bacteriostatic activity and effects on bacterial motility but retained cytoprotective function . These results suggest that human tear fluid can protect corneal epithelial cells against P . aeruginosa virulence mechanisms in a manner not dependent upon bacteriostatic activity or effects on bacterial motility . Whether overlapping tear film components are involved in these defense functions is to be determined. Am J Physiol Lung Cell Mol Physiol, 2003 Oct, 285(4), L847 - 53 Epub 2003 Jun 20. Clarithromycin inhibits overproduction of muc5ac core protein in murine model of diffuse panbronchiolitis; Kaneko Y et al.; Long-term treatment of macrolide antibiotics is considered an effective treatment for diffuse panbronchiolitis (DPB) . Although hypersecretion is a common feature of this disease, and it is known that macrolides inhibit mucin production, the mechanism of the effect on mucin production is unclear . The aim of our study was to determine the production of muc5ac core protein, a major core protein of mucin in airway secretion, and the effect of clarithromycin treatment on such production in a mouse model mimicking DPB . Alcian blue-periodic acid-Schiff-positive cells were detected in the lungs of Pseudomonas aeruginosa-infected mice . Western blots of these mice showed muc5ac glycoprotein at day 1 and increased progressively from day 4 to day 14 after inoculation of bacteria . Clarithromycin (10 mg . kg-1 . day-1 for 7 days) significantly reduced the muc5ac expression at both the mRNA and protein levels . To investigate the role of molecules upstream in muc5ac regulation, we examined the role of mitogen-activated protein kinase . Extracellular signal-regulated kinase 1/2 phosphorylation increased in the infected lung and decreased after treatment . Our results suggest that overproduction of muc5ac plays an important role in the pathogenesis of DPB and that clinical improvement following macrolide therapy seems to involve, at least in part, its inhibition of mucin overproduction, through modulation of intracellular signal transduction. J Hosp Infect, 2003 Jun, 54(2), 158 - 60 Emergence of nosocomial Pseudomonas aeruginosa colonization/infection in pregnant women with preterm premature rupture of membranes and in their neonates; Casetta A et al.; The epidemiology, risk factors, maternal and neonatal outcomes of nosocomial Pseudomonas aeruginosa acquisition in preterm premature rupture of membranes were analysed . Of 63 women receiving antibiotic prophylaxis with co-amoxiclav, 11 acquired P . aeruginosa vaginal carriage with a median delay of 15 days (6-42) i.e . an incidence of 8.94 per 1000 days of expectant management . Five neonates born to 11 positive mothers were colonized or infected, three of whom died of fulminant sepsis . The duration of antibiotic treatment and multiple pregnancy were identified as independent risk factors . The epidemiological investigation revealed a vertical transmission between mothers and neonates, and suggested selective pressure of antibiotic treatment. Org Lett, 2003 Jun 26, 5(13), 2215 - 7 Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade; Dorrestein PC et al.; The pyoverdine chromophore is formed in a reaction involving a two-electron oxidation, a conjugate addition, and a second two-electron oxidation . This oxidative cascade can be carried out with polyphenol oxidase (PPO), MnO(2), and cell-free extracts from Pseudomonas aeruginosa . {reaction: see text} Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8484 - 9 Epub 2003 Jun 18. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa; Wolfgang MC et al.; Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of causing a variety of life-threatening human infections . The genetic basis for preferential infection of certain immunocompromised patients or individuals with cystic fibrosis by P . aeruginosa is not understood . To establish whether variation in the genomic repertoire of P . aeruginosa strains can be associated with a particular type of infection, we used a whole-genome DNA microarray to determine the genome content of 18 strains isolated from the most common human infections and environmental sources . A remarkable conservation of genes including those encoding nearly all known virulence factors was observed . Phylogenetic analysis of strain-specific genes revealed no correlation between genome content and infection type . Clusters of strain-specific genes in the P . aeruginosa genome, termed variable segments, appear to be preferential sites for the integration of novel genetic material . A specialized cloning vector was developed for capture and analysis of these genomic segments . With this capture system a site associated with the strain-specific ExoU cytotoxin-encoding gene was interrogated and an 80-kb genomic island carrying exoU was identified . These studies demonstrate that P . aeruginosa strains possess a highly conserved genome that encodes genes important for survival in numerous environments and allows it to cause a variety of human infections . The acquisition of novel genetic material, such as the exoU genomic island, through horizontal gene transfer may enhance colonization and survival in different host environments. Vet Microbiol, 2003 Jul 17, 94(3), 237 - 43 Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis; Hammer AS et al.; Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI . A total of 212 isolates of P . aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia . Isolates from mink were separated into 34 distinct clones by PFGE subtyping . All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak . P . aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other . However, PFGE types of strains causing clusters of farm outbreaks changed from year to year . These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P . aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment. Shock, 2003 Jul, 20(1), 46 - 51 Cepharanthin, an alkaloid from Stephania cepharantha, inhibits increased pulmonary vascular permeability in an ovine model of sepsis; Murakami K et al.; Sepsis is a life-threatening event when it occurs in patients suffering from smoke inhalation injury . Pneumonia is one of the