Pseudomonas aeruginosa

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 100–193 kDa in size . We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100–108 kDa), medium (122–151 kDa), and large (181–193 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 levels among the patients . CONCLUSIONS: Inhaled fluticasone therapy, despite being an effective anti-inflammatory agent, has no significant effect on eNO production, either at individual time points or over the entire 52-week profile, in bronchiectasis . It appears that eNO might not reflect the extent of airway inflammation in bronchiectasis.

Cytokine, 2005 Jan 7, 29(1), 18 - 23
Interleukin-8 in whole blood and clinical status in cystic fibrosis; Schmitt-Grohe S et al.; Cytokines and polymorphonuclear leukocytes play a key role in immune mediated inflammation in progressive pulmonary damage due to cystic fibrosis . The aim of this study is to establish a simple measure of the host's propensity to secrete inflammatory cytokines and to correlate this with clinical status . Patients (n=44, median age 16 years) with the DeltaF 508 mutation (homozygous) were grouped according to their Shwachman score: Patients with mild disease (Shwachman score 71-100 points, group A, n=22, median FEV(1) 79%) were compared with those with more severe disease (Shwachman score 41-55 points, group B, n=22, median FEV(1) 55%) and age-matched controls (group C, n=22, median FEV(1) 102%) . Whole blood was stimulated with 5 ng of lipopolysaccharide (LPS) . Interleukin-8 (IL-8) was measured by chemiluminescent immunometric assay (DPC, Bad Nauheim, Germany) . Though there was a significant difference at baseline for IL-8 (median group A/B/C 6.1/30.5/5.8 pg/ml; p<0.001), there was no significant difference after stimulation . Moreover, in Pseudomonas aeruginosa positive (Psa+) patients (n=26) there was a significant negative correlation (r=-0.539; p<0.004) between baseline IL-8 and FEV(1) (%) . Clinical course and lung function (in Psa+) correlate with IL-8 levels.

Inorg Chem, 2004 Dec 13, 43(25), 7926 - 33
Role of cofactors in folding of the blue-copper protein azurin; Wittung-Stafshede P; Many proteins in living cells coordinate cofactors, such as metal ions, to attain their activity . Since the cofactors in such cases often can interact with their corresponding unfolded polypeptides in vitro, it is important to unravel how cofactors modulate protein folding . In this review, I will discuss the role of cofactors in folding of the blue-copper protein Pseudomonas aeruginosa azurin . In the case of both copper (Cu(II) and Cu(I)) and zinc (Zn(II)), the metal can bind to unfolded azurin . The residues involved in copper (Cu(II) and Cu(I)) coordination in the unfolded state have been identified as Cys112, His117, and Met121 . The affinities of Cu(II), Cu(I), and Zn(II) are all higher for the folded than for the unfolded azurin polypeptide, resulting in metal stabilization of the native state as compared to the stability of apo-azurin . Cu(II), Zn(II), and several apo forms of azurin all fold in two-state kinetic reactions with roughly identical polypeptide-folding speeds . This suggests that the native-state beta-barrel topology, not cofactor interactions or thermodynamic stability, determines azurin's folding barrier . Nonetheless, copper binds much more rapidly (i.e., 4 orders of magnitude) to unfolded azurin than to folded azurin . Therefore, the fastest route to functional azurin is through copper binding before polypeptide folding; this sequence of events may be the relevant biological pathway.

Vaccine, 2004 Dec 6, 22 Suppl 1, S44 - 8
Prophylaxis and therapy of Pseudomonas aeruginosa infection in cystic fibrosis and immunocompromised patients; Lang AB et al.; Pseudomonas aeruginosa is an opportunistic bacterium responsible for chronic lung infection in cystic fibrosis patients, as well as nosocomial infections in immunocompromised patients . An O-polysaccharide-toxin A conjugate vaccine was evaluated for prophylaxis of P . aeruginosa in cystic fibrosis patients . Vaccination proved to be useful in preventing and/or delaying infection . Fully human monoclonal antibodies (mAb) against P . aeruginosa O-polysaccharides were developed for the treatment of immunocompromised patients in whom active immunoprophylaxis is not applicable . Characterisation of the mAb revealed high antigen specificity and avidity, as well as excellent efficacy in relevant in vitro and in vivo systems, permitting future clinical evaluation.

