Curr Opin Otolaryngol Head Neck Surg, 2005 Feb, 13(1), 60 - 6 Pediatric sinusitis: when do we operate? Lieser JD, Derkay CS. PURPOSE OF REVIEW: The role of surgery in the treatment of pediatric sinusitis is still in evolution . This review of recent literature highlights developments in the study of pediatric sinusitis, particularly as it pertains to surgical intervention . RECENT FINDINGS: There is growing support in the literature for adenoidectomy as a first-line surgical intervention for chronic rhinosinusitis in children when maximal medical management fails . Maxillary sinus aspiration or middle meatal culture can be performed at the same sitting to facilitate directed antibiotic therapy . Intravenous antibiotics seem to be a promising alternative to functional endoscopic sinus surgery (FESS), especially in younger children . Current literature continues to support FESS as a safe and effective treatment for pediatric sinus disease . Previous notions that FESS may inhibit midfacial growth have been challenged by several recent studies . There is no clear consensus regarding timing of FESS for chronic rhinosinusitis . However, the current literature seems to support FESS when maximal medical therapy, adenoidectomy, and culture-directed systemic antibiotics have all failed with persistence of sinonasal disease, when anatomic abnormalities predispose to chronic rhinosinusitis by obstructing normal sinonasal drainage pathways, in sinonasal polyposis to facilitate application of topical steroids, as an adjunct to desensitization in aspirin-sensitive patients, when orbital or intracranial complications of sinonasal disease occur, and in selected cystic fibrosis patients to improve quality of life and facilitate application of topical antibiotics with activity against Pseudomonas aeruginosa . SUMMARY: Although the current literature lends some additional clarity to the indications for surgical intervention in pediatric chronic rhinosinusitis, additional research is still needed to elucidate appropriate timing for surgery and more specific indications. Biophys J . 2005 Jan 14; {Epub ahead of print} The conversion of active to latent plasminogen activator inhibitor-1 is an energetically silent event; Boudier C et al.; ABSTRACT Plasminogen activator inhibitor-1 (PAI-1) is a proteinase inhibitor, which plays a key role in the regulation of fibrinolysis . It belongs to the serpins, a family of proteins that behave either as proteinase inhibitors or proteinase substrates, both reactions involving limited proteolysis of the reactive center loop and insertion of part of this loop into beta-sheet A . Titration calorimetry shows that the inhibition of tissue-type plasminogen and pancreatic trypsin are exothermic reactions with DeltaH = -20.3, and -22.5 kcal.mol-1, respectively . The Pseudomonas aeruginosa elastase-catalyzed reactive center loop cleavage and inactivation of the inhibitor is also exothermic (DeltaH = -38.9 kcal.mol-1) . The bacterial elastase also hydrolyses peptide-bound PAI-1 in which acetyl-TVASSSTA, the octapeptide corresponding to the P14-P7 sequence of the reactive center loop is inserted into beta-sheet A of the serpin with DeltaH = -4.0 kcal.mol-1 . In contrast, DeltaH = 0 for the spontaneous conversion of the metastable active PAI-1 molecule into its thermodynamically stable inactive (latent) conformer although this conversion also involves loop/sheet insertion . We conclude that the active to latent transition of PAI-1 is an entirely entropy-driven phenomenon. J Surg Res, 2005 Jan, 123(1), 109 - 17 Protamine sulfate reduces the susceptibility of thermally injured mice to Pseudomonas aeruginosa infection(1); Haynes A 3rd et al.; BACKGROUND: In this study, we investigated the ability of protamine sulfate, at sub-bactericidal dosing, to interfere with the in vivo virulence of Pseudomonas aeruginosa (PAO1) during burn wound infection . MATERIALS AND METHODS: The study was conducted using the murine model of thermal injury . Preliminary experiments determined a protocol for administration of protamine sulfate that had no in vivo bactericidal effects . Based on this, the effect of local injection of protamine sulfate on the in vivo virulence of PAO1 was assessed using these parameters: (1) the percent mortality among PAO1-infected, thermally injured mice; (2) the local proliferation and spread of PAO1 within the infected burned tissue; (3) the systemic spread of PAO1 within thermally injured/infected mice; and (4) the local cytokine response elicited by PAO1 thermally injured/infected mice . RESULTS: Injection of protamine sulfate into the thermally injured tissue of PAO1-infected/thermally injured mice significantly decreased the percent mortality and inhibited the systemic dissemination of PAO1 microorganisms to the liver and spleen . It had no effect, however, on the ability of the bacteria to proliferate and spread within the thermally injured tissue . It also was determined that protamine sulfate was ineffective at preventing mouse death at the dose administered if injected intramuscularly instead of directly into burned tissue . Protamine sulfate reduced the expression of the proinflammatory cytokines IL-6 and LIF in the injured/infected tissue . Heparan sulfate given in conjunction with protamine sulfate returned mortality levels to those of untreated mice . CONCLUSIONS: Our results suggest that: (1) local injection of sub-bactericidal doses of protamine sulfate reduces the virulence of P . aeruginosa; (2) this effect is due to interference with the systemic rather than local spread of P . aeruginosa; and (3) local application of protamine sulfate may have potential as supportive therapy for prevention of systemic P . aeruginosa infection in severely burned patients. Ther Umsch, 2004 Dec, 61(12), 715 - 9 {An auto-iatrogenic disease}; Reinhart WH; A 55-year-old practitioner from an island in the northern sea felt an increasing hypersensitivity of his entire body to various ambient and nutritional allergens and toxics . He started to treat himself with increasing doses of glucocorticoids and moved to a southern climate in Lanzarote and later on to the Swiss mountains in the grisons . On admission to our hospital in December he was in a disastrous psychotic condition, trying to cool down his body by laying naked on his bed at ambient temperatures around the freezing point . He had consumed on average 250 mg prednisone daily over weeks . As we found out later his personal assistant travelling with him was giving him glucocorticoids through the infusion during his hospital stay . He developed a necrotizing septic phlebitis at the infusion site followed by a Pseudomonas aeruginosa sepsis with fatal multiorgan failure . This case illustrates the dangers of self-treatment by doctors and the difficulties in treating a physician. J Antimicrob Chemother . 2005 Jan 13; {Epub ahead of print} Is there a pharmacodynamic need for the use of continuous versus intermittent infusion with ceftazidime against Pseudomonas aeruginosa? An in vitro pharmacodynamic model; Alou L et al.; In order to explore the pharmacodynamic need for continuous versus intermittent (three times a day) administration of ceftazidime in critically ill patients, a pharmacokinetic computerized device was used to simulate concentrations of ceftazidime in human serum after 6 g/day . Efficacy was measured as the capability of simulated concentrations over time to reduce initial inoculum against four strains of Pseudomonas aeruginosa . MICs of the strains matched NCCLS breakpoints: one susceptible strain (MIC=8 mg/L), two intermediate strains (MIC=16 mg/L) and one resistant strain (MIC=32 mg/L) . Cmax was 119.97+/-2.53 mg/L for intermittent bolus and Css (steady-state concentration) was 40.38+/-0.16 mg/L for continuous infusion . AUC0-24 was similar for both regimens ( approximately 950 mg.h/L) . Inhibitory quotients were three times higher for the intermittent administration whereas t > MIC was higher for continuous infusion (100%) versus intermittent administration (99.8%, 69% and 47.6% for the susceptible, intermediate and resistant strains, respectively) . Against the susceptible and intermediate strains, no differences were found between both regimens with >/= 3 log10 reduction from 8 to 24 h . Against the resistant strain, only the continuous infusion achieved this bactericidal activity in the same time period, minimizing the differences between resistant and susceptible strains . Significantly higher initial inoculum reduction at 32 h was obtained for the continuous versus the intermittent administration (83.35% versus 38.40%, respectively) . These results stress the importance of optimizing t >MIC, even at peri-MIC concentrations, of ceftazidime against resistant strains . Local prevalence of resistance justifies, on a pharmacodynamic basis, electing for continuous infusion versus intermittent administration. Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 900 - 6 Annexin II is a novel receptor for Pseudomonas aeruginosa; Kirschnek S et al.; Infections with Pseudomonas aeruginosa (P . aeruginosa) are critical in ventilated and poly-traumatized patients . Most important, these bacteria cause frequent and chronic pulmonary infections in patients with cystic fibrosis . Therefore, identification of molecular mechanisms that mediate the infection of mammalian cells with P . aeruginosa is urgently required . Here, we aimed to identify novel receptors that are involved in internalization of P . aeruginosa into mammalian epithelial cells . Employing SDS-PAGE purification and mass spectrometry we demonstrate that annexin II specifically binds to P . aeruginosa . The significance of the interaction of annexin II with P . aeruginosa for the infection of mammalian cells is indicated by the finding that neutralization of the ligands on P . aeruginosa by incubation of the bacteria with recombinant, soluble annexin II prevents internalization of P . aeruginosa into human epithelial cells. Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 650 - 5 Forceful large-scale expression of "problematic" membrane proteins; Mokhonova EI et al.; We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches . The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host . This enabled us to enhance the expression level of a toxic membrane protein 30-50 times compared with expression in the native state and to obtain 3-5mg of a highly purified functionally active protein per liter of culture . We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example . The amplified MexY was correctly folded in the cytoplasmic membrane of the E . coli without forming inclusion bodies . This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce. Clin Microbiol Infect, 2005 Jan, 11(1), 73 - 6 IMPs, VIMs and SPMs: the diversity of metallo-beta-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in a Brazilian hospital; Sader HS et al.; Pseudomonas aeruginosa isolates (n = 183), collected from bacteraemic patients hospitalised in Sao Paulo Hospital (Brazil) during 2000-2001, were screened for susceptibility to antimicrobial agents . The polymyxins were the most active compounds (100% susceptibility), followed by amikacin and cefepime (59.0%), meropenem (57.4%), and imipenem and gentamicin (55.2%) . Imipenem-resistant isolates were ribotyped and screened for production of metallo-beta-lactamases (MBLs) by PCR with primers for bla(IMP), bla(VIM) and bla(SPM) . MBL production was detected in 36 isolates (19.7% of the entire collection; 43.9% of the imipenem-resistant isolates) and the MBLs included SPM-1-like (55.6%), VIM-2-like (30.6%) and IMP-1-like (8.3%) enzymes. Clin Microbiol Infect, 2005 Jan, 11(1), 71 - 3 Isolation of an amikacin-resistant Escherichia coli strain after tobramycin treatment of previous recurrent episodes of respiratory tract infections caused by Pseudomonas aeruginosa; Ruiz J et al.; Amikacin-resistant Escherichia coli strains are isolated rarely from clinical samples . In the present study, investigation of an amikacin-resistant clinical isolate of E . coli demonstrated the presence of two class 1 integrons carrying the aacA4 gene plus the aacA7 gene, and the dfrA17 gene plus the aadA5 gene, respectively . Resistance to amikacin in this E . coli isolate was related to the presence of both aacA4 and aacA7. Can J Microbiol, 2004 Sep, 50(9), 751 - 766 Comparison of S-layer secretion genes in freshwater caulobacters; Iuga M et al.; Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus . All elaborate a crystalline protein surface (S) layer made up of protein monomers 100&ndash;193 kDa in size . We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100&ndash;108 kDa), medium (122&ndash;151 kDa), and large (181&ndash;193 kDa) . All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C . crescentus, indicating that the S-layer proteins were all secreted by a type I system . The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals . Cross-expression studies showed that the 6 strains recognized secretion signals from C . crescentus and Pseudomonas aeruginosa and similarly that C . crescentus was able to secrete the S-layer protein C-terminus of 1 strain examined . Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion . Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size. Crit Care Med, 2005 Jan, 33(1), 46 - 53 Invasive approaches to the diagnosis of ventilator-associated pneumonia: A meta-analysis; Shorr AF et al.; OBJECTIVE:: Ventilator-associated pneumonia remains a major challenge in the intensive care unit . The role for invasive diagnostic methods (e.g., bronchoscopy) remains unclear . We hypothesized that invasive testing would alter antibiotic management in patients with ventilator-associated pneumonia but would not necessarily alter mortality . DESIGN:: Meta-analysis of randomized, controlled trials of invasive diagnostic strategies in suspected ventilator-associated pneumonia and a separate pooled analysis of prospective, observational studies of the effect of invasive cultures on antibiotic utilization in ventilator-associated pneumonia . SETTING:: NA . PATIENTS:: Subjects enrolled in the various clinical trials identified . INTERVENTIONS:: None . MEASUREMENTS AND MAIN RESULTS:: We identified four randomized, controlled trials that included 628 patients . The overall quality of these studies was moderate (median Jadad score of 5) and there was both clinical and statistical heterogeneity among these trials . Ventilator-associated pneumonia was confirmed bronchoscopically in 44-69% of participants, with Pseudomonas aeruginosa and Staphylococcus aureus being the most frequently isolated pathogens . Most subjects (90.3%) received adequate antibiotics; however, in one trial there was a significant difference between the invasive and noninvasive arms with respect to this factor . Overall, an invasive approach did not alter mortality (odds ratio 0.89, 95% confidence interval 0.56-1.41) . Invasive testing, though, affected antibiotic utilization (odds ratio for change in antibiotic management after invasive sampling, 2.85, 95% confidence interval 1.45-5.59) . Five prospective observational studies examined invasive testing and included 635 subjects . These reports confirm that invasive sampling leads to modifications in the antibiotic regimen in more than half of patients (pooled estimate for rate of alteration in antibiotic prescription, 50.3%, 95% confidence interval 35.9-64.6%) . CONCLUSIONS:: Few trials have systematically examined the impact of diagnostic techniques on outcomes for patients suspected of suffering from ventilator-associated pneumonia . Invasive strategies do not alter mortality . Invasive approaches to ventilator-associated pneumonia affect antibiotic use and prescribing. J Mol Biol, 2005 Feb 4, 345(5), 1059 - 70 Epub 2004 Dec 21. Tracking the evolution of porphobilinogen synthase metal dependence in vitro; Frere F et al.; Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes . Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+) . Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme . Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli . Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution . The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+) . The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination . Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site . The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites . Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive . The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity . Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity . Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges. Biotechnol Bioeng . 2005 Jan 7; {Epub ahead of print} Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system; Karig D et al.; Small changes in transcriptional activity often significantly affect phenotype but are not detectable in vivo by conventional means . To address this problem, we present a technique for detecting weak transcriptional responses using signal-amplifying genetic circuits . We apply this technique to reveal previously undetectable log phase responses of several Rhl quorum sensing controlled (qsc) promoters from Pseudomonas aeruginosa . Genetic circuits with Rhl promoters and transcriptional amplification components were built and tested in Escherichia coli . This enabled us to isolate the behavior of the promoters under study from Las and quinolone interactions . To amplify qsc promoter responses to acyl-homoserine lactones (AHL), the highly efficient lambda repressor gene was placed downstream of several Rhl promoters and coupled to a fluorescent reporter under the control of the lambda P((R)) promoter . With amplification, up to approximately 100-fold differences in fluorescence levels between AHL induced and noninduced cultures were observed for promoters whose responses were otherwise not detectable . In addition, the combination of using signal amplification and performing experiments in E . coli simplified the analysis of AHL signal crosstalk . For example, we discovered that while a C4HSL/RhlR complex activates both qscrhlA and qscphzA1, a 3OC12HSL/RhlR complex activates qscphzA1 but not qscrhlA in our system . This crosstalk information is particularly important since one of the potential uses of amplification constructs is for the detection of specific quorum sensing signals in environmental and clinical isolates . Furthermore, the process of decomposing networks into basic parts, isolating these components in a well-defined background, and using amplification to characterize both crosstalk and cognate signal responses embodies an important approach to understanding complex genetic networks . (c) 2004 Wiley Periodicals, Inc. J Burn Care Rehabil, 2005 January/February, 26(1), 53 - 56 Therapeutic Efficacy of Three Silver Dressings in an Infected Animal Model; Heggers J et al.; The organic salt AgNO3 has been available as a topical armamentarium to the medical arena for centuries and for burns for the past 60 years . Thirty-five (1968) years later, Charles Fox introduced and popularized a new topical agent known as silver sulfadiazine . More recently, several new slow-release silver dressings came to the forefront . Acticoat(R) (Smith & Nephew, Largo, FL) Silverlon(R) (Argentum, Lakemont, GA) & Silvasorb(R) (Medline Industries, Inc, Mundelein, IL) . Because the standard of care is to change dressings daily, our study focused in on weekly dressing changes as a cost-containment issue . Sprague-Dawley rats received a standard contact burn (20% TBSA) . On day 3, the wound was excised and infected with Pseudomonas aeruginosa and Staphylococcus aureus at 5.0 x 10 cfu/ml . The animals were divided into four groups (n = 5 each group): untreated control, Acticoat(R) group, Silvasorb(R) group, and Silverlon(R) group . The dressings remained on the wounds for 10 days when the wounds were quantitatively assessed . Mean wound counts of the control ranged from 1.2 x 10 to 6.5 x 10 for P . aeruginosa and S . aureus, respectively . Acticoat(R) dressing counts for both organisms were 0 and 1.8 x 10 (median alpha); Silvasorb(R) was 0 and 6.3 x 10 and Silverlon was 1.5 x 10 x 7.4 x 10 (median), Acticoat(R) and Silvasorb(R) were both significantly lower (P < .05) than the control for P . aeruginosa, and Acticoat(R) was significantly lower (P < .05) than the control for S . aureus . Although counts for Silvasorb(R) (M) appear significantly lower than the controls for S . aureus, the numbers were not sufficient to be significant . However, Silverlon(R) did achieve a slight significance . These preliminary data suggest that weekly dressing changes with these new silver dressings are feasible and economically and medically congruous. Crit Care Med, 2004 Nov, 32(11), 2293 - 9 Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU; Pankhaniya RR et al.; OBJECTIVE:: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system . The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2 . We examined whether the acute lung injury caused by cytotoxic P . aeruginosa was dependent on the patatin-like phospholipase domain of ExoU . DESIGN:: Laboratory investigation using an established mouse model for P . aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality . SETTING:: University experimental research laboratory . SUBJECTS:: Balb/c mice . INTERVENTIONS:: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein . The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay . Second, the same site-directional mutation was introduced into the exoU gene of P . aeruginosa PA103 . Mice were intratracheally infected with either a wild-type P . aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU . Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively . MEASUREMENTS AND MAIN RESULTS:: Recombinant ExoU had lysophospholipase A activity . Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity . Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice . CONCLUSION:: Virulent P . aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU. Respir Res . 2005 Jan 6;6(1):2 {Epub ahead of print} Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa; Dagenais A et al.; BACKGROUND: The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains . As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the alpha,beta and gamma subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase . METHODS: Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains . The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection . RESULTS: The infection of the different mouse strains evoked regulation of alpha and beta ENaC mRNA . Following Pseudomonas instillation, the expression of alpha ENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p< 0.05) . The relative expression of beta ENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p< 0.05) . No significant modulation of gamma ENaC mRNA was detected although the general pattern of expression of the subunit was similar to alpha and beta subunits . No modulation of alpha1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded . The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains . CONCLUSIONS: These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs. Infect Control Hosp Epidemiol, 2004 Dec, 25(12), 1083 - 9 An outbreak of Pseudomonas aeruginosa pneumonia and bloodstream infection associated with intermittent otitis externa in a healthcare worker; Zawacki A et al.; OBJECTIVES: To investigate an outbreak of Pseudomonas aeruginosa pneumonia and bloodstream infection among four neonates, determine risk factors for infection, and implement preventive strategies . DESIGN: Retrospective case finding; prospective surveillance cultures of patients, personnel, and environmental sites; molecular typing by pulsed-field gel electrophoresis; and a matched case-control study . PATIENTS AND SETTING: Neonates in the level-III neonatal intensive care unit of a tertiary-care pediatric institution . INTERVENTIONS: Cohorting of patients with positive results for P . aeruginosa, work restrictions for staff with positive results, implementation of an alcohol-based hand product, review of infection control policies and procedures, and closure of the unit until completion of the investigation . RESULTS: Seven (4%) of 190 environmental cultures and 5 (3%) of 178 cultures of individual healthcare workers' hands grew P . aeruginosa . All four outbreak isolates and one previous bloodstream isolate were genotypically identical, as were the P . aeruginosa isolates from the hands and external auditory canal of a healthcare worker with intermittent otitis externa . Four of 5 case-patients versus 5 of 15 matched control-patients had been cared for by this healthcare worker (P = .05) . The healthcare worker was treated and no further cases occurred . CONCLUSIONS: These findings suggest that a healthcare worker with intermittent otitis externa may have caused this cluster of fatal P . aeruginosa infections, adding the external ear to the list of colonized body sites that may serve as a source of potentially pathogenic organisms. Zh Mikrobiol Epidemiol Immunobiol, 2004 