most frequent sources of infection in sepsis . Activated leukocytes likely play a role in the pathogenesis of sepsis . Cepharanthin is a biscoclaurine alkaloid that reportedly inhibits the activation of neutrophils . In this study, we investigated the effects of cephranthin on a post-smoke inhalation model of sepsis in sheep . Female sheep (n = 15) were surgically prepared for the study . After 5 days recovery from the operative procedures, tracheostomy was performed in all animals and 48 breaths of cotton smoke (<40 degrees C) were given via a modified bee smoker under halothane anesthesia . After smoke insufflation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into the airway using a bronchoscope . All of the animals were mechanically ventilated with 100% O(2) . Cepharanthin (1.3 mg/kg/h) was infused in five sheep continuously beginning 1 h after the insult and thereafter for the remainder of the 24-h study period . Control animals (n = 6) were treated with 5% dextrose as a vehicle control . Cepharanthin significantly attenuated changes in lung histology as well as in lung wet/dry weight ratio . An in vitro study revealed that cepharanthin inhibited the release of neutrophil elastase from isolated neutrophils stimulated with either formyl-methyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate with an IC(50) of 60 microM . Cepharanthin also inhibited the fMLP-induced increase in intracellular calcium levels of neutrophils . This result indicates cepharanthin inhibits protein kinase C or a more downstream signaling pathway in neutrophil activation . In conclusion, cepharanthin attenuates acute lung injury and septic shock after smoke inhalation in sheep. Diagn Microbiol Infect Dis, 2003 Jun, 46(2), 131 - 7 In vitro evaluation of the activity of two doses of Levofloxacin alone and in combination with other agents against Pseudomonas aeruginosa; Burgess DS et al.; P . aeruginosa is one of the most difficult to treat pathogens that generally requires combination therapy to prevent the development of resistance . This study evaluated the in vitro activity of two concentrations of levofloxacin (modeled for the 500 mg and 750 mg daily dose) in combination with ceftazidime, cefepime, piperacillin/tazobactam, imipenem, and tobramycin against P . aeruginosa . MICs and time-kill studies were performed against 12 non-duplicate clinical isolates of P . aeruginosa . The percent susceptible for levofloxacin, ceftazidime, cefepime, piperacillin/tazobactam, imipenem, and tobramycin were 67%, 58%, 58%, 67%, 75%, and 100%, respectively . Tobramycin was the most active single agent, killing and maintaining > or =99.9% killing over a 24 h period against all isolates . Levofloxacin 4 microg/mL(750 mg/day) alone reached 99.9% killing and maintain this killing over the time period more often than levofloxacin 2 microg/mL (500 mg/day) . No combination was antagonistic and all combinations with tobramycin were indifferent . Overall, levofloxacin 2 microg/mL plus a beta-lactam was synergistic (65%) more often than levofloxacin 4 microg/mL combinations (46%) . This was not unexpected due to the increased activity of levofloxacin 4 microg/mL . However, levofloxacin 4 microg/mL combinations maintained a > or =99.9% killing over the entire 24 h period more often than levofloxacin 2 microg/mL combinations (94% vs 83%) . The findings from this work suggest that levofloxacin 750 mg/day in combination with another agent active against P . aeruginosa may prove to be clinically beneficial and superior to combinations using lower doses of levofloxacin . In vivo studies are needed to evaluate the clinical significance of these findings. Isr Med Assoc J, 2003 May, 5(5), 329 - 32 Saliva secretion and oral flora in prolonged nasogastric tube-fed elderly patients; Leibovitz A et al.; BACKGROUND: In a previous study we showed that prolonged nasogastric tube feeding is associated with pathogenic oral flora . OBJECTIVE: To reexamine the impact of prolonged nasogastric tube feeding on the oral microbiota and to explore the salivary flow and composition in elderly patients in long-term care . METHODS: We compared a group of elderly patients fed by nasogastric tube with a control group of elderly patients in long-term care who are fed orally . Bacteriologic studies were performed by culturing samples from the oropharynx . Saliva studies included quantitative and biochemical analysis of basal and stimulated salivary flow . RESULTS: Bacteriologic studies performed in 90 patients revealed a significantly higher prevalence of gram-negative bacteria in nasogastric tube-fed patients (73% vs . 13%, P < 0.001) . It is emphasized that Pseudomonas aeruginosa and Klebsiella pneumoniae were commonly and exclusively isolated from the oral flora of the nasogastric tube-fed patients (P < 0.001, P < 0.05) . In the saliva studies performed on 23 nasogastric tube-fed and 21 control patients, basal and stimulated salivary flow was not significantly different in the two groups, however the ratio of stimulated to basal flow was reduced in the nasogastric tube-fed group (P < 0.05) . Significant differences were also found in the concentrations of sodium, amylase, phosphor and magnesium . Noteworthy was the concentration of uric acid, the main non-enzymatic antioxidant of saliva, which was significantly lower in nasogastric-tube fed patients (P < 0.002) . CONCLUSIONS: These findings suggest that prolonged nasogastric tube feeding is associated with pathologic colonization of the oroparynx and with alterations in the saliva that are related to the risk of aspiration pneumonia . Further research is called for, as well as a thorough revision of the existing oral cleansing procedures in these patients. Tex Heart Inst J, 2003, 30(2), 137 - 9 Pseudomonas aeruginosa pseudoaneurysm of the ascending aorta after coronary artery bypass graft surgery; Schmitt TM et al.; Mycotic pseudoaneurysms of the ascending aorta are rare in patients undergoing coronary artery bypass graft surgery and are usually caused by Staphylococcus aureus . We describe a patient with a mycotic pseudoaneurysm of the ascending aorta at the proximal vein graft anastomosis site after coronary artery bypass grafting . Cultures from the saphenous vein harvest site and from the aneurysm sac obtained intraoperatively during repair of the pseudoaneurysm grew Pseudomonas aeruginosa . Treatment included femorofemoral bypass and hypothermic circulatory arrest with in situ patch repair . The patient was given ceftazidime and gentamicin intravenously for 2 weeks, then ceftazidime alone for 6 weeks . Thereafter, he began taking ciprofloxacin orally for chronic suppression . He was doing well at 18-month follow-up. Biol Pharm Bull, 2003 Jun, 26(6), 902 - 4 Comparative study of protective effects of chitin, chitosan, and N-acetyl chitohexaose against Pseudomonas aeruginosa and Listeria monocytogenes infections in mice; Okawa Y et al.; We conducted a comparative study of the protective effects of chitin, chitosan, and N-acetyl chitohexaose (NACOS-6) against mice infected intravenously or intraperitoneally with Pseudomonas aeruginosa and Listeria monocytogenes . Mice pretreated with chitin, chitosan, and NACOS-6 showed resistance to intraperitoneal infections by both microbes . Only mice pretreated with chitin and chitosan showed resistance to intravenous infections by both microbes . The number, active oxygen generation, and myeloperoxidase activity of peritoneal exudate cells (PEC) in the chitin, chitosan, and NACOS-6-treated mice were greater than those of the untreated mice . Also, these PEC factors from mice pretreated with chitin and chitosan were greater than those from the NACOS-6-treated mice. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7889 - 94 Epub 2003 Jun 13. Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ; Cordell SC et al.; SulA halts cell division in Escherichia coli by binding to the major component of the division machinery FtsZ . We have solved the crystal structure of SulA alone and in complex with FtsZ from Pseudomonas aeruginosa . SulA is expressed when the SOS response is induced . This is a mechanism to inhibit cell division and repair DNA in the event of DNA damage . FtsZ is a tubulin-like protein that forms polymers, with the active-site GTPase split across two monomers . One monomer provides the GTP-binding site and the other, through its T7 loop nucleotide hydrolysis . Our structures show that SulA is a dimer, and that SulA inhibits cell division neither by binding the nucleotide-binding site nor by inducing conformational changes in FtsZ . Instead, SulA binds the T7 loop surface of FtsZ, opposite the nucleotide-binding site, blocking polymer formation . These findings explain why GTP hydrolysis and polymer turnover are required for SulA inhibition. J Biol Chem, 2003 Aug 29, 278(35), 32794 - 800 Epub 2003 Jun 13. Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins; Sun J et al.; Pseudomonas aeruginosa ExoT is a type III cytotoxin that functions as an anti-internalization factor with an N-terminal RhoGAP domain and a C-terminal ADP-ribosyltransferase domain . Although ExoT RhoGAP stimulates actin reorganization through the inactivation of Rho, Rac, and Cdc42, the function of the ADP-ribosylation domain is unknown . The present study characterized the mammalian proteins that are ADP-ribosylated by ExoT, using two-dimensional SDS-PAGE and matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) analysis . ExoT ADP-ribosylated two cytosolic proteins in cell lysates upon type III delivery into cultured HeLa cells . MALDI-TOF mass spectrometry analysis identified the two proteins as Crk-I and Crk-II that are Src homology 2-3 domains containing adaptor proteins, which mediate signal pathways involving focal adhesion and phagocytosis . ExoT ADP-ribosylated recombinant Crk-I at a rate similar to the ADP-ribosylation of soybean trypsin inhibitor by ExoS . ExoS did not ADP-ribosylate Crk-I . ADP-ribosylation of Crk-I may be responsible for the anti-phagocytosis phenotype elicited by ExoT in mammalian cells. Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 801 - 5 Streptomyces speibonae sp . nov., a novel streptomycete with blue substrate mycelium isolated from South African soil; Meyers PR et al.; An actinomycete with blue substrate mycelium was isolated from a soil sample in Cape Town, South Africa, and designated strain PK-Blue(T) . The colour of the substrate mycelium was not sensitive to changes in pH . The organism produced hairy spores in Spirales-type spore chains . Chemical taxonomy indicated that it belonged to the genus Streptomyces . Strain PK-Blue(T) produced no diffusible pigments other than melanin, grew at 45 degrees C, did not degrade adenine and exhibited no antibacterial activity against Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa . Analysis of its 16S rRNA gene sequence and the results of physiological tests showed that strain PK-Blue(T) (= DSM 41797(T) = ATCC BAA-411(T)) represents the type strain of a novel species of Streptomyces, for which the name Streptomyces speibonae sp . nov . is proposed. Indian J Med Res, 2002 Dec, 116, 264 - 7 A preliminary study on metallo-beta-lactamase producing Pseudomonas aeruginosa in hospitalized patients; Navaneeth BV et al.; Metallo beta-lactamase (MBL) producing Pseudomonas aeruginosa is an emerging threat and a cause of concern for the physicians treating such infections . The present study was undertaken to know the resistance pattern of P . aeruginosa to beta-lactamase inhibitors and carbapenems, and to detect the presence of MBL among resistant isolates to both groups of antibiotics . Between June-November 2001, 50 P . aeruginosa isolates from clinical specimens were tested for susceptibility to beta-lactamase inhibitors and carbapenems by Kirby-Bauer disc diffusion method . Isolates resistant to both groups of antibiotics were screened for the presence of MBLs by disc diffusion method using 2-mercaptoethanol . Of the 50 isolates, 6 (12%) were resistant to both beta-lactamase inhibitors and carbapenems . All 6 isolates were MBL producers were resistant to all the antibiotics tested . Resistance to piperacillin-tazobactam, cefoperazone-sulbactam and ticarcillin-clavulanic acid was 12, 20 and 36 per cent respectively . Resistance of 12 per cent each was noted to imipenem and meropenem respectively . This is to the best of our knowledge the first report of MBL producing P . aeruginosa from India and suggests the need for early detection, notification and control of spread. J Antimicrob Chemother, 2003 Jul, 52(1), 116 - 9 Epub 2003 Jun 12. Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program; Walsh TR et al.; As part of the SENTRY Antimicrobial Surveillance Program, an imipenem-resistant Pseudomonas aeruginosa strain (81-11963A) was isolated from the blood culture of a female neonate institutionalized at the local children's hospital in Warsaw, Poland . Cloning of an imipenem resistance determinant revealed it to be a VIM-2 metallo-beta-lactamase, but sequence analysis of DNA adjacent to blaVIM-2 revealed it to have a unique gene context . Downstream of the blaVIM-2 gene resides an aacA4 gene encoding the AAC(6')-Ib aminoglycoside acetyltransferase . The integron containing blaVIM-2 shows high similarity to that reported from In58 in France but was novel in that it possessed a gene cassette with a 59 truncated base element only 19 base pairs (bp) long, consisting of a conserved core site and an inverse core site separated by only 5 bp . This appears to be the first report of a metallo-beta-lactamase gene arising from a pathogenic strain in Eastern Europe. EMBO J, 2003 Jun 16, 22(12), 2959 - 69 The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU; Sato H et al.; Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system . Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections . To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae . ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype . Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU . The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences . Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity . ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids . ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid . Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract . From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors. Hum Gene Ther, 2003 May 20, 14(8), 729 - 47 Safety and efficiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo; Johnson LG et al.; We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability . Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after lumenal application of vehicle, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), sodium caprate (C10), or sodium laurate (C12) . Lung toxicity was assessed after tracheobronchial instillation to murine airways and the relative ability of these agents to enhance in vivo adenoviral gene transfer was evaluated . Lumenal C12 increased LDH release in vitro, but C10 and EGTA did not . Increased levels of interleukin 8 (IL-8) were secreted from EGTA-pretreated cystic fibrosis HAE cells after apical application of Pseudomonas aeruginosa (10(8) CFU/ml), whereas IL-8 secretion from C10- and C12-pretreated cells was not different from controls . In vivo toxicity studies demonstrated no effect of EGTA, C10, or C12 on weight gain, lung edema, or bronchoalveolar lavage fluid (BALF) albumin . EGTA increased BALF cell counts, neutrophils, and murine (m) macrophage inflammatory protein 2, mKC, mIL-6, and mIL-1 beta levels . C10 had no effect on BALF cell counts or LDH, but increased murine tumor necrosis factor alpha . C12 increased BALF LDH, neutrophils, and mIL-6 levels . Histopathological analysis revealed mild focal lung inflammation more frequently in the EGTA, C10, and C12 groups than in vehicle controls, with greater intensity in the C12 group relative to the other groups . C10 and C12 also increased airway responsiveness to methacholine challenge compared with control and EGTA groups . Adenoviral gene transfer to murine trachea in vivo was enhanced more efficiently by C10 than by C12 or EGTA . Thus, the different toxicities may permit the selection of agents that enhance gene transfer with minimal adverse effects. J Biol Chem, 2003 Aug 29, 278(35), 32733 - 43 Epub 2003 Jun 10. Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa . PlcH is a multifunctional enzyme; Luberto C et al.; Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide . Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified . Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa . SM synthase-like activity was found secreted in the culture medium of P . aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli . From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns . After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa . Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity . We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P . aeruginosa . First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH . Second, purified PlcH showed SM synthase activity . Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P . aeruginosa . Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity . Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide . This SM synthase did not have reverse transferase activity . In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase. J Infect, 2003 May, 46(4), 207 - 14 Quorum sensing and its relevance to infectious diseases; Donabedian H; Quorum sensing allows bacteria to detect the density of their own species and alter their metabolism to take advantage of this density . Quorum sensing is used by a wide variety of bacteria including human pathogens . Quorum sensing genes are important for the pathogenic potential of Pseudomonas aeruginosa and Staphylococcus aureus, as well as other invasive bacteria . An understanding of quorum sensing may lead to new therapeutic strategies. Res Microbiol, 2003 May, 154(4), 269 - 75 Myoviridae bacteriophages of Pseudomonas aeruginosa: a long and complex evolutionary pathway; Krylov V et al.; Recently we have accomplished the entire DNA sequence of bacteriophage phiKZ, a giant virus infecting Pseudomonas aeruginosa . The 280334-bp of phiKZ genome is a linear, circularly permutated and terminally redundant, AT-rich dsDNA molecule that contains no sites for NotI, PstI, SacI, SmaI, XhoI and XmaIII endonucleases . Limited homology to other bacteriophages on the DNA and protein levels indicated that phiKZ represents a distinct branch of the Myoviridae