Appl Environ Microbiol, 2004 Dec, 70(12), 7404 - 12
Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism; Renninger N et al.; The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains . Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium . The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation . Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions . Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane . TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell . Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.

J Antimicrob Chemother, 2005 Jan, 55(1), 22 - 30 Epub 2004 Dec 1.
Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa; Kugelberg E et al.; OBJECTIVES: Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNA gyrase/topoisomerase IV, or cause activation of various efflux systems . We have analysed the effect of quinolone resistance caused by DNA gyrase/topoisomerase IV mutations on bacterial fitness . METHODS: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified . Mutant fitness was determined by measuring growth rates in vitro . Mutants with reduced growth rates were serially passaged to obtain growth-compensated mutants . The level of DNA supercoiling was determined by isolating plasmid DNA from the susceptible, resistant and compensated mutants and comparing the topoisomer distribution patterns by gel electrophoresis in the presence of chloroquine . RESULTS: Low-level resistance (4-48 mg/L) was caused by single mutations in gyrA or gyrB . Among these strains, three out of eight mutants showed lower fitness, whereas high-level resistant (>256 mg/L) mutants with double mutations in gyrA and parC, parE, nfxB or unknown genes all showed a reduced fitness . Slow-growing resistant mutants with a gyrA mutation had decreased DNA supercoiling . After serial passage in laboratory medium, mutant fitness was increased by compensatory mutation(s) that restored supercoiling to normal levels . The compensatory mutation(s) was not located in any of the genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) that were expected to affect supercoiling . CONCLUSIONS: Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P . aeruginosa.

J Antimicrob Chemother, 2005 Jan, 55(1), 57 - 60 Epub 2004 Dec 1.
Fluoroquinolone consumption and resistance in haematology-oncology patients: ecological analysis in two university hospitals 1999-2002; Kern WV et al.; OBJECTIVES: To compare rates of in vitro fluoroquinolone resistance of bacterial isolates obtained from inpatients of two haematology-oncology services with high and low fluoroquinolone consumption . METHODS: Two hospitals with consistently high (A) and low (B) fluoroquinolone use in their haematology-oncology services between the years 1999 and 2002 were identified in a hospital antibiotic use surveillance project . Rates of in vitro resistance to fluoroquinolones in inpatients of the services were determined for Escherichia coli and coagulase-negative staphylococcal bloodstream isolates, and also for Pseudomonas aeruginosa and Staphylococcus aureus isolates from any site . RESULTS: Fluoroquinolone resistance of E . coli was significantly higher in hospital A than in hospital B, but there was no such correlation between fluoroquinolone use and resistance rates for P . aeruginosa and staphylococci . CONCLUSION: The impact of antibiotic consumption on the prevalence of resistance may differ widely between different pathogens . Interventions using ecological analyses of the relationship between hospital antibiotic use and resistance need to consider pathogen-specific dynamics in the emergence and control of bacterial resistance.

J Antimicrob Chemother, 2005 Jan, 55(1), 61 - 70 Epub 2004 Dec 1.
Italian metallo-{beta}-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme; Toleman MA et al.; OBJECTIVES: As part of the SENTRY Antimicrobial Surveillance Programme, 383 non-replicative randomly collected Pseudomonas aeruginosa isolates were collected during 1999-2002 . These strains originated from three geographically distinct hospitals within Italy: Genoa (Northern Italy); Rome and Catania (Sicily), and were further studied to identify the prevalence of metallo-beta-lactamase (MbetaL) alleles across Italy and to determine their genetic details . METHODS: Multidrug-resistant (MDR) strains were identified by MIC analysis followed by genotyping and PCR-based strategies . RESULTS: Initial MIC analysis identified 31 MDR isolates that displayed an Etest MbetaL-positive phenotype . Of these, 25 produced either the MbetaL VIM-1 or IMP-13 as detected by PCR and sequencing . VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome . MbetaL-producing isolates were found at all Italian SENTRY sites and together amounted to 6.5% of all P . aeruginosa isolates . Genetic analysis indicated that many strains contained multiple integrons and identified two novel MbetaL integrons, one from the site in Genoa and one from Sicily . Integrons identical in structure and sequence to In70, the first identified and characterized bla(VIM)-containing integron from Verona, were found in isolates with distinct ribotypes at the Roman and Sicilian sites indicating that this integron has recently disseminated across Italy . All 25 MbetaL-producing isolates were genetically linked in that all isolates contained Tn5051 sequences and all harboured the insertion sequence IsPa7 which may be involved in the mobilization of these resistance alleles . CONCLUSIONS: Taken together, these results indicate that Italy has a nationwide problem of MDR P . aeruginosa produced by mobile MbetaL genes.