Nov-Dec, (6), 6 - 10 {Sensitivity of nosocomial microflora circulating in a transplantation clinic to medicinal bacteriophages}; Clonal diversity of metallo-beta-lactamase-possessing Pseudomonas aeruginosa in geographically diverse regions of Japan; Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438540, JapanThe aim of this study was to determine the distribution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in Japan and to investigate the molecular characteristics of resistance gene cassettes including the gene encoding this enzyme . A total of 594 nonduplicate strains of P . aeruginosa isolated from 60 hospitals throughout Japan in 2002 were evaluated . This study indicated that although the prevalence of imipenem-resistant P . aeruginosa has not increased compared to that found in previous studies, clonal distribution of the same strain across Japan is evident. J Immunol, 2005 Jan 15, 174(2), 1097 - 103 Lipoprotein I, a TLR2/4 Ligand Modulates Th2-Driven Allergic Immune Responses; Revets H et al.; Asthma is an inflammatory lung disease that is initiated and directed by Th2 and inhibited by Th1 cytokines . Microbial infections have been shown to prevent allergic responses by inducing the secretion of the Th1 cytokines IL-12 and IFN-gamma . In this study, we examined whether administration of lipoprotein I (OprI) from Pseudomonas aeruginosa could prevent the inflammatory and physiological manifestations of asthma in a murine model of OVA-induced allergic asthma . OprI triggered dendritic cells to make IL-12 and TNF-alpha, with subsequent IFN-gamma production from T cells . OprI stimulation of dendritic cells involved both TLR2 and TLR4 . Intranasal coadministration of OprI with OVA allergen resulted in a significant decrease in airway eosinophilia and Th2 (IL-4 and IL-13) cytokines and this effect was sustained after repeated allergen challenge . The immediate suppressive effect of OprI (within 2 days of administration) was accompanied by an increase in Th1 cytokine IFN-gamma production and a significant, but transient infiltration of neutrophils . OprI did not redirect the immune system toward a Th1 response since no increased activation of locally recruited Th1 cells could be observed upon repeated challenge with allergen . Our data show for the first time that a bacterial lipoprotein can modulate allergen-specific Th2 effector cells in an allergic response in vivo for a prolonged period via stimulation of the TLR2/4 signaling pathway. Acta Pharm, 2004 Dec, 54(4), 331 - 8 Healing potential of Anogeissus latifolia for dermal wounds in rats; Govindarajan R et al.; Wound healing potential of ethanolic extract of Anogeissus latifolia bark (ALE) for treatment of dermal wounds in rats was studied on excision and incision wound models . HPTLC of the total extract was recorded for the purpose of standardization . Various parameters of incision wound, viz . epithelization period, scar area, tensile strength and hydroxyproline measurements along with wound contraction, were used to evaluate the effect of A . latifolia on wound healing . The results obtained indicate that A . latifolia accelerates the wound healing process by decreasing the surface area of the wound and increasing the tensile strength . Nitrofurazone ointment was used as a positive control . Complete epithelization was observed within 15 days with ALE . Measurements of the healed area and the hydroxyproline level were in agreement . Antibacterial activity of ALE was studied against Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) compared to erythromycin and tetracycline . Moderate activity was observed against all organisms . The present study provides a scientific rationale for the traditional use of Anogeissus latifolia in the management of skin diseases such as sores, boils and itching. Zhonghua Nei Ke Za Zhi, 2004 Nov, 43(11), 853 - 6 {The effect of antioxidant treatment in pneumonia of granulocytopenic rats.}; Xu JF et al.; OBJECTIVE: To evaluate the therapeutic role of antioxidant intervention in granulocytopenia rats with pseudomonas aeruginosa pneumonia . METHODS: 90 male Sprague-Dawley rats were randomly divided into two groups . Group A: the control granulocytopenia group, in which the granulocytopenia was induced by using cyclophosphamide and cortisone acetate . Group B: the antioxidant intervention group, in which the granulocytopenia model was the same as group A, while peritoneal injection of NAC 150 mg.kg(-1).d(-1)half an hour after the immunocompromised model was reproduced, and the injection was continued for a consecutive 7 days, and NAC was injected once more half an hour before bacterial tracheal inoculation . The model of pulmonary infection was established by using standard a strain of pseudomonas aeruginosa . The time-course of the following was observed 0 h before bacterial inoculation, and 6 h, 9 h, 24 h, 48 h and 72 h after infection: the peripheral white blood cells, mortality, oxidant/antioxidant indexes, bacterial burden of lung tissue homogenate, pulmonary vascular permeability and lung wet/dry weight ratio, and the pulmonary histopathological changes . RESULTS: The peripheral white blood cells of both groups were less than 4 x 10(9)/L . The concentration of superoxide dismutase in both serum and lung tissue in group B were higher than that in group A, while concentration of malondialdehyde in group B was lower than that in group A . Pulmonary vascular permeability and lung wet/dry ratio of group B were much lower than that of group A . There was no difference in bacterial burden of lung tissue between the two groups . Group B showed a lower mortality than group A (16.3% vs 23.4%) . Lung histopathological observation showed that lung injury, pulmonary congestion and hemorrhage were more serious or obvious in group A as compared with group B . Apoptotic bodies were found in the lung epithelial cells of Group A . CONCLUSIONS: Antioxidant intervention can alleviate lung injury in the granulocytopenia rats with pseudomonas aeruginosa pneumonia . It may become an important subsidiary approache to pneumonia in granulocytopenia patients. Pediatr Pulmonol, 2005 Feb, 39(2), 141 - 9 Aerosol treatment with MNEI suppresses bacterial proliferation in a model of chronic Pseudomonas aeruginosa lung infection; Woods DE et al.; Neutrophil elastase is present at high levels in airway fluid of patients with cystic fibrosis (CF), and is responsible for considerable inflammatory damage . Human monocyte/neutrophil elastase inhibitor (MNEI), a 42-kDa serpin protein, is an effective inhibitor of neutrophil elastase, cathepsin G, and proteinase-3, related proteases released from inflammatory neutrophils . We hypothesized that recombinant MNEI would reduce inflammatory damage and enhance bacterial clearance from the lung in an animal model of chronic Pseudomonas aeruginosa infection . In vitro studies showed that MNEI causes dose-dependent inhibition of the activity of rat neutrophil elastase . Recombinant MNEI was administered daily by aerosolization to rats previously inoculated with agar beads containing P . aeruginosa to initiate chronic infection . Administered MNEI was partially recovered in lavage fluid of treated rats as a 66-kDa complex with protease indicative of in vivo inhibition of elastase or a related protease . Aerosol treatment with MNEI significantly decreased the extent of inflammatory injury, quantified as the histopathology score . MNEI, which had no bactericidal effect on P . aeruginosa in vitro, significantly enhanced clearance of bacteria from infected rat lungs . The reduction of histopathology scores and enhancement of bacterial killing were evident 6 hr after a single aerosol treatment with MNEI . These findings indicate an important function of MNEI in protecting innate antimicrobial defense . Similar results were previously obtained for aerosolized prolastin (alpha1-antitrypsin), indicating that enhanced bacterial clearance by MNEI is due to inhibition of neutrophil protease . These findings demonstrate the value of this nonantibiotic protease inhibitor as an adjunct for the treatment and prevention of the infection component of CF lung disease . Pediatr Pulmonol . 2005;39:141-149 . (c) 2005 Wiley-Liss, Inc. Pediatr Pulmonol, 2005 Feb, 39(2), 135 - 40 Immunoglobulin and IgG subclass levels in a regional pediatric cystic fibrosis clinic; Garside JP et al.; The aim of this study was to report serum immunoglobulin (Ig) and IgG subclass levels in a large pediatric population with cystic fibrosis, and relate these to measures of disease severity . Total immunoglobulin levels were measured in 154 patients, and IgG subclass levels were measured in 136 patients and compared to age-related normal population data and to levels reported in previously published studies of children with cystic fibrosis . Clinical data were also collected: genotype; height, weight, and BMI standard deviation scores; FEV(1) (as percent predicted); Shwachmann-Kulczycki (S-K) and Northern chest X-ray scores; and Pseudomonas aeruginosa infection status . The clinical well-being of patients with hypo- or hyper-gammaglobulinemia was compared with age- and sex-matched control patients who had normal levels of gammaglobulin . IgG subclass levels were measured, and the results were compared with previous studies . Eleven patients had hypergammaglobulinemia (7.8% compared with 0-69% in the published literature) . Patients with hypergammaglobulinemia had lower FEV(1) percent-predicted values, and worse S-K and Northern chest X-ray scores than controls . Three patients had hypogammaglobulinemia (1.9% compared with 0-10.8% in the published literature) . There was no difference in any clinical parameter between controls and those with hypogammaglobulinemia . Nineteen patients (14%) had low levels of IgG1, and 40 patients (29%) had low levels of IgG2 . The low percentage of patients with abnormally high immunoglobulin levels probably reflects the improved respiratory status of today's children with CF . The low percentage of those with low IgG probably reflects better nutritional status . The finding of worse lung function and clinical scores in patients with hypergammaglobulinemia agrees with the published literature . The high percentage of patients with low IgG2 was unexpected and was not previously reported . The clinical significance of this in patients with CF is unknown . Pediatr Pulmonol . 2005;39:135-140 . (c) 2005 Wiley-Liss, Inc. J Biochem (Tokyo), 2004 Nov, 136(5), 607 - 15 Activation of SoxR-Dependent Transcription in Pseudomonas aeruginosa; Kobayashi K et al.; The SoxR protein of Escherichia coli responds to redox signals by activating the transcription of soxS, which encodes another transcription activator that directly stimulates oxidative stress genes . In contrast, Pseudomonas aeruginosa has an open reading frame (ORF) encoding a putative protein homologous to E . coli SoxR, but not to SoxS . Instead of a soxS homolog, ORFs encoding an unknown hypothetical protein and soxR are arranged divergently with their 5' ends separated by a 78 bp region containing a sequence homologous to the SoxR-binding soxS promoter . In this study, we report the overproduction and purification of SoxR from P . aeruginosa to investigate the mechanism of gene activation by SoxR . The spectroscopic properties of the purified SoxR protein indicate that it contains a redox active iron-sulfur {2Fe-2S} cluster . Redox titration of the SoxR protein revealed a midpoint potential of -290 mV . The SoxR protein specifically binds a fragment of the SoxS promoter-like region in a concentration-dependent fashion, as shown by both gel mobility shift and fluorescence polarization assays . The purified SoxR stimulates the in vitro transcription of the gene encoding the hypothetical protein in P . aeruginosa . This activity was lost following reduction of the SoxR {2Fe-2S} clusters . The levels of mRNA in the hypothetical protein increased in paraquat-treated cells . These results indicate that P . aeruginosa SoxR is a direct transcriptional activator of the hypothetical protein, and suggest that SoxR proteins may play multiple regulatory roles as a transcription factor in addition to its protective role in oxidative stress. Biol Proced Online, 2004, 6, 268 - 276 Epub 2004 Dec 30. Construction of a bacterial autoinducer detection system in mammalian cells; Shiner EK et al.; Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within a population . QS systems in Gram negative bacteria consist of transcription factors of the LuxR family and their acyl homoserine lactone (AHL) ligands . We describe here a method for examining QS signaling systems in mammalian cells that uses engineered LuxR-type proteins from the opportunistic pathogen, Pseudomonas aeruginosa, which can function as AHL-dependent transcription factors . The engineered proteins respond to their cognate ligands and display sequence specific DNA binding properties . This system has several potential biotechnological and biological applications . It may be used to characterize any LuxR-type protein, screen animal and plant cell extracts or exudates for compounds that mimic or interfere with AHL signaling or to screen different cell types for AHL inactivating activities. J Bacteriol, 2005 Jan, 187(2), 771 - 7 Evidence for Two Flagellar Stators and Their Role in the Motility of Pseudomonas aeruginosa; Toutain CM et al.; Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility . In this study, we present evidence that P . aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria . Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility. J Bacteriol, 2005 Jan, 187(2), 554 - 66 Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture; Mashburn LM et al.; Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis . Often these infections are not caused by colonization with P . aeruginosa alone but instead by a consortium of pathogenic bacteria . Little is known about growth and persistence of P . aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P . aeruginosa pathogenesis and physiology . In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P . aeruginosa in monoculture and in coculture with Staphylococcus aureus . Monoculture results indicate that approximately 5% of all P . aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls . Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments . The presence of S . aureus caused decreased transcription of P . aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S . aureus increases usable iron for P . aeruginosa in this environment . We propose a model where P . aeruginosa lyses S . aureus and uses released iron for growth in low-iron environments. Pediatr Pulmonol . 2004 Dec 30;39(2):141-149 {Epub ahead of print} Aerosol treatment with MNEI suppresses bacterial proliferation in a model of chronic Pseudomonas aeruginosa lung infection; Woods DE et al.; Neutrophil elastase is present at high levels in airway fluid of patients with cystic fibrosis (CF), and is responsible for considerable inflammatory damage . Human monocyte/neutrophil elastase inhibitor (MNEI), a 42-kDa serpin protein, is an effective inhibitor of neutrophil elastase, cathepsin G, and proteinase-3, related proteases released from inflammatory neutrophils . We hypothesized that recombinant MNEI would reduce inflammatory damage and enhance bacterial clearance from the lung in an animal model of chronic Pseudomonas aeruginosa infection . In vitro studies showed that MNEI causes dose-dependent inhibition of the activity of rat neutrophil elastase . Recombinant MNEI was administered daily by aerosolization to rats previously inoculated with agar beads containing P . aeruginosa to initiate chronic infection . Administered MNEI was partially recovered in lavage fluid of treated rats as a 66-kDa complex with protease indicative of in vivo inhibition of elastase or a related protease . Aerosol treatment with MNEI significantly decreased the extent of inflammatory injury, quantified as the histopathology score . MNEI, which had no bactericidal effect on P . aeruginosa in vitro, significantly enhanced clearance of bacteria from infected rat lungs . The reduction of histopathology scores and enhancement of bacterial killing were evident 6 hr after a single aerosol treatment with MNEI . These findings indicate an important function of MNEI in protecting innate antimicrobial defense . Similar results were previously obtained for aerosolized prolastin (alpha1-antitrypsin), indicating that enhanced bacterial clearance by MNEI is due to inhibition of neutrophil protease . These findings demonstrate the value of this nonantibiotic protease inhibitor as an adjunct for the treatment and prevention of the infection component of CF lung disease . Pediatr Pulmonol . 2005;39:141-149 . (c) 2005 Wiley-Liss, Inc. Pediatr Pulmonol . 2004 Dec 30;39(2):135-140 {Epub ahead of print} Immunoglobulin and IgG subclass levels in a regional pediatric cystic fibrosis clinic; Garside JP et al.; The aim of this study was to report serum immunoglobulin (Ig) and IgG subclass levels in a large pediatric population with cystic fibrosis, and relate these to measures of disease severity . Total immunoglobulin levels were measured in 154 patients, and IgG subclass levels were measured in 136 patients and compared to age-related normal population data and to levels reported in previously published studies of children with cystic fibrosis . Clinical data were also collected: genotype; height, weight, and BMI standard deviation scores; FEV(1) (as percent predicted); Shwachmann-Kulczycki (S-K) and Northern chest X-ray scores; and Pseudomonas aeruginosa infection status . The clinical well-being of patients with hypo- or hyper-gammaglobulinemia was compared with age- and sex-matched control patients who had normal levels of gammaglobulin . IgG subclass levels were measured, and the results were compared with previous studies . Eleven patients had hypergammaglobulinemia (7.8% compared with 0-69% in the published literature) . Patients with hypergammaglobulinemia had lower FEV(1) percent-predicted values, and worse S-K and Northern chest X-ray scores than controls . Three patients had hypogammaglobulinemia (1.9% compared with 0-10.8% in the published literature) . There was no difference in any clinical parameter between controls and those with hypogammaglobulinemia . Nineteen patients (14%) had low levels of IgG1, and 40 patients (29%) had low levels of IgG2 . The low percentage of patients with abnormally high immunoglobulin levels probably reflects the improved respiratory status of today's children with CF . The low percentage of those with low IgG probably reflects better nutritional status . The finding of worse lung function and clinical scores in patients with hypergammaglobulinemia agrees with the published literature . The high percentage of patients with low IgG2 was unexpected and was not previously reported . The clinical significance of this in patients with CF is unknown . Pediatr Pulmonol . 2005;39:135-140 . (c) 2005 Wiley-Liss, Inc. Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 1122 - 7 Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2-; Coyle CL et al.; Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2- have been made . The reaction was found to follow a second-order rate law: k 4.2 x 10(3) M(-1) s(-1) {25 degrees, micro0.1 M, pH 7.0 (phosphate)}; deltaH+/+ 3.2 kcal/ mol; AS+/+ -30 cal/mol-deg . The electrostatics-corrected self-exchange rate constant (k11 corr) calculated for cytochrome c551 based on the Fe(EDTA)2- cross reaction is 2 M(-1) s(-1), as compared to a value of 6 M(-1) s(-1) for horse heart cytochrome c . The close correspondence of the two k11 corr values is taken as an indication that the two proteins employ very similar electron transfer mechanisms in their reactions with Fe(EDTA)(2-) . It is proposed that this mechanism involves reagent contact, but little protein conformational change, at the partially exposed heme edge. J Am Soc Nephrol . 2004 Dec 29; {Epub ahead of print} Randomized, Double-Blind Trial of Antibiotic Exit Site Cream for Prevention of Exit Site Infection in Peritoneal Dialysis Patients; Bernardini J et al.; Infection is the Achilles heel of peritoneal dialysis . Exit site mupirocin prevents Staphylococcus aureus peritoneal dialysis (PD) infections but does not reduce Pseudomonas aeruginosa or other Gram-negative infections, which are associated with considerable morbidity and sometimes death . Patients from three centers (53% incident to PD and 47% prevalent) were randomized in a double-blinded manner to daily mupirocin or gentamicin cream to the catheter exit site . Infections were tracked prospectively by organism and expressed as episodes per dialysis-year at risk . A total of 133 patients were randomized, 67 to gentamicin and 66 to mupirocin cream . Catheter infection rates were 0.23/yr with gentamicin cream versus 0.54/yr with mupirocin (P = 0.005) . Time to first catheter infection was longer using gentamicin (P = 0.03) . There were no P . aeruginosa catheter infections using gentamicin compared with 0.11/yr using mupirocin (P < 0.003) . S . aureus exit site infections were infrequent in both groups (0.06 and 0.08/yr; P = 0.44) . Peritonitis rates were 0.34/yr versus 0.52/yr (P = 0.03), with a striking decrease in Gram-negative peritonitis (0.02/yr versus 0.15/yr; P = 0.003) using gentamicin compared with mupirocin cream, respectively . Gentamicin use was a significant predictor of lower peritonitis rates (relative risk, 0.52; 95% confidence interval, 0.29 to 0.93; P < 0.03), controlling for center and incident versus prevalent patients . Gentamicin cream applied daily to the peritoneal catheter exit site reduced P . aeruginosa and other Gram-negative catheter infections and reduced peritonitis by 35%, particularly Gram-negative organisms . Gentamicin cream was as effective as mupirocin in preventing S . aureus infections . Daily gentamicin cream at the exit site should be the prophylaxis of choice for PD patients. Invest Ophthalmol Vis Sci, 2005 Jan, 46(1), 248 - 51 Polyphosphate Kinase 1 and the Ocular Virulence of Pseudomonas aeruginosa; Parks QM et al.; PURPOSE: To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa . METHODS: Using a mouse model of infection, P . aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence . These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment . RESULTS: The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P < 0.016) . Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea . However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P < 0.006) than either the wild-type or restored strain . Furthermore, the PPK1-deficient mutant produced significantly less (P < 0.022) pyocyanin . CONCLUSIONS: PPK1 is essential for a successful ocular infection by P . aeruginosa . The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response. Invest Ophthalmol Vis Sci, 2005 Jan, 46(1), 88 - 95 Lumican regulates corneal inflammatory responses by modulating fas-fas ligand signaling; Vij N et al.; PURPOSE: The authors have previously shown that apoptosis of stromal cells is downregulated in the lumican-null mouse and that this may be due to disruption of Fas-Fas ligand (FasL) signaling . The present study was undertaken to investigate the role of lumican in regulating Fas and its impact on inflammation and healing of corneal injuries . METHODS: Apoptosis was determined by measuring caspase-3/7 activity in corneal extracts . Protein and RNA levels of Fas were estimated by immunoblot analysis and RT-PCR, respectively . Circular and incisional stromal wounds were exposed to Pseudomonas aeruginosa LPS, and healing was assessed by (1) observing wound closure with fluorescence and bright-field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining for macrophages (F4/80) and neutrophils (NIMP-R14), (3) measuring myeloperoxidase (MPO) levels by ELISA to quantify neutrophils, and (4) measuring proinflammatory cytokines by ELISA . RESULTS: Lum(-/-)-injured corneas showed significantly lower caspase-3/7 activity (apoptosis) . Lum(-/-)-wounded corneas showed delayed healing, reduced recruitment of macrophages and neutrophils, lower MPO levels, and no induction of the proinflammatory cytokines TNFalpha and IL1beta . The Fas protein level, before and after wounding, was dramatically lower in Lum(-/-)- compared with Lum(+/+)-injured cornea . However, Fas mRNA levels were comparable in both genotypes, suggesting regulation of Fas at the protein level . Moreover, a solid-state binding assay and coimmunoprecipitation of FasL and lumican suggested binding of FasL to lumican . CONCLUSIONS: The data suggest that lumican binds FasL and facilitates induction of Fas . Poor signaling through Fas-FasL in lumican deficiency leads to impaired induction of inflammatory cytokines and corneal healing. Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 309 - 14 Epub 2004 Dec 27. Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones; Kaufmann GF et al.; Bacteria use small diffusible molecules to exchange information in a process called quorum sensing . An important class of autoinducers used by Gram-negative bacteria is the family of N-acylhomoserine lactones . Here, we report the discovery of a previously undescribed nonenzymatically formed product from N-(3-oxododecanoyl)-L-homoserine lactone; both the N-acylhomoserine and its novel tetramic acid degradation product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, are potent antibacterial agents . Bactericidal activity was observed against all tested Gram-positive bacterial strains, whereas no toxicity was seen against Gram-negative bacteria . We propose that Pseudomonas aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria . Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization . These findings merit new attention such that other previously identified autoinducers be reevaluated for additional biological functions. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 287 - 95 GeneChip expression analysis of the VqsR regulon of Pseudomonas aeruginosa TB; Juhas M et al.; Two interlinked quorum sensing circuits, las and rhl, which control pathogenesis of Pseudomonas aeruginosa are further modulated by numerous regulators, including VqsR (virulence and quorum sensing regulator) . High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type and VqsR mutant in ABC minimal medium . The expression of a large group of metabolic genes, ECF sigma factors as well as of many quorum-sensing genes previously not assigned to VqsR-regulon was found to be affected by the disruption of vqsR. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 209 - 216 The Pseudomonas aeruginosa PA14 type III secretion system is expressed but not essential to virulence in the Caenorhabditis elegans-P . aeruginosa pathogenicity model; Wareham DW et al.; The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P . aeruginosa pathogenesis . Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans . Although C . elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence . This was despite the TTSS being actively expressed during C . elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR . However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C . elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity . A fuller understanding of the mechanism of resistance to type III attack in C . elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man. FEMS Microbiol Lett, 2005 Jan 1, 242(1), 161 - 7 Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance; Murakami K et al.; The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions . We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance . The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem . For imipenem, the survival was approximately 40 times lower . Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant . Our results indicate that rpoS gene is involved in tolerance to antibiotics in P . aeruginosa during the stationary phase and heat stress . However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene. Colloids Surf B Biointerfaces, 2005 Jan 15, 40(1), 25 - 9 Linear alkyl benzene sulphonate (LAS) degradation by immobilized Pseudomonas aeruginosa under low intensity ultrasound; Lijun X et al.; We studied the LAS degradation of immobilized Pseudomonas aeruginosa with low-intensity ultrasonic and the influence of original LAS concentration, pH, rotary velocity and different conditions of low-intensity ultrasonic irradiation on the degradation of LAS . In our experiment, the degradation rate of LAS was the main index . We found that low-intensity ultrasonic irradiation could improve the metabolism of microorganism cells and promote the LAS biodegradation of immobilized cells . In the experiment, 50mg/l LAS were used to simulate wastewater, and low-intensity ultrasonic was considered . We found the influence was obvious, and the optimal degradation rate was acquired when the conditions of ultrasonic were frequency 24kHz, power 8W, stimulation time 5s, intermissive time 30s, and total time 10min . The LAS degradation rate of immobilized cells with ultrasonic were respectively 40% and 9.5% higher than that of the suspending cells and immobilized cells without irradiation. J Hosp Infect, 2005 Feb, 59(2), 102 - 7 Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces; Panagea S et al.; We conducted an environmental survey in the Liverpool adult cystic fibrosis (CF) centre in order to determine the extent of environmental contamination with an epidemic strain of Pseudomonas aeruginosa that colonizes most CF patients in Liverpool, and to identify possible reservoirs and routes of cross-infection . In addition, we studied the survival of this strain on dry surfaces, compared with that of other CF P . aeruginosa strains, to explore factors that might contribute to its high transmissibility . Samples were collected from staff, patients and the environment (drains, bath tubs, showers, dry surfaces, respiratory equipment and air) in the inpatient ward and outpatient clinic . P . aeruginosa strains were tested using a new polymerase chain reaction amplification assay specific for the Liverpool epidemic strain (LES) . LES was isolated from patients' hands, clothes and bed linen . Environmental contamination with LES was only detected in close proximity to colonized patients (external surfaces of their respiratory equipment, and spirometry machine tubing and chair) and was short-lived . No persistent environmental reservoirs were found . LES was detected in the majority of air samples from inside patients' rooms, the ward corridor and the outpatient clinic . Survival of LES on dry surfaces was significantly longer than that for some other strains tested, but not compared with other strains shown not to be transmissible . Improved environmental survival on its own, therefore, cannot explain the high transmissibility of this epidemic strain . Our study suggests that airborne dissemination plays a significant role in patient-to-patient spread of LES, and confirms the need to segregate those patients colonized by epidemic P . aeruginosa strains from all other CF patients. J Hosp Infect, 2005 Feb, 59(2), 96 - 101 Risk factors for imipenem-resistant Pseudomonas aeruginosa: a comparative analysis of two case-control studies in hospitalized patients; Zavascki AP et al.; Risk factors for acquisition of imipenem-resistant Pseudomonas aeruginosa by hospitalized patients were assessed at a tertiary care hospital . Two case-control studies with different control groups were used . In Study 1, patients with imipenem-resistant P . aeruginosa (IRPA) (case group) were compared with patients selected at random from the same unit . In Study 2, the case group was compared with patients with imipenem-susceptible P . aeruginosa (ISPA) . Ninety-three patients with IRPA and 93 control patients were included in Study 1, and 93 IRPA patients and 65 patients with ISPA were included in Study 2 . Carbapenem treatment {odds ratio (OR) 5.82}, mechanical ventilation (OR 3.22) and hospital admission in the previous year (OR 2.59) were associated with IRPA in Study 1 . An interaction between carbapenem and vancomycin was found to be a significant risk factor for IRPA (OR for carbapenem in patients with vancomycin use 43.71) . In Study 2, carbapenem exposure (OR 12.82) and renal failure (OR 5.00) were associated with IRPA . Our study confirmed that carbapenem exposure is the main risk factor for IRPA, and found that the use of both carbapenem and vancomycin can increase this effect. J Hosp Infect, 2005 Feb, 59(2), 83 - 9 Impact of restricting fluoroquinolone prescription on bacterial resistance in an intensive care unit; Aubert G et al.; The purpose of this study was to assess the effect of reducing prescription of fluoroquinolones in an intensive care unit (ICU) upon bacterial resistance, particularly as regards Pseudomonas aeruginosa . For six months between January 2001 and June 2001, administration of fluoroquinolones was kept to a minimum . A bacteriological screening of patients was performed to assess the incidence of fluoroquinolone-resistant bacteria . There was a 75.8% restriction in prescriptions of fluoroquinolones . There was no significant change in bacterial ecology between the periods preceding (12 months) and following (12 months) restriction . There was a significant recovery of sensitivity of P . aeruginosa to ciprofloxacin (P</=0.01), with a decrease in resistant strains from 71.3% in the pre-restriction period to 52.4% in the post-restriction period . Regarding clinical data, no significant differences were noted between the pre-restriction and the post-restriction periods, except for the number of cases of ventilator-associated pneumonia with P . aeruginosa resistant to ciprofloxacin . This study demonstrated the possibility of introducing rotation of antibiotics in an ICU. Med Mal Infect, 2004 Feb, 34(2), 62 - 9 {A 7-year survey of strains identified in blood cultures in a clinical hematology unit}; Elouennass M et al.; GOAL: This study had for aim to analyze the epidemiology of strains identified in blood cultures (hopital d'instruction des armees Percy, Clamart, France, hematology unit) to compare the rate of identified micro-organisms with literature data, and to search for a possible correlation between antibiotherapy management and evolution of resistance profiles . MATERIAL AND METHODS: All the micro-organisms (N = 690) collected over seven years (January 1996 to December 2002), from blood cultures of hospitalized patients in conventional and sterile sector were studied . RESULTS: Gram positive cocci rate (GPC) was 62.6% and Gram negative bacilli (GNB) 31.3% . Evolution in time showed a decrease of GPC and an increase of GNB, notably the non fermenting Gram negative bacilli, leading to an equal rate by 2001-2002 . The most frequently identified species were Staphylococcus epidermidis (36.4%), Escherichia coli (8.7%), Pseudomonas aeruginosa (6.8%), and Staphylococcus aureus (4.9%) . The rate of methicillin-resistant staphylococci was 63.6% . Fifty-five percent of E . coli strains had a penicillinase phenotype . Pseudomonas aeruginosa resistance was 8.5, 8.5, 6.4 and 8.5%, respectively for ceftazidime, piperacillin-tazobactam, imipenem, and amikacin . CONCLUSION: This study showed a tendency to inversion of former bacteremia epidemiology with increasing negative Gram bacilli . It justifies the antibiotherapy protocols adopted in the hematology unit. Microb Pathog, 2004 Dec, 37(6), 313 - 22 Epub 2004 Dec 08. Polarisation of type III translocation by Pseudomonas aeruginosa requires PcrG, PcrV and PopN; Sundin C et al.; Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell . We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PcrV, PopB and PopD . To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants . In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased . When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium . This confirms that ExoS translocation by P . aeruginosa occurs by a polarised mechanism . In contrast, high levels of ExoS were recovered in the tissue culture medium when cells were infected with pcrG, pcrV and popN mutants . Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions . This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells. Infect Immun, 2005 Jan, 73(1), 661 - 5 Tissue inhibitor of metalloproteinase 1 regulates resistance to infection; Lee MM et al.; Tissue inhibitor of metalloproteinase 1 (TIMP-1)-deficient mice are resistant to Pseudomonas aeruginosa corneal infections . Corneas healed completely in TIMP-1-deficient mice, and infections were cleared faster in TIMP-1-deficient mice than in wild-type littermates . Genetic suppression studies using matrix metalloproteinase (MMP)-deficient mice showed that MMP-9, MMP-3, and MMP-7 but not MMP-2 or MMP-12 are needed for resistance . Increased resistance was also seen during pulmonary infections . These results identify a novel pathway regulating infection resistance. Infect Immun, 2005 Jan, 73(1), 638 - 43 Characterization of an ExoS Type III translocation-resistant cell line; Rucks EA et al.; Pseudomonas aeruginosa ExoS is a type III-secreted type III-secreted, bifunctional protein that causes diverse effects on eukaryotic cell function . The coculture of P . aeruginosa strains expressing ExoS with HL-60 myeloid cells revealed the cell line to be resistant to the toxic effects of ExoS . Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (TPA) rendered the cell line sensitive to ExoS . To understand the cellular basis for the alteration in sensitivity, undifferentiated and TPA-differentiated HL-60 cells were compared for differences in bacterial adherence, type III secretion induction, and ExoS translocation . These comparisons found that ExoS was translocated more efficiently in TPA-differentiated HL-60 cells than in undifferentiated cells . The studies support the ability of eukaryotic cells to influence P . aeruginosa TTS at the level of membrane translocation. Infect Immun, 2005 Jan, 73(1), 573 - 82 Functional regions of the Pseudomonas aeruginosa cytotoxin ExoU; Rabin SD et al.; ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system . To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system . Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing . Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus . Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity . Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity . To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells . These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence . Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU. Infect Immun, 2005 Jan, 73(1), 444 - 52 Use of phage display to identify potential Pseudomonas aeruginosa gene products relevant to early cystic fibrosis airway infections; Beckmann C et al.; Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis . Treatment of established infections is difficult, even with microbiologically active agents . Thus, prevention of infection is an important goal of management . Isolates from cystic fibrosis patients appear to originate from the environment but adapt to the milieu of the airway of the cystic fibrosis patient and evolve toward a common phenotype . Identification of the antigens expressed early in infection may lead to novel targets for vaccine development . Immunogenic peptides were identified in a J404 random nonapeptide phage display library with serum from cystic fibrosis patients obtained within the first year of P . aeruginosa infection . One hundred sixty-five reactive clones were verified by plaque lift assays, and their inserts were sequenced . The sequenced nonapeptides were compared with the published sequence of strain PAO1, identifying homologies to 76 genes encoding outer membrane and secreted proteins . The majority of these were proteins involved in small-molecule transport, membrane structural proteins, and secreted factors . An in silico analysis was performed that suggested that the occurrence of multiple matches to predominantly outer membrane and secreted proteins was not attributable to random chance . Finally, gene expression array data from early isolates of P . aeruginosa from cystic fibrosis patients was compared with the results from phage display analysis . Eleven outer membrane and secreted proteins were common between the two data sets . These included genes involved in iron acquisition, antibiotic efflux, fimbrial biogenesis, and pyocin synthesis . These results demonstrate the feasibility and validity of this novel approach and suggest potential targets for future development. Antimicrob Agents Chemother, 2005 Jan, 49(1), 461 - 3 Pharmacodynamic evaluation of extending the administration time of meropenem using a Monte Carlo simulation; Lomaestro BM et al.; A Monte Carlo simulation demonstrated that 1 g of meropenem (MEM) every 8 h (q8h) (3-h infusion) has a higher target attainment rate against Pseudomonas aeruginosa than either 500 mg of MEM q8h (3-h infusion) or 0.5 g of imipenem-cilastatin (I-C) q6h (1-h infusion) . For other pathogens, 500 mg of MEM q8h was equivalent or superior to I-C. Antimicrob Agents Chemother, 2005 Jan, 49(1), 451 - 3 Characterization of In100, a new integron carrying a metallo-{beta}-lactamase and a carbenicillinase, from Pseudomonas aeruginosa; Quinteira S et al.; In100, a new integron carrying a carbapenemase gene (bla(VIM-2)) associated with a carbenicillinase (blaP1b) and aminoglycoside resistance genes (aacA4 and aadA2), was detected in a Pseudomonas aeruginosa clinical isolate . The particular gene cassette organization of In100 seems to reflect the evolution of antibiotic usage in therapeutics. Antimicrob Agents Chemother, 2005 Jan, 49(1), 316 - 22 De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity; Deslouches B et al.; Comparison of human immunodeficiency virus lentiviral lytic peptide 1 with other host-derived peptides indicates that antimicrobial properties of membrane-active peptides are markedly influenced by their cationic, hydrophobic, and amphipathic properties . Many common themes, such as Arg composition of the cationic face of an amphipathic helix and the importance of maintaining the hydrophobic face, have been deduced from these observations . These studies suggest that a peptide with these structural properties can be derived de novo by using only a few strategically positioned amino acids . However, the effects of length and helicity on antimicrobial activity and selectivity have not been objectively evaluated in the context of this motif . To address these structure-function issues, multimers of a 12-residue lytic base unit (LBU) peptide composed only of Arg and Val residues aligned to form idealized amphipathic helices were designed . Bacterial killing assays and circular dichroism analyses reveal a strong correlation between antibacterial activity, peptide length, and propensity to form a helix in solvent mimicking the environment of a membrane . Increasing peptide length beyond two LBUs (24-residue peptides) resulted in no appreciable increase in antimicrobial activity . Derivatives (WLBU) of the LBU series were further engineered by substituting Trp residues in the hydrophobic domains . The 24-residue WLBU2 peptide was active at physiologic NaCl concentrations against Staphylococcus aureus and mucoid and nonmucoid strains of Pseudomonas aeruginosa . Further, WLBU2 displayed the highest antibacterial selectivity of all peptides evaluated in the present study by using a coculture model of P . aeruginosa and primary human skin fibroblasts . These findings provide fundamental information toward the de novo design of an antimicrobial peptide useful for the management of infectious diseases. J Immunol, 2005 Jan 1, 174(1), 404 - 10 Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection; Toliver-Kinsky TE et al.; Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells . This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms . Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection . Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P . aeruginosa . Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice . Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection . These data indicate that Flt3L can increase the resistance of mice to a P . aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function. J Biol Chem . 2004 Dec 15; {Epub ahead of print} Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors; Jarvis TC et al.; DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria . The components and functions of E . coli DNA polymerase III holoenzyme have been studied extensively . Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa . We have expressed and purified the processivity factor (beta), single-stranded DNA binding protein, a complex containing the polymerase (alpha) and exonuclease (epsilon) subunits and the essential components of the DnaX complex (tau(3) delta delta') . Efficient primer elongation requires the presence of alpha epsilon, beta and tau(3) delta delta' . Pseudomonas aeruginosa alpha epsilon can substitute completely for E . coli Pol III in E . coli holoenzyme reconstitution assays . Pseudomonas beta and tau(3) delta delta' exhibit a ten-fold lower activity relative to their E . coli counterparts in E . coli holoenzyme reconstitution assays . Although the Pseudomonas counterpart to the E . coli psi subunit was not apparent in sequence similarity searches, addition of purified E . coli chi and psi (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas tau(3) delta delta' complex approximately 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D338 - 43 Pseudomonas aeruginosa Genome Database and PseudoCAP: facilitating community-based, continually updated, genome annotation; Winsor GL et al.; Using the Pseudomonas aeruginosa Genome Project as a test case, we have developed a database and submission system to facilitate a community-based approach to continually updated genome annotation . Researchers submit proposed annotation updates through one of three web-based form options which are then subjected to review, and if accepted, entered into both the database and log file of updates with author acknowledgement . In addition, a coordinator continually reviews literature for suitable updates, as we have found such reviews to be the most efficient . Both the annotations database and updates-log database have Boolean search capability with the ability to sort results and download all data or search results as tab-delimited files . To complement this peer-reviewed genome annotation, we also provide a linked GBrowse view which displays alternate annotations . Additional tools and analyses are also integrated, including PseudoCyc, and knockout mutant information . We propose that this database system, with its focus on facilitating flexible queries of the data and providing access to both peer-reviewed annotations as well as alternate annotation information, may be a suitable model for other genome projects wishing to use a continually updated, community-based annotation approach . The source code is freely available under GNU General Public Licence. FEMS Immunol Med Microbiol, 2005 Jan 1, 43(1), 29 - 35 Population genetic analysis of Pseudomonas aeruginosa using multilocus sequence typing; Vernez I et al.; To study the population genetic structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing scheme . The sequences of internal fragments of seven housekeeping genes were obtained for 34 P . aeruginosa isolates from patients hospitalized in five different European cities . Twenty-six different allelic profiles were identified . The mean allelic diversity was 0.854 (range: 0.606-0.978), which was about six times greater than the results obtained with the multilocus enzyme electrophoresis method . Linkage disequilibrium was measured with the index of association . An index of 1.95+/-0.24 was calculated when all the strains were considered . This index was 1.76+/-0.27 when only one strain per sequence type was considered . Both results were different from 0, indicating linkage among loci, which means that the population structure of our set of P . aeruginosa isolates is clonal . The clonal structure of the population was also suggested by the congruence of the topology of the different trees obtained from the seven housekeeping genes . These results are in contrast to previous studies, finding a non clonal population structure . Since a small number of isolates was analyzed in this study, there might be a bias of selection which includes the possibility that they belong to widely disseminated epidemic clones . Another possibility is that recombination did not occurred homogeneously throughout the genome of P . aeruginosa, so that part of it has a clonal structure, while the remaining part of the genome is more frequently subject to recombination. Int J Pharm, 2005 Jan 6, 288(1), 131 - 40 Epub 2004 Nov 18. A chemical method of gentamicin bonding to gelatine-sealed prosthetic vascular grafts; Ginalska G et al.; Our aim was to develop a new method of chemical binding of gentamicin to vascular prostheses made of poly(ethylene terephthalate) (PET) fibres and covered with pig gelatine . We estimated (with the HPLC method) the immobilization yield, which equalled 76 or 8% depending on the concentration of the antibiotic used and the amount of gentamicin bound to the prosthesis (1.08-20.6mg/g of prosthesis) . The antibiotic was coupled in two modes: stable covalent binding or weak adhesion . The results confirmed that only a small quantity of the antibiotic (1.03-3.09%) was bound by adsorption . The modification of the prosthesis surface with immobilized gentamicin was visualized with a scanning microscope (SEM) . Bacteriostatic properties of bound gentamicin were verified against different concentrations (cfu) of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains . We have found lack of growth of these pathogen strains in Luria-Bertani (LB) medium containing pieces of gentamicin-coupled prosthesis during at least 28 days of the experiment . Contrary to that, a control medium containing pieces of prosthesis only soaked with gentamicin allowed a constant growth of bacteria. Eur J Biochem, 2004 Dec, 271(23-24), 4968 - 77 Structure of the core oligosaccharide of a rough-type lipopolysaccharide of Pseudomonas syringae pv . phaseolicola; Zdorovenko EL et al.; The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv . phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional (1)H-, (13)C- and (31)P-NMR spectroscopy . Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either l-rhamnose or 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), respectively . Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues . The inner core of P . syringae is distinguished by a high degree of phosphorylation of l-glycero-d-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups . The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa . The outer core composition and structure may be useful as a chemotaxonomic marker for the P . syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas. Eur J Clin Microbiol Infect Dis, 2004 Oct, 23(10), 765 - 71 Comparison of the Micronaut Merlin automated broth microtiter system with the standard agar dilution method for antimicrobial susceptibility testing of mucoid and nonmucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients; Balke B et al.; The aim of this study was to compare a commercially available automated broth microdilution system (Merlin; Micronaut, Germany) with the standard agar dilution method for susceptibility testing of pulmonary isolates from cystic fibrosis patients . Accurate susceptibility testing of bacterial isolates from cystic fibrosis patients is known to pose problems . Although commercially available automated test systems could facilitate susceptibility testing of such isolates in routine diagnostics, these systems have not been recommended thus far . However, a pilot study recently indicated that the Merlin system, which is based on an endpoint measurement rather than on growth curves, might be applicable in the susceptibility testing of isolates from cystic fibrosis patients . In the present study, the Merlin system was further evaluated using an extended panel of nonmucoid and mucoid Pseudomonas aeruginosa isolates . The results showed that the MICs obtained with the Merlin system tended to be lower than those obtained with the agar dilution method, a finding that became increasingly apparent when mucoid Pseudomonas aeruginosa strains were tested . The correlation coefficients (r values) of the MIC results for all strains tested were between 0.6 and 0.8 for five of the seven antimicrobial agents, with r values exceeding 0.8 for only meropenem and ciprofloxacin . However, since the overall rate of serious discrepancies was within an acceptable range, the Merlin system appears to be applicable for routine clinical use in susceptibility testing of P . aeruginosa isolates from cystic fibrosis patients. Cornea, 2005 Jan, 24(1), 51 - 8 Trends in contact lens-related corneal ulcers; Mah-Sadorra JH et al.; PURPOSE:: To analyze the frequency of contact lens-related corneal ulcers and its relationship to the type of contact lens and care . METHODS:: Charts of 376 patients with corneal ulcers seen at the Cornea Service of Wills Eye Hospital from July 1, 1999 to December 31, 2002 were retrospectively reviewed . All patients with contact lens-related corneal ulcers were identified, and information regarding cultures, lens type, usage, and cleaning was obtained . RESULTS:: Of the 376 cases, 113 (30%) were related to contact lens use . The contact lens history was recorded in 83 of 113 cases (73%) . The soft daily wear frequent replacement lens was the most common lens type associated with corneal ulcers (n = 36/83, 43%) . Corneal cultures were performed in 71 of 113 cases (63%) and were positive in 51 of 71 cases (72%) . The most common microorganism involved was Pseudomonas aeruginosa (n = 17/51, 33%) . The frequency of contact lens-related corneal ulcers from 1999-2002 (n = 113/376, 30%) was significantly greater than that from years 1996-1999 (n = 37/299, 12%) at our institution (P < 0.05) . CONCLUSION:: There was a significant increase in the number of contact lens-related corneal ulcers between 1999 and 2002 compared with previous years (P < 0.05) . The contact lens type most frequently associated with corneal ulcers was the soft daily-wear frequent-replacement contact lens (43%) . Contact lens-related corneal ulcers continue to be a serious problem despite a shift in the market to the use of frequent-replacement daily-wear contact lenses and advances in contact lens technology. Ann Dermatol Venereol, 2004 Nov, 131(11), 975 - 8 {Pseudomonas eccrine hidradenitis in a child revealing acute lymphoblastic leukemia}; Laffitte E et al.; INTRODUCTION: We report the case of a Pseudomonas (P.) aeruginosa eccrine hidradenitis in a child, or a "Pseudomonas Hot Foot Syndrome", revealing an acute lymphoblastic leukemia . OBSERVATION: A 10 year-old girl consulted for the sudden onset of painful and necrotic palmoplantar nodules in a context of fever and shivering . Histology of a cutaneous biopsy found necrosis of the eccrine glands and, on culture, P . aeruginosa . The blood count revealed pancytopenia and the myelogram acute lymphoblastic leukemia . All the hemocultures and other microbiological samples were negative . The cutaneous signs had appeared 48 hours after bathing in an aquatic amusement park . Diagnosis of Pseudomonas eccrine hidradenitis, or "Pseudomonas Hot Foot Syndrome" was retained, although the local sanitary authorities were not able to demonstrate P . aeruginosa contamination of the water in the park . COMMENTS: Lesions evoking juvenile Pseudomonas aeruginosa eccrine hidradenitis without obvious traumatic factor must lead to the search for P . aeruginosa contamination from water and the subsequent sanitary and epidemiological consequences . Furthermore, severe P . aeruginosa cutaneous infections in children must also lead to the search for an underlying immunosuppression and notably an acute leukemic process. Ann Plast Surg, 2004 Dec, 53(6), 570 - 6 Use of the vastus lateralis muscle flap with a grooving procedure in the surgical treatment of chronic osteomyelitis of the femur; Necmioglu S et al.; Severe femoral fractures may be associated with devascularization of cortical bone, soft-tissue loss, and significant morbidity . After surgical treatment of these femoral fractures, chronic infection may ensue and requires additional reconstructive procedures . Local muscle flap coverage is used to treat chronic osteomyelitis . A new procedure-the vastus lateralis muscle flap with grooving of the femoral shaft-was used for the treatment of chronic osteomyelitis of the femoral shaft . The authors present 6 patients with chronic osteomyelitis of the femur who were treated with a vastus lateralis muscle flap . Five of the patients were male and the other was female . The average age of the patients was 33.8 years (range, 17-54 years) . All patients experienced infection during the early postoperative period . Drainage of abscess, debridement, sequestrectomy repair of fistula, and mini fenestration were performed at least 3 times, and antibiotics were administered several times . During the operations, tissue samples were evaluated for bacterial cultivation . Staphylococcus aureus was seen in 4 patients, S . epidermidis in 1 patient, and Pseudomonas aeruginosa in the remaining patient . A vastus lateralis muscle flap with grooving of the infected femoral shaft is presented . The authors have not encountered a recurrence of infection during a minimum 3.9 years of follow-up . They think this technique is an alternative to the current techniques for the surgical treatment of chronic osteomyelitis of the femur. J Bacteriol, 2005 Jan, 187(1), 366 - 75 Identification of a Dichelobacter nodosus ferric uptake regulator and determination of its regulatory targets; Parker D et al.; The expression of iron regulated genes in bacteria is typically controlled by the ferric uptake regulator (Fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, and virulence . We have identified a fur homologue in Dichelobacter nodosus, the causative agent of ovine footrot, and shown that it complements an Escherichia coli fur mutant . Homology modeling of the D . nodosus Fur protein with the recently solved crystal structure of Fur from Pseudomonas aeruginosa indicated extensive structural conservation . As Southern hybridization analysis of different clinical isolates of D . nodosus indicated that the fur gene was present in all of these strains, the fur gene was insertionally inactivated to determine its functional role . Analysis of these mutants by various techniques did not indicate any significant differences in the expression of known virulence genes or in iron-dependent growth . However, we determined several Fur regulatory targets by two-dimensional gel electrophoresis coupled with mass spectrometry . Analysis of proteins from cytoplasmic, membrane, and extracellular fractions revealed numerous differentially expressed proteins . The transcriptional basis of these differences was analyzed by using quantitative reverse transcriptase PCR . Proteins with increased expression in the fur mutant were homologues of the periplasmic iron binding protein YfeA and a cobalt chelatase, CbiK . Down-regulated proteins included a putative manganese superoxide dismutase and ornithine decarboxylase . Based on these data, it is suggested that in D . nodosus the Fur protein functions as a regulator of iron and oxidative metabolism. J Mol Evol, 2004 Dec, 59(6), 725 - 37 Evolutionary analysis of the two-component systems in Pseudomonas aeruginosa PAO1; Chen YT et al.; Gene organization and functional motif analyses of the 123 two-component system (2CS) genes in Pseudomonas aeruginosa PAO1 were carried out . In addition, NJ and ML trees for the sensor kinases and the response regulators were constructed, and the distances measured and comparatively analyzed . It was apparent that more than half of the sensor-regulator gene pairs, especially the 2CSs with OmpR-like regulators, are derivatives of a common ancestor and have most likely co-evolved through gene pair duplication . Several of the 2CS pairs, especially those with NarL-like regulators, however, appeared to be relatively divergent . This is supportive of the recruitment model, in which a sensor gene and regulator gene with different phylogenetic history are assembled to form a 2CS . Correlation of the classification of sensor kinases and response regulators provides further support for these models . Upon comparison of the phylogenetic trees comprised of sensors and regulators, we have identified six congruent clades, which represent the group of the most recently duplicated 2CS gene pairs . Analyses of the congruent 2CS pairs of each of the clades revealed that certain paralogous 2CS pairs may carry a redundant function even after a gene duplication event . Nevertheless, comparative analysis of the putative promoter regions of the paralogs suggested that functional redundancy could be prevented by a differential control . Both codon usage and G+C content of these 2CS genes were found to be comparable with those of the P . aeruginosa genome, suggesting that they are not newly acquired genes. Crit Care Med, 2004 Dec, 32(12), 2502 - 7 Host response to intratracheally instilled bacteria in ventilated and nonventilated rats; Brackenbury AM et al.; OBJECTIVE: Pneumonia occurs in approximately 7% of hospitalized patients . Susceptibility to certain bacteria such as Pseudomonas aeruginosa increases in critically ill patients, particularly those requiring mechanical ventilation . Previous studies investigating this susceptibility have used injurious modes of ventilation . The objective of this study was to evaluate the host's response to intratracheal instillation of P . aeruginosa in the setting of noninjurious mechanical ventilation and compare this with normal, spontaneously breathing animals receiving bacteria . DESIGN: Randomized, controlled in vivo animal study . SETTING: Research laboratory at a university-affiliated institution . SUBJECTS: Adult male Sprague-Dawley rats . INTERVENTIONS: Rats were randomized into four groups: spontaneously breathing given saline, spontaneously breathing given bacteria, mechanically ventilated given saline, and mechanically ventilated given bacteria . The ventilation strategy used involved low stretch (tidal volume of 8 mL/kg) with a positive end-expiratory pressure of 5 cm H2O . MEASUREMENTS AND MAIN RESULTS: Lung compliance, bacterial recovery, surfactant, total