family . In this work, we analyzed a group of six P . aeruginosa phages (Lin68, Lin21, PTB80, NN, EL, and RU), which are morphologically similar to phiKZ, have similar genome size and low G+C content . All phages have a broad host range among P . aeruginosa strains, and they are resistant to the inhibitory action of many P . aeruginosa plasmids . The analysis of the genomic DNA by restriction enzymes and DNA-DNA hybridization shows that phages are representative of three phiKZ-like species: phiKZ-type (phiKZ, Lin21, NN and PTB80), EL-type (EL and RU) and Lin68 which has a shorter tail than other phages . Except for related phages EL and RU, all phiKZ-like phages have identical N-terminal amino acid sequences of the major capsid protein . Random genome sequencing shows that the EL and RU phages have no homology to the phiKZ-like phages on DNA level . We propose that the phiKZ, Lin21, NN, PTB80 and Lin68 phages can be included in a new phiKZ genus, and that the EL and RU phages belong to a separate genus within the Myoviridae family . Based on the resistance to many restriction enzymes and the transduction ability, there are indications that over the long pathway of evolution, the phiKZ-like phages probably inherited the capacity to infect different bacterial species. Infect Genet Evol, 2002 May, 1(3), 237 - 42 Whole genome fingerprinting and genotyping of multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from endophthalmitis patients in India; Ahmed N et al.; Genome sequence-based fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for fingerprinting and subtyping 42 multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from post-surgical endophthalmitis . The FAFLP profiles derived from EcoRI/MseI restricted fragments differentiated clinical isolates and were found to be extremely reproducible . Seventeen different amplification patterns (amplitypes) were observed for all the 42 isolates from 11 different patients . Also, we were able to genotype the isolates based on the differential amplification of a total of 31 FAFLP markers representing genomic fragments from the P . aeruginosa chromosome . This data appears to provide clues to the genetic diversity of endopthalmitis associated strains and could be converted into digitised fingerprints suitable for electronic manipulations and archiving. Arch Bronconeumol, 2003 Jun, 39(6), 274 - 82 {Second consensus report on the use of antimicrobial agents in exacerbations of chronic obstructive pulmonary disease}; Alvarez F et al.; Aware of the importance of chronic obstructive pulmonary disease (COPD), a panel of experts belonging to the Spanish Society of Respiratory Medicine and Thoracic Surgery (SEPAR), the Spanish Society of Chemotherapy (SEQ) and the Spanish Society of Family and Community Medicine (SEMFyC) issued a statement of consensus in 2000 to serve as the basis for adequate antibiotic control of the disease . Three years later, in accordance with significant scientific progress made in this area, the statement has been thoroughly revised . The new paper in fact constitutes a second consensus statement on the use of antibiotics in COPD exacerbations . When several scientific associations expressed interest in joining the project and contributing to it the Spanish Society of Emergency Medicine (SEMES), the Spanish Society of General Medicine (SEMG) and the Spanish Society of Rural and General Medicine (SEMERGEN) their incorporation led SEPAR and SEMFyC to change the structure of the statement and certain aspects of its content . Additionally, a new group of antibiotics, the ketolides, has joined the therapeutic arsenal . Telithromycin, the single representative of the group for the moment, can be considered not only an alternative treatment but even the drug of choice in certain clinical settings that are analyzed in the new statement . Those developments, along with others, such as the increasingly recognized action of levofloxacin against Pseudomonas aeruginosa and the steady action of amoxicillin with clavulanic acid when administered at recommended doses every 8 hours, provide new antimicrobial therapeutic protocols for COPD . Finally, the statement includes a scientific analysis of other groups of antimicrobial agents (macrolides, oral cephalosporins, etc.) and guidelines for both primary care physicians and specialists to follow when prescribing them. J Indian Med Assoc, 2002 Nov, 100(11), 664 - 6 Contact lens wear and microbial keratitis; Ahuja M; Common types of contact lens are hard, rigid gas-permeable or soft lenses . Most lenses are worn on a daily basis . Cosmetic lenses are worn for non-medical indications . Microbial keratitis, a rare but most significant complication is discussed in this article . Pseudomonas aeruginosa and staphylococci are the most common organisms cause infective keratitis . Fungi and acanthamoeba are also responsible . The causes of increased susceptibility to infection are poor lens hygiene, adhesion of bacteria to lens surface and hypoxia . Preventing measures to be taken while wearing contact lens are discussed in a nutshell . Ocular pain, conjunctival infection, photophobia, epiphora and reduced vision are some of the symptoms of corneal infection . Diagnostic laboratory investigations are to be carried out immediately when a microbial corneal ulcer is suspected . Acanthamoeba can be cultured from corneal scrapes . Immunologically based fluorescein labelling techniques appear to be more sensitive than simple staining . The treatment consists of medical and surgical intervention . Corneal thinning, descemetocele formation and perforation are possible complications. Eur Respir J, 2003 Jun, 21(6), 1040 - 5 Induction of type-IIA secretory phospholipase A2 in animal models of acute lung injury; Attalah HL et al.; The aim of this study was to evaluate the presence of type-II secretory phospholipase A2 (sPLA2-IIA) in alveolar space and its possible role in the destruction of surfactant in three rat models of acute lung injury . Alveolar instillation of either lipopolysaccaride or live Pseudomonas aeruginosa resulted in a significant increase in lung oedema and in a decrease in static compliance of the respiratory system together