Proteins . 2004 Nov 30; {Epub ahead of print}
High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 A resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches; Mitchell EP et al.; PA-IIL is a fucose-binding lectin from Pseudomonas aeruginosa that is closely related to the virulence factors of the bacterium . Previous structural studies have revealed a new carbohydrate-binding mode with direct involvement of two calcium ions (Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Perez S, Wu AM, Gilboa-Garber N, Imberty A . Structural basis for selective recognition of oligosaccharides from cystic fibrosis patients by the lectin PA-IIL of Pseudomonas aeruginosa . Nat Struct Biol 2002;9:918-921) . A combination of thermodynamic, structural, and computational methods has been used to study the basis of the high affinity for the monosaccharide ligand . A titration microcalorimetry study indicated that the high affinity is enthalpy driven . The crystal structure of the tetrameric PA-IIL in complex with fucose and calcium was refined to 1.0 A resolution and, in combination with modeling, allowed a proposal to be made for the hydrogen-bond network in the binding site . Calculations of partial charges using ab initio computational chemistry methods indicated that extensive delocalization of charges between the calcium ions, the side chains of the protein-binding site and the carbohydrate ligand is responsible for the high enthalpy of binding and therefore for the unusually high affinity observed for this unique mode of carbohydrate recognition . Proteins 2005 . (c) 2005 Wiley-Liss, Inc.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Nov, 24(11), 1299 - 300, 1303
{Investigation of cross-infection of Pseudomonas aeruginosa in an intensive care unit.}; Lai FC et al.; OBJECTIVE: To investigate the factors responsible for Pseudomonas aeruginosa nosocomial cross-infection in the intensive care unit (ICU) and provide effective measure for the prevention and management . METHODS: The homology of 7 Pseudomonas aeruginosa strains isolated from the patients in the ICU and the environment was examined by biological, serological, drug-resistance and plasmid analysis . RESULTS: The results of serological and plasmid analyses had good consistency, which demonstrated that the 7 Pseudomonas aeruginosa strains originated from solution in the oxygen humidifier, with uniform serological and plasmid type, and belonged to the same clone with cross-infection by contact between the patients or nursing staff and the objects in the ICU . CONCLUSION: Serological and plasmid analyses are more practical for identifying the sources of Pseudomonas aeruginosa cross-infection, which can be prevented by strict disinfection of the instrument and supervision of the nursing staff in the ICU.

Trends Mol Med, 2004 Dec, 10(12), 599 - 606
The role of pyocyanin in Pseudomonas aeruginosa infection; Lau GW et al.; Pyocyanin (PCN) is a blue redox-active secondary metabolite that is produced by Pseudomonas aeruginosa . PCN is readily recovered in large quantities in sputum from patients with cystic fibrosis who are infected by P . aeruginosa . Despite in vitro studies demonstrating that PCN interferes with multiple cellular functions, its importance during clinical infection is uncertain . This is partially caused by the difficulty in defining the contribution of PCN among the numerous virulence factors produced by P . aeruginosa during infection . In addition, few cellular pathways that are affected by PCN are known . This review briefly highlights recent advances that might clarify the role of PCN in P . aeruginosa pathogenesis.

Fitoterapia, 2004 Dec, 75(7-8), 758 - 9
Antibacterial activity of the essential oil from Ferula gummosa seed; Eftekhar F et al.; Antibacterial activity of Ferula gummosa essential oil was studied against bacterial laboratory ATCC standards using the disk diffusion method . The results showed activity against Gram(+) bacteria and Escherichia coli . Little antibacterial activity was found against Pseudomonas aeruginosa.