cells, and cytokine concentrations in the lung lavage were analyzed after 4 hrs . Results showed that neither ventilation nor bacteria alone altered lung function, although the combination of ventilation and Pseudomonas significantly decreased arterial oxygenation and lung compliance . Increases in lavage cell counts, cytokines, and surfactant were observed in both groups administered bacteria compared with animals given saline . However, there were no significant differences in bacterial recovery, cell counts, cytokines, and surfactant measurements in the groups given bacteria . CONCLUSIONS: These data suggest that bacterial instillation with low-stretch ventilation had a significant effect on lung function but did not alter the inflammatory response to a bacterial challenge over this time course compared with spontaneously breathing animals. Crit Care Med, 2004 Dec, 32(12), 2491 - 5 Role of atrial natriuretic peptide in pulmonary permeability and vasoregulation in ovine sepsis; Stubbe HD et al.; OBJECTIVE: Atrial natriuretic peptide is regarded as an important regulator of pulmonary vasomotor tone and permeability . This study investigated the role of atrial natriuretic peptide in sepsis-associated pulmonary pathophysiology . DESIGN: Prospective experimental investigation . SETTING: Laboratory at a university hospital . SUBJECTS: Twelve awake, chronically instrumented sheep . INTERVENTIONS: The sheep were instrumented with lung lymph fistulas and received a continuous infusion with live Pseudomonas aeruginosa for 48 hrs . After 40 hrs, the atrial natriuretic peptide-receptor antagonist HS-142-1 was continuously infused in the HS-124-1 group (3 mg/kg/hr, n = 6) for 8 hrs, whereas the control group received the carrier (n = 6) . MEASUREMENTS AND MAIN RESULTS: Lung lymph flow was markedly elevated in response to sepsis after 40 hrs in both groups . Atrial natriuretic peptide-receptor blockade further increased lymph flows by 41 +/- 17% (41 hrs) up to 64 +/- 20% (44 hrs, p < .05) in the presence of normal permeability to protein . Although mean pulmonary artery pressure increased (p < .05 vs . 40 hrs), capillary pressure remained unaffected . Despite identical fluid balances in both groups, cardiovascular filling variables significantly increased in the HS-142-1 group . This was associated with increasing cardiac index and mean arterial pressure (p < .05 vs . 40 hrs) . In the control group, all variables remained constant between 41 and 48 hrs . CONCLUSION: Blockade of atrial natriuretic peptide receptors increases pulmonary transvascular fluid flux independent of changes in permeability to protein in chronic ovine sepsis . Atrial natriuretic peptide may therefore play a protective role for the alveolar-capillary barrier during sepsis. J Antimicrob Chemother . 2004 Dec 8; {Epub ahead of print} Liposome-mediated gentamicin delivery: development and activity against resistant strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients; Mugabe C et al.; OBJECTIVES: Chronic pulmonary infection by Pseudomonas aeruginosa in cystic fibrosis patients is virtually impossible to eradicate by means of existing free antibiotics . We sought to assess the antibacterial activities of liposomal gentamicin against clinical isolates of P . aeruginosa . METHODS: Gentamicin was encapsulated into liposomes with different lipid compositions (1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol in the molar ratio of 2:1 by sonication . The in vitro stability of liposome-encapsulated gentamicin was studied over a 48 h period at 4 and 37 degrees C in PBS and at 37 degrees C in pooled plasma . The MICs of free and liposomal gentamicin for clinical isolates of P . aeruginosa were assessed by broth dilution . RESULTS: The encapsulation efficiency of all liposomal preparations was 4%-5.18% of the initial amount of the drug in solution . The liposomes retained 60%-70% of the encapsulated gentamicin for 48 h when they were incubated in normal human pooled plasma or PBS at 4 or 37 degrees C . The MICs of liposomal gentamicin for all clinical isolates of P . aeruginosa were lower than the MICs of free gentamicin . Importantly, liposomal gentamicin altered the susceptibilities of these clinical isolates from gentamicin resistant to either intermediate or susceptible . CONCLUSIONS: Taken together, these data indicate that liposomal gentamicin formulations could be more effective than the free drug in controlling pulmonary infections due to P . aeruginosa. Curr Eye Res, 2004 Oct-Nov, 29(4-5), 225 - 33 Regulation of Pseudomonas aeruginosa corneal infection in IL-1beta converting enzyme (ICE, caspase-1) deficient mice; Thakur A et al.; Purpose . Antibody neutralization studies have shown that in Pseudomonas aeruginosa corneal infection, IL-1beta is critical to regulation of the host inflammatory response, but mechanisms remain undetermined . To elucidate these mechanisms, caspase-1 knockout (ICE(-/-)) mice, that do not release mature IL-1beta after endotoxin challenge, were tested . Methods . Clinical scores, MPO activity (for PMN quantitation), bacterial plate count, semiquantitative RT-PCR, ELISA and TUNEL staining were used to characterize the inflammatory response after infection in knockout and C57BL/6 (B6) wild type mice . Results . Clinical scores were significantly reduced in ICE(-/-) vs . B6 mice at 3, 5 and 7 days postinfection (p.i.) . The decreased inflammatory response of ICE(-/-) mice was striking at 1 day p.i., and bacterial load also was significantly reduced in the cornea of the knockout mice at 3-7 days p.i . Knockout mice exhibited significantly increased mRNA and protein levels for IL-1Ra, the physiological regulator of IL-1 activity, and in addition, a significant increase in the number of apoptotic cells were quantitated in the corneal epithelium of ICE(-/-) vs . B6 mice at 1 day p.i . Conclusions . These data provide evidence that bacterial infection in the cornea of ICE(-/-) mice induces a reduced inflammatory response by: reduction in PMN and cytokines and chemokines that attract these cells to the cornea; enhanced apoptotic cell death in the infected epithelium; and increased IL-1Ra levels . The data also confirm the importance of IL-1 regulation in this model and suggest that ICE inhibition may be an attractive ancillary therapeutic strategy to control the host response to this pathogen. Prostaglandins Leukot Essent Fatty Acids, 2005 Jan, 72(1), 41 - 7 The significance of oxidant/antioxidant balance for the pathogenesis of experimental sepsis by multidrug-resistant Pseudomonas aeruginosa; Koussoulas V et al.; Objective: The significance of lipid peroxidation as an independent factor leading to sepsis by multidrug-resistant Pseudomonas aeruginosa . Design experimental study Methods: Twenty-six rabbits were applied . They were divided into two groups; A (n=6) comprising controls, and B (n=20) comprising animals infected by the injection of 1x10(8)cfu/kg inoculum of the test pathogen into the left inner jugular vein . Six rabbits of group B were followed-up to estimate survival; all of the remaining were sacrificed . Blood was sampled for the determination of serum malondialdehyde (MDA) by the thiobarbiturate assay, total antioxidant status (TAS) by a chromogenic assay, tumor necrosis factor alpha by a bioassay on fibrosarcoma L929 cell line, and endotoxins (LPS) by the QCL-1000 LAL assay . Results: Mean survival of group B was 60.0+/-15.8h . MDA was significantly higher in group B compared to group A at 30, 60, 120 and 150min . TAS was statistically decreased in group B compared to group A at 30 and 60min . Increases of MDA in group B were followed by reciprocal decreases of TAS (P of correlation <0.001) . Hemodynamic instability was recorded in group B compared to group A 160min after bacterial challenge . Conclusions: Early alterations of oxidant/antioxidant balance occur in experimental sepsis by multidrug-resistant P . aeruginosa followed by hemodynamic instability . Results highlight the perspective of the administration of antioxidants as immunomodulatory treatment of sepsis in animal studies. J Clin Microbiol, 2004 Dec, 42(12), 5783 - 92 Genome diversity of Pseudomonas aeruginosa isolates from cystic fibrosis patients and the hospital environment; Finnan S et al.; Pseudomonas aeruginosa is a gram-negative rod that is ubiquitous in nature . P . aeruginosa is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts . In cystic fibrosis patients, P . aeruginosa is the leading cause of death . In this study, the evolutionary genetic relationships among 17 P . aeruginosa isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone groEL . The P . aeruginosa isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere . Phylogenetically, clinical and environmental isolates clustered together with one another on the mdh gene tree . At the groEL locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments . Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase . Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions . Among the clinical isolates examined, the 15 virulence regions were variably present . The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions . Of the four genomic islands (the flagellum island and PAGI-1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI-1, -2, and -3 were absent from all isolates tested . Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen. J Clin Microbiol, 2004 Dec, 42(12), 5644 - 9 Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa; Curran B et al.; A multilocus sequence typing (MLST) scheme has been developed for Pseudomonas aeruginosa which provides molecular typing data that are highly discriminatory and electronically portable between laboratories . MLST data confirm the data from previous studies that suggest that P . aeruginosa is best described as nonclonal but as having an epidemic population . The index of association was 0.17, indicating a freely recombining population; however, there was evidence of clusters of closely related strains or clonal complexes among the members of this population . It is apparent that the sequence types (STs) from single isolates, representing each of the present epidemic clones in the United Kingdom from Liverpool, Manchester, and the West Midlands, are not closely related to each other . This suggests distinct evolutionary origins for each of these epidemic clones in the United Kingdom . Furthermore, these clones are distinct from European clone C . Comparison of the results of MLST with those of toxA typing and serotyping revealed that strains with identical STs may possess different toxA types and diverse serotypes . Given that recombination is important in the population of P . aeruginosa, the lack of a linkage between toxA type and serotype is not surprising and reveals the strength of the MLST approach for obtaining a better understanding of the epidemiology of P . aeruginosa. FEBS Lett, 2004 Dec 3, 578(1-2), 5 - 9 A model of a transmembrane drug-efflux pump from Gram-negative bacteria; Fernandez-Recio J et al.; In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins . A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA . The pump displaces drugs vectorially from the bacterium using proton electrochemical force . Crystal structures are available for TolC and AcrB from E . coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa . Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport. Int J Tuberc Lung Dis, 2004 Nov, 8(11), 1301 - 7 Exhaled nitric oxide in bronchiectasis: the effects of inhaled corticosteroid therapy; Tsang KW et al.; SETTING: While exhaled nitric oxide (eNO) levels are reduced by inhaled corticosteroid therapy in asthma, such treatment effect is unclear in bronchiectasis . DESIGN: Stable non-smoking bronchiectasis patients were randomised to receive either fluticasone (1 mg/daily) or identical placebo via the Accuhaler device . RESULTS: Sixty non-smoking patients (38 women; mean age 56.4 +/- 12.7 years) were recruited . Of these, half received inhaled fluticasone and half placebo therapy . eNO was measured using a chemiluminescence analyser at 0, 4, 12, 24, 36 and 52 weeks . There was no significant difference in eNO levels between fluticasone and placebo patients over the study period . There was no correlation between baseline eNO with age, FEV1, FVC, 24 h sputum volume or number of bronchiectatic segments . Patients with Pseudomonas aeruginosa (PA) infection, but not their counterparts, displayed a correlation between 0- and 52-week eNO levels . PA infection was associated with significantly lower eNO