with alveolar-neutrophil influx as compared with healthy control rats . The upregulation of messenger ribonucleic acid and sPLA2-IIA by the lung was evident . This was associated with surfactant degradation and a decrease in large:small ratio of surfactant aggregates in bacteria-instilled rats . A negative correlation between compliance and sPLA2-IIA activity in bronchoalveolar lavage fluid was shown . By contrast, during alpha naphthylthiourea-induced injury, neither alveolar-neutrophil influx nor increase in sPLA2-IIA activity was observed . Additional experiments in rats treated with a specific inhibitor of type-II secretory phospholipase A2 activity (3 acetamine-1-benzyl-2 ethylindolyl-5 oxy; propane phosphonic acid (LY311727)) demonstrated no improvement in physiological parameters despite a biochemical effect, suggesting that its activity is only one of the multiple factors involved in the pathophysiology of lung injury. Eur Respir J, 2003 Jun, 21(6), 932 - 8 Pseudomonas aeruginosa adherence to human basement membrane collagen in vitro; Tsang KW et al.; The mechanisms for Pseudomonas aeruginosa colonisation in the airways of patients with bronchiectasis and cystic fibrosis are poorly understood . P . aeruginosa could evade mucociliary clearance by adhering to the basement membrane at areas denuded of intact respiratory epithelium . The authors have developed an in vitro model to study P . aeruginosa adherence to human basement membrane type-IV collagen by using scanning electron microscopy . P . aeruginosa adherence density was determined as the number of P . aeruginosa per 20 microscope fields (2,000x) to log inocular size after incubation at 37 degrees C for 45 min . The presence of phytohaemagglutinin (PHA)-E, which binds specifically to D-galactose-beta1-4-D-N-acetylglucosamine, significantly reduced P . aeruginosa adherence density compared with control . The presence of heparin and calcium also significantly reduced P . aeruginosa adherence density . P . aeruginosa adherence was not affected by the presence of proline, trans-hydroxyproline, glycine, galactose, N-acetylneuraminic acid, N-acetylglucosamine or Arachis hypogea . Pseudomonas aeruginosa adherence probably acts via recognition of the D-galactose-beta1-4-D-N-acetylglucosamine sequence on type-IV collagen and this process could be inhibited by heparin and calcium . As persistent Pseudomonas aeruginosa colonisation is detrimental to patients with cystic fibrosis and bronchiectasis and there is currently no effective treatment for its eradication, these results could lead to novel therapy for persistent Pseudomonas aeruginosa infection. Biochemistry, 2003 Jun 17, 42(23), 7238 - 44 Function of the MexB efflux-transporter divided into two halves; Eda S et al.; The MexA-MexB-OprM efflux pump exports structurally and functionally diverse xenobiotics and confers multi-drug resistance on Pseudomonas aeruginosa cells . The MexB transporter traverses the inner membrane twelve times, bears two large periplasmic domains and has two homologous tandem repeats . To test whether two homologous halves of MexB function independently or interdependently, the protein was divided medially into two halves, each consisting of six amino- and carboxyl-proximal transmembrane segments . When two halves of MexB were coexpressed from independent open reading frames, the cells lacking chromosomal mexB exhibited restored antibiotic resistance at a level close to that in the cells producing a full-length MexB . In contrast, MexB protein containing either an amino- or carboxyl-half fragment failed to transport antibiotics . To test whether the amino- and carboxyl-proximal halves were present in a complex, we purified the histidine-tagged carboxyl-proximal half molecule using nickel-chelate chromatography from the cells that coexpressed two halves . The results showed that the nonhistidine-tagged amino-proximal half was co-purified with the carboxyl-proximal half, thereby indicating that the amino-proximal half fragment was tightly associated with the carboxyl-proximal half molecule . These findings suggest that the presence of both amino- and carboxyl-halves of MexB in a complex is essential for transport activity. Crit Care Med, 2003 Jun, 31(6), 1630 - 7 Sepsis from Pseudomonas aeruginosa pneumonia decreases intestinal proliferation and induces gut epithelial cell cycle arrest; Coopersmith CM et al.; OBJECTIVES: To evaluate whether the up-regulation in sepsis-induced gut epithelial apoptosis is balanced by an increase in intestinal proliferation and to assess mechanisms affecting the gut's regenerative response to overwhelming infection . DESIGN: Prospective, randomized, controlled study . SETTING: Animal laboratory in a university medical center . INTERVENTIONS: Mice were subjected to intratracheal injection of Pseudomonas aeruginosa and killed between 1.5 and 24 hrs after induction of pneumonia-induced sepsis to assess for gut epithelial proliferation and cell division and for apoptosis . Animals were compared with sham-operation controls, septic transgenic mice that overexpress Bcl-2 throughout their small intestinal epithelium, and septic p53-/- mice . MEASUREMENTS AND MAIN RESULTS: Proliferation and cell division were assessed by measuring S-phase and M-phase cells in intestinal crypts . The number of S-phase cells showed a progressive decline at all time points measured, with a 5-fold decrease in proliferation between control animals and septic mice 24 hrs after intratracheal injection of pathogenic bacteria (p <.0001) . In contrast, cells in M-phase remained constant for the first 12 hrs after the onset of sepsis, but increased nearly 50% at 24 hrs after instillation of P . aeruginosa (p <.005) . Both the decrease in S-phase cells and the increase in M-phase cells were partially suppressible in Bcl-2 overexpressors, but cellular proliferation and division were similar between septic p53-/- and p53+/+ mice . Crypt apoptosis was increased at all time points, with maximal death occurring between 12 and 24 hrs . CONCLUSIONS: Sepsis from P . aeruginosa pneumonia induces a p53-independent decrease in gut epithelial proliferation . Despite an increase in sepsis-induced intestinal