J Microbiol Methods, 2005 Jan, 60(1), 21 - 30
Optimisation of the fluorescein diacetate antibacterial assay; Wanandy S et al.; The fluorescein diacetate (FDA) antibacterial assay relies on the cleavage of fluorescein diacetate by metabolically active bacteria . The recent finding that microbiological media can lead to significant levels of cleavage has reduced the reliability of the assay . Using the nucleophilic scavengers N-ethylmaleimide and maleic anhydride, we have demonstrated that this abiotic cleavage is most likely due to nucleophiles such as cysteine and histidine commonly present in the media . To increase the reliability of the assay we have modified the original assay conditions to include use of dilute medium (peptone 0.2% w/v, yeast extract 0.1% w/v and NaCl 0.1% w/v) in a non-nucleophilic buffer and overnight incubation of the medium after addition of antibacterial agents . The optimised fluorescein diacetate assay has been used to determine the MIC of gentamicin, tetracycline and chloramphenicol for Escherichia coli, Staphyloccocus aureus and Pseudomonas aeruginosa and gave quantitative results that were reproducible and consistent with published data.

Crit Care, 2004 Dec, 8(6), R491 - 4 Epub 2004 Dec.
Case report: greater meningeal inflammation in lumbar than in ventricular region in human bacterial meningitis; Naija W et al.; Differences in the composition of ventricular and lumbar cerebrospinal fluid (CSF) based on single pairs of samples have previously been described . We describe a patient that developed post-surgical recurrent meningitis monitored by daily biochemical and bacteriological CSF analysis, simultaneously withdrawn from lumbar space and ventricles . A 20-year-old Caucasian man was admitted to the ICU after a resection of a chordoma that extended from the sphenoidal sinus to the anterior face of C2 . CSF was continuously leaking into the pharyngeal cavity after surgery, and three episodes of recurrent meningitis, all due to Pseudomonas aeruginosa O12, occurred . Our case showed permanent ventricular-to-lumbar CSF gradients of leukocytes, protein and glucose that were increased during the acute phase of meningitis, with the greatest amplitude being observed when bacteria were present in both ventricular and lumbar CSF . This might suggest a greater extent of meningeal inflammation in the lumbar than in the ventricular region . Our case also showed that the increase in intravenous antibiotics (cefepim from 8 to 12 g/day and ciprofloxacine from 1.2 to 2.4 g/day) led to an increase in concentration in plasma but not in CSF.

Infect Control Hosp Epidemiol, 2004 Nov, 25(11), 997 - 1000
Should electronic faucets be recommended in hospitals?
Chaberny IF, Gastmeier P.
Microbiological examinations of electronic faucets newly installed in a hospital kitchen revealed high bacteria counts and Pseudomonas aeruginosa during a 6-month period of observation . Our data suggest that the use of electronic faucets poses a potential risk for nosocomial infection in high-risk areas of hospitals.

J Chemother, 2004 Oct, 16(5), 437 - 41
Antimicrobial susceptibility patterns in Pseudomonas aeruginosa: data from a multicenter Intensive Care Unit Surveillance Study (ISS) in the United States; Friedland I et al.; The objectives of this study were to analyze susceptibility rates and patterns in Pseudomonas aeruginosa isolates from patients in intensive care units (ICU) . A total of 2209 isolates in 1995/1996 and 2672 in 2001/2002 were tested at United States sites participating in the ICU Surveillance Study . In both periods, of the agents tested, amikacin was the most active and ciprofloxacin, the least . Resistance to common antipseudomonal agents tested increased from 1995/1996 to 2001/2002; the rise was least for amikacin (2%) and greatest for ciprofloxacin (16%) . The proportion of isolates susceptible to all six antipseudomonal agents tested since 1996 decreased from 60.4% to 48.9% in 2001/2002 . Examination of MIC distributions for the two periods showed that for some drugs, e.g . imipenem and ceftazidime, the populations of susceptible and resistant isolates remained distinct, although the resistant population increased . For other drugs, e.g . amikacin and piperacillin-tazobactam, the MIC distribution shifted upward over time . The categorical agreement between agents of the same or like classes for isolates tested in 2001/2002 was highest for ciprofloxacin and levofloxacin (93.2%, with 1.2% major errors) and lowest for the aminoglycosides (81.3%, with 10.2% major errors) . We can conclude that resistance to antipseudomonal agents among ICU isolates of P . aeruginosa, especially fluoroquinolones, is increasing . The resistance rate for some antipseudomonal agents may not accurately reflect shifts in the MIC distribution curve.

Transpl Int . 2004 Nov 24; {Epub ahead of print}
The incidence and importance of bacterial contaminants of cadaveric renal perfusion fluid; Wakelin SJ et al.; Infections represent a significant risk in the postoperative transplant recipient . The perfusion fluid used to perfuse and preserve the kidneys prior to transplantation represents a potential medium in which organisms can grow . The aim of this study was to determine the incidence and clinical relevance of bacterial contamination of perfusion fluid . A total of 4 centres participated in the study and 269 perfusion fluid samples were taken for microbiological analysis . Organisms were isolated from 38 out of 218 (17.4%) perfusion fluid samples taken prior to allograft implantation and 23 out of 51 (45%) samples taken at procurement . Low virulence organisms predominated although Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were also isolated . Although infective complications were not seen in the allograft recipients, given the frequency with which contamination occurs and the variation in unit antibiotic protocols, we recommend the routine culturing of perfusion fluid to ensure that any potentially significant organisms are identified and treated appropriately.

Braz J Infect Dis, 2004 Aug, 8(4), 267 - 71 Epub 2004 Aug.
Carbapenem-resistant Pseudomonas aeruginosa outbreak in an intensive care unit of a teaching hospital; Gales AC et al.; The genetic similarity of carbapenem-resistant Pseudomonas aeruginosa strains isolated in the Hospital Universitario Sao Francisco, Braganca Paulista, Sao Paulo, Brasil, was evaluated by pulsed field gel electrophoresis (PFGE) . A unique clone was detected among 5 of 7 isolates, suggesting that cross-contamination might have played a role in the spread of carbapenem-resistant P . aeruginosa strains . Interestingly, a similar PFGE pattern was encountered in a P . aeruginosa strain isolated from Hospital Sao Paulo that was used as a PFGE control.

J Leukoc Biol . 2004 Nov 24; {Epub ahead of print}
Intratracheal administration of liposomal clodronate accelerates alveolar macrophage reconstitution following fetal liver transplantation; Everhart MB et al.; To facilitate study of alveolar macrophages in vivo, we developed a method to rapidly and efficiently replace resident alveolar macrophages with macrophages of a different (donor) genotype . Chimeric mice were generated by lethal irradiation followed by fetal liver transplantation (FLT) using green fluorescent protein (GFP) transgenic reporter mice as donors . Kinetics of peripheral blood monocyte (PBM) and alveolar macrophage reconstitution was determined 4 and 10 weeks post-FLT by quantifying the percentage of GFP+ cells . To enhance the recruitment of donor monocytes into the lung after FLT, mice were treated with intratracheal administration of liposomal clodronate to deplete host alveolar macrophages at 6 weeks post-FLT . PBM reconstitution occurred by 4 weeks after FLT (85.7+/-1.6% of CD11b+/Gr-1+ monocytes were GFP+), and minimal alveolar macrophage repopulation was observed (9.5% GFP+) . By 10 weeks following FLT, 48% of alveolar macrophages were GFP+ by immunostaining of macrophages on lung tissue sections, and 55.1 +/- 1.6% of lung lavage macrophages were GFP+ by fluorescein-activated cell sorter analysis . Clodronate treatment resulted in a significant increase in GFP+ alveolar macrophages 10 weeks after FLT . By immunostaining, 90% of macrophages were GFP+ on lung tissue sections and 87.5 +/- 1.1% GFP+ in lung lavage (compared with GFP-transgenic controls) . The ability of newly recruited alveolar macrophages to clear Pseudomonas aeruginosa and activate nuclear factor-kappaB in response to Eschericia coli lipopolysaccharide demonstrated normal macrophage function . Optimizing this methodology provides an important tool for the study of specific genes and their contribution to alveolar macrophage function in vivo.