apoptosis, there is no compensatory increase in intestinal epithelial proliferation, and there is evidence of a cell cycle block with an accumulation of cells in M-phase . Decreasing gut apoptosis by overexpression of Bcl-2 is associated with a partial reversal of the effect of sepsis on the cell cycle. Int J Antimicrob Agents, 2003 Jun, 21(6), 562 - 7 Target site bacterial killing of cefpirome and fosfomycin in critically ill patients; Zeitlinger MA et al.; We employed an in-vivo pharmacokinetic/in-vitro pharmacodynamic method to simulate bacterial killing in plasma and the interstitium of skeletal muscle tissue after intravenous administration of 2 g of cefpirome and 8 g of fosfomycin alone and in combination to patients with sepsis . Interstitial antimicrobial concentrations were determined by use of in-vivo microdialysis . CFU/ml of Staphylococcus aureus (ATCC 29213) and Pseudomonas aeruginosa (clinical isolate) decreased by approximately 2log(10) for plasma and muscle tissue 6 h after cefpirome and fosfomycin administration compared with the baseline, respectively . The simulation of plasma and tissue pharmacokinetics for the combined administration of these antibiotics resulted in complete eradication of S . aureus within 5 h after drug exposure . No bacterial re-growth occurred in any of the simulations within 6 h . The in-vitro simulation of in-vivo plasma and tissue pharmacokinetics of cefpirome and fosfomycin has shown that both antimicrobial agents kill S . aureus and P . aeruginosa strains effectively after single dose administration . This effect was most pronounced by the combined use of these antimicrobial agents . Therefore, this data corroborates antimicrobial strategies of simultaneous administration of cefpirome and fosfomycin in patients with severe soft tissue infection. Biotechnol Prog, 2003 May-Jun, 19(3), 1038 - 44 Ultrasound increases the rate of bacterial cell growth; Pitt WG et al.; Ultrasound was employed to increase the growth rate of bacterial cells attached to surfaces . Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli cells adhered to and grew on a polyethylene surface in the presence of ultrasound . It was found that low-frequency ultrasound (70 kHz) of low acoustic intensity (<2 W/cm(2)) increased the growth rate of the cells compared to growth without ultrasound . However, at high intensity levels, cells were partially removed from the surface . Ultrasound also enhanced planktonic growth of S . epidermidis and other planktonic bacteria . It is hypothesized that ultrasound increases the rate of transport of oxygen and nutrients to the cells and increases the rate of transport of waste products away from the cells, thus enhancing their growth. Appl Opt, 2003 Jun 1, 42(16), 3005 - 8 Absorption coefficient imaging by near-field scanning optical microscopy in bacteria; de Paula AM et al.; We present a method for obtaining a position-dependent absorption coefficient from near-field scanning optical transmission microscopy . We show that the optical transmission intensity can be combined with the topography, resulting into an absorption coefficient that simplifies the analysis of different materials within a sample . The method is tested with the dye rhodamine 6G, and we show some analysis in biological samples such as bacteria KIebsiella pneumoniae and Pseudomonas aeruginosa . The calculated absorption coefficient images show important details of the bacteria, in particular for P . aeruginosa, in which membrane vesicles are clearly seen. Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 310 - 7 Seven GC-rich microbial genomes adopt similar codon usage patterns regardless of their phylogenetic lineages; Chen LL et al.; Seven GC-rich (group I) and three AT-rich (group II) microbial genomes are analyzed in this paper . The seven microbes in group I belong to different phylogenetic lineages, even different domains of life . The common feature is that they are highly GC-rich organisms, with more than 60% genomic GC content . Group II includes three bacteria, which belong to the same subdivision as Pseudomonas aeruginosa in group I . The genomic GC content of the three bacteria is in the range of 26-50% . It is shown that although the phylogenetic lineages of the organisms in group I are remote, the common feature of highly genomic GC content forces them to adopt similar codon usage patterns, which constitutes the basis of an algorithm using a set of universal parameters to recognize known genes in the seven genomes . The common codon usage pattern of function known genes in the seven genomes is GGS type, where G, G, and S are the bases of G, non-G, and G/C, respectively . On the contrary, although the phylogenetic lineages of the three bacteria in group II are quite close, the codon usage patterns of function known genes in these genomes are obviously distinct . There are no universal parameters to identify known genes in the three genomes in group II . It can be deduced that the genomic GC content is more important than phylogenetic lineage in gene recognition programs . We hope that the work might be useful for understanding the common characteristics in the organization of microbial genomes. Spine, 2003 Jun 1, 28(11), E209 - 11 Distant skip level discitis and vertebral osteomyelitis after caudal epidural injection: a case report of a rare complication of epidural injections; Yue WM et al.; STUDY DESIGN: A case report of distant discitis and vertebral osteomyelitis involving skip levels after caudal epidural steroid injection . OBJECTIVES: To report and investigate the occurrence of distant infective discitis and vertebral osteomyelitis involving skip levels after epidural injection . SUMMARY OF THE BACKGROUND DATA: Distant discitis and vertebral osteomyelitis is a serious but rare complication after epidural injection . A case involving skip levels and without the occurrence of epidural abscess formation has apparently not been previously reported in the literature . METHODS: An elderly woman presenting with clinical, radiologic, and magnetic resonance imaging evidence of spinal canal stenosis involving L3/4 and L4/5 levels and degenerative spondylolisthesis of the L4/5 level was given