Ann Pharmacother, 2005 Jan, 39(1), 39 - 44 Epub 2004 Nov 23.
Inhaled Tobramycin in Non-Cystic Fibrosis Patients with Bronchiectasis and Chronic Bronchial Infection with Pseudomonas aeruginosa; Drobnic ME et al.; BACKGROUND: Non-cystic fibrosis (CF) patients with bronchiectasis usually develop chronic bronchial infection with Pseudomonas aeruginosa (PA) that is related to worsening lung function and increased morbidity and mortality . OBJECTIVE: To determine whether direct aerosol delivery of tobramycin to the lower airways may control infection and produce only low systemic toxicity . METHODS: A double-blind, placebo-controlled crossover trial involving 30 patients was conducted to determine the clinical effectiveness and safety of 6-month tobramycin inhalation therapy . Patients received 300 mg of aerosolized tobramycin or placebo twice daily in 2 cycles, each for 6 months, with a one-month washout period . The number of exacerbations, number of hospital admissions, number of hospital admission days, antibiotic use, pulmonary function, quality of life, tobramycin toxicity, density of PA in sputum, emergence of bacterial resistance, and emergence of other opportunistic bacteria were recorded . RESULTS: The number of admissions and days of admission (mean +/- SD) during the tobramycin period (0.15 +/- 0.37 and 2.05 +/- 5.03) were lower than those during the placebo period (0.75 +/-1.16 and 12.65 +/- 21.8) (p < 0.047) . A decrease in PA density in sputum was associated with tobramycin administration in the analysis of the first 6-month cycle (p = 0.038) . No significant differences were observed in the number of exacerbations, antibiotic use, pulmonary function, and quality of life . The emergence of bacterial resistance and other bacteria did not differ between the 2 periods of study . Inhaled tobramycin was associated with bronchospasm in 3 patients, but not with detectable ototoxicity or nephrotoxicity . CONCLUSIONS: Aerosol administration of high-dose tobramycin in non-CF bronchiectatic patients for endobronchial infection with PA appears to be safe and decreases the risk of hospitalization and PA density in sputum . Nevertheless, pulmonary function and quality of life are not improved, and the risk of bronchospasm is appreciable.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4713 - 7
n-6 polyunsaturated fatty acids enhance the activities of ceftazidime and amikacin in experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa; Giamarellos-Bourboulis EJ et al.; Recent in vitro and ex vivo studies disclosed an enhancement of the activity of antimicrobials on multidrug-resistant Pseudomonas aeruginosa by n-6 polyunsaturated fatty acids (PUFAS); therefore their effect was evaluated in experimental sepsis in 60 rabbits . Solutions of gamma-linolenic acid (GLA) and arachidonic acid (AA) were administered intravenously with ceftazidime and amikacin in rabbits with sepsis caused by one multidrug-resistant isolate . Therapy was started after bacterial challenge in five groups comprising 12 animals in each group: A, normal saline; B, antimicrobials; C, 99% ethanol and antimicrobials; D, GLA and antimicrobials; and E, AA and antimicrobials . Blood was sampled for the estimation of levels of endotoxins in serum (lipopolysaccharide), leukocytes, tumor necrosis factor alpha (TNF-alpha) and antimicrobials . Animals were sacrificed 210 min after bacterial challenge for tissue cultures . All animals had considerable endotoxemia and evolved leukopenia . The number of viable cells in blood, lung, and mesenteric lymph nodes was significantly reduced in groups D and E compared to that in other groups . Levels of antimicrobials in serum were inadequate to achieve bacterial killing due to the level of resistance . n-6 PUFAs did not influence TNF-alpha . It is concluded that intravenous coadministration of n-6 PUFAs and antimicrobials enhanced antimicrobial bacterial killing in experimental sepsis caused by multidrug-resistant P . aeruginosa.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4693 - 702
Integron carrying a novel metallo-beta-lactamase gene, blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6')-30/aac(6')-Ib': report from the SENTRY Antimicrobial Surveillance Program; Mendes RE et al.; Since January 2002 Pseudomonas sp . strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-beta-lactamase production, and their resistance determinants have been analyzed . Pseudomonas aeruginosa index strain 101-4704, which harbors a novel bla(IMP) variant, bla(IMP-16), was isolated in April 2002 from a 60-year-old man in Brasilia, Brazil . bla(IMP-16) was found on the chromosome of the P . aeruginosa index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%) . Sequence analysis revealed that bla(IMP-16) was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes . Downstream of bla(IMP-16) resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely, aac(6')-30, which was fused with aac(6')-Ib' . The amino acid sequence of the aac(6')-30 putative protein showed the most identity (52.7%) to the sequence of AAC(6')-29b described previously . The fourth gene cassette constituted aadA1 . The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins . The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower kcat/Km value for imipenem (0.36 microM(-1) . s(-1)) . This report further emphasizes the spread of metallo-beta-lactamase genes and their close association with various aminoglycoside resistance genes.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4654 - 61
Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase; Castanheira M et al.; As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant Pseudomonas aeruginosa clinical isolates were detected with metallo-beta-lactamase (MbetaL) activity . The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany . The resistant determinant was isolated amplifying the region between the integrase and the aacA4 gene cassette . Sequencing revealed a novel MbetaL gene, designated bla(GIM-1) . Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively . The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MbetaL family . Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-beta-lactamase inhibitors . bla(GIM-1) was found on a 22-kb nontransferable plasmid . The new MbetaL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an aacA4 cassette downstream of the MbetaL gene that appeared to be truncated with bla(GIM-1) . The aacA4 was followed by an aadA1 gene cassette that was interrupted by a copy of the IS1394 . This integron also carried an oxacillinase gene, bla(OXA-2), before the 3'-CS region . GIM-1 appears to be a unique MbetaL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MbetaL enzymes to be characterized.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4606 - 10
National surveillance of antimicrobial resistance in Pseudomonas aeruginosa isolates obtained from intensive care unit patients from 1993 to 2002; Obritsch MD et al.; Nosocomial infections caused by Pseudomonas aeruginosa in critically ill patients are often difficult to treat due to resistance to multiple antimicrobials . The purpose of this study was to evaluate antimicrobial resistance among P . aeruginosa isolates from intensive care unit patients in the United States from 1993 to 2002 by using the Intensive Care Unit Surveillance Study database . Over the 10-year period, susceptibility of 13,999 nonduplicate isolates of P . aeruginosa was analyzed . From 1993 to 2002, nationwide increases in antimicrobial resistance were greatest for ciprofloxacin, imipenem, tobramycin, and aztreonam . Rates of multidrug resistance (resistance to > or =3 of the following drugs: ceftazidime, ciprofloxacin, tobramycin, and imipenem) increased from 4% in 1993 to 14% in 2002 . The lowest dual resistance rates were observed between aminoglycosides or fluoroquinolones with piperacillin-tazobactam while the highest were for those that included beta-lactams and ciprofloxacin . Ongoing surveillance studies are crucial in monitoring antimicrobial susceptibility patterns and selecting empirical treatment regimens.

J Mol Biol, 2004 Dec 10, 344(5), 1183 - 97
A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a Transcription inhibitor and co-activator of bacteriophage T4; Pineda M et al.; Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase . The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence . In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence . The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters . Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4 . We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex . Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding . This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap . We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y . We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters . We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors . In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA . We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.

J Environ Sci (China), 2004, 16(5), 797 - 801
Experimental study on Cr (V) reduction by Pseudomonas aeruginosa; Liu YG et al.; Investigation on Cr(V) reduction was conducted using Pseudomonas aeruginosa . The study demonstrated that the Cr(VI) can be effectively reduced to Cr(III) by Pseudomonas aeruginosa . The effects of the factors affecting Cr(VI) reduction rate including carbon source type, pH, initial Cr(VI) concentration and amount of cells inoculum were thoroughly studied . Malate was found to yield maximum biotransformation, followed by succinate and glucose, with the reduction rate of 60.86%, 43.76% and 28.86% respectively . The optimum pH for Cr(VI) reduction was 7.0, with reduction efficiency of 61.71% being achieved . With the increase of initial Cr(VI) concentration, the rate of Cr(VI) reduction decreased . The reduction was inhibited strongly when the initial Cr(VI) concentration increased to 157 mg/L . As the amount of cells inoculum increased, the rate of Cr(VI) reduction also increased . The mechanism of Cr(VI) reduction and final products were also analysed . The results suggested that the soluble enzymes appear to be responsible for Cr(VI) reduction by Pseudomonas aeruginosa, and the reduced Cr(III) was not precipitated in the form of Cr(OH)3.

Infect Immun, 2004 Dec, 72(12), 6969 - 77
Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung; Shaver CM et al.; Pseudomonas aeruginosa uses a type III secretion system to promote development of severe disease, particularly in patients with impaired immune defenses . While the biochemical and enzymatic functions of ExoU, ExoS, and ExoT, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood . Since ExoU and ExoS are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection . In the work described here, several isogenic mutants of a bacterial strain that naturally secretes ExoU, ExoS, and ExoT were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia . Measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of ExoU had the greatest impact on virulence while secretion of ExoS had an intermediate effect and ExoT had a minor effect . It is of note that these results conclusively show for the first time that ExoS is a virulence factor . Infection with isogenic mutants secreting wild-type ExoS, ExoS defective in GTPase-activating protein (GAP) activity, or ExoS defective in ADP-ribosyltransferase activity demonstrated that the virulence of ExoS was largely dependent on its ADP-ribosyltransferase activity . The GAP activity of this protein had only a minor effect in vivo . The relative virulence associated with each of these type III effector proteins may have important prognostic implications for patients infected with P . aeruginosa.

Infect Immun, 2004 Dec, 72(12), 6892 - 901
Gamma interferon does not enhance clearance of Pseudomonas aeruginosa but does amplify a proinflammatory response in a murine model of postseptic immunosuppression; Murphey ED et al.; Patients that have suffered a major injury may sustain a period of immunocompromise and altered Th1/Th2 cytokine balance that can predispose them to opportunistic infections . Pseudomonas aeruginosa is frequently a causative organism for nosocomial infections in critically ill patients and is associated with high mortality . We previously mimicked this clinical scenario by challenging mice with P . aeruginosa 5 days after a cecal ligation and puncture (CLP) procedure . Mice that were subjected to CLP had reduced ability to clear bacteria, significantly lower gamma interferon (IFN-gamma) concentrations in plasma, and significantly elevated levels of interleukin 10 (IL-10) in plasma in response to the Pseudomonas challenge compared to uninjured control mice . We investigated the significance of the alteration in IFN-gamma by administering recombinant IFN-gamma to post-CLP mice at the time of Pseudomonas challenge and by challenging IFN-gamma knockout (IFN-gamma KO) mice with Pseudomonas . Administration of IFN-gamma to post-CLP mice attenuated IL-10 secretion and enhanced IL-12 secretion but did not improve bacterial clearance or survival after Pseudomonas challenge . Furthermore, IFN-gamma KO mice had significantly higher plasma IL-10 concentrations but did not exhibit impaired bacterial clearance or increased mortality following Pseudomonas challenge . These data indicate that systemic administration of IFN-gamma effectively reverses alterations in immune function that are commonly associated with immunosuppression in critically injured mice but does not improve bacterial clearance or survival following Pseudomonas challenge . Further, endogenous IFN-gamma does not appear to contribute significantly to early clearance of Pseudomonas bacteremia, nor does it affect the mortality rate after a lethal Pseudomonas challenge.

Water Res, 2004 Dec, 38(20), 4313 - 22
Bioremediation of crystal violet using air bubble bioreactor packed with Pseudomonas aeruginosa; El-Naggar MA et al.; Seven water and sediment samples were collected and tested for decolorizing crystal violet . Pseudomonas aeruginosa was the most effective isolate for dye decolorization . The LC(50) of the crystal violet (115 mg/l) was measured using Artemia salina as a biomarker . The effect of different heavy metals on crystal violet decolorization was investigated . Cd(2+) and Fe(3+) ions showed marginal enhancement of the decolorization process, the rate was 1.35 mg/l/h compared to 1.25 mg/l/h for the control . Phenol and m-cresol showed no effect on crystal violet decolorization, meanwhile p-cresol and p-nitrophenol reduced the decolorization rate to 1.07 and 0.01 mg/l/h, respectively . P . aeruginosa cells were immobilized by entrapment in agar-alginate beads . The beads were cultivated and reused in Erlenmeyer flask and in an air bubble column bioreactor and they enhanced the crystal violet decolorization rate to 3.33 and 7.5 mg/l/h, respectively.

J Food Prot, 2004 Nov, 67(11), 2555 - 9
Detection and identification of bacteria in a juice matrix with Fourier transform-near infrared spectroscopy and multivariiate analysis; Rodriguez-Saona LE et al.; The use of Fourier transform-near infrared (FT-NIR) spectroscopy combined with multivariate pattern recognition techniques was evaluated to address the need for a fast and senisitive method for