an epidural injection of steroids and lignocaine via the caudal route . A month later, she presented with worsened low back pain, elevated serum acute phase reactants, and plain radiographic evidence of L4/5 infective discitis . Magnetic resonance imaging and microbiologic examination of computed tomographically guided biopsy specimens confirmed infective discitis involving L2/3 and L4/5 intervertebral levels, together with adjacent vertebral osteomyelitis . RESULTS: The patient was successfully treated with antibiotics targeted at Pseudomonas aeruginosa, which was isolated in the culture of the biopsy specimens . Follow-up improvements in the clinical condition, serum acute phase reactants levels, radiographs, and magnetic resonance imaging were noted . CONCLUSIONS: Distant discitis and vertebral osteomyelitis involving skip levels and without the occurrence of epidural abscess formation is a serious but rare complication after epidural injection. J Microbiol Methods, 2003 Aug, 54(2), 239 - 47 Measurement of chlorite dismutase activities in perchlorate respiring bacteria; Xu J et al.; Chlorite dismutase (CD) catalyzes the disproportionation of chlorite to chloride (ClO(2)(-)-->Cl(-)+O(2)) and is present in bacteria capable of cell respiration using perchlorate or chlorate . The activity of this enzyme has previously been measured by monitoring oxygen evolution using a Clark-type dissolved oxygen (DO) probe . We demonstrate here, using two other methods to measure CD activity (a chloride-specific electrode and ion chromatography (IC)) via chloride production, that the DO probe method underestimates dismutation rates . Of the three methods, the chloride probe was the easiest to use and did not require extensive sample handling or post-experimental analysis . Using the chloride electrode method, we determined whole cell rate constants (V(max)=64 U/mg DW, K(m)=0.17 mM) for the chlorate-grown suspensions of Dechlorosoma sp . strain KJ . We compared the CD activities of strain KJ at a fixed chlorite concentration (0.6 mM) to four other perchlorate respiring bacteria (PRB), and to one non-PRB (Pseudomonas aeruginosa) . Chlorate-grown cultures of the five PRB strains had CD activities ranging from 25 to 50 U/mg of cell dry weight (DW), while aerobically grown cultures of the PRB had much lower CD activities (0.5-4 U/mg DW) . To our knowledge, this is the first systematic comparison of the different methods to measure CD activities, and the first comparison of CD activities of different PRBs. Pathol Biol (Paris), 2003 Apr, 51(3), 135 - 42 {Evaluation of a E-test method to detect bactericidy of beta lactam-aminoglycoside associations against Pseudomonas aeruginosa isolates from cystic fibrosis}; Massoni-Cristante S et al.; BACKGROUND: Pulmonary infectious exacerbations with Pseudomonas aeruginosa are the major problem for patients with cystic fibrosis . Emergence of multi-resistant mucoid strains leads to complicate the choice of antibiotherapy . Therefore, synergic and bactericidal treatment must be used . Then, it is interesting to estimate the bactericidal activity of antibiotics associations in order to optimise the treatment . The aim of this work is to describe a new method of bactericidal antibiotics combinations by superimposing 2 E-test strips and to compare results with those obtained with a broth bactericidal method chosen as reference method . OBSERVATIONS: Twenty strains of P . aeruginosa (13 mucoid and 7 non mucoid) were selected from expectorants of cystic fibrosis children . Four antibiotics combinations were tested (ceftazidime/tobramycine, cefepime/tobramycine, ceftazidime/amikacine, cefepime/amikacine) . Two antibiotics combinations by superimposing E-test strips techniques were used: maximal concentration on maximal concentration (C(max)/C(max)) and minimal inhibitory concentration on minimal inhibitory concentration (MIC/MIC) . The comparison of results between killing curves and superposition of E-test strips (C(max)/C(max)) show 88% agreements, 4% major discrepancies specially with mucoid strains and 8% minor discrepancies . The Cmax/Cmax method seems to give better results than MIC/MIC method . CONCLUSION: The superposition of E-test strips method is an attractive method: it is rapid, easy to use and well correlated to broth bactericidal method. Trends Microbiol, 2003 May, 11(5), 195 - 200 Genomics of pyoverdine-mediated iron uptake in pseudomonads; Ravel J et al.; Pyoverdines (PVDs) are complex siderophores produced by members of the fluorescent Pseudomonas . They comprise a dihydroxyquinoline fluorescent chromophore joined to a peptide of remarkably variable length and composition . In Pseudomonas aeruginosa, PVDs also function as signal molecules for the production of virulence factors . Genes responsible for the biosynthesis, excretion, uptake and regulation of these high-affinity siderophores are located either at a single locus or at up to three different loci in the genomes of the four pseudomonads analyzed . The peptide backbone of PVD is assembled by non-ribosomal peptide synthetases (NRPSs) and modified by accessory enzymes in the cytoplasm, and probably the periplasm . Regulation of PVD production and uptake depends on two extracytoplasmic sigma factors (ECF-sigmas), PvdS and FpvI, together with one anti-sigma, FpvR. Thorax, 2003 Jun, 58(6), 525 - 7 Identification of airborne dissemination of epidemic multiresistant strains of Pseudomonas aeruginosa at a CF centre during a cross infection outbreak; Jones AM et al.; BACKGROUND: Chronic Pseudomonas aeruginosa infection is a major cause of morbidity and mortality for individuals with cystic fibrosis (CF) . P aeruginosa cross infection outbreaks have recently been reported at CF holiday camps and specialist centres . The mechanism of cross infection is unknown . A study was performed to look for the presence of epidemic strains of P aeruginosa in the environment of a CF centre during a cross infection outbreak and to examine their potential modes of spread between patients . METHODS: Microbiological sampling of the environment of the CF facility was performed, including room air sampling . Individual P aeruginosa strains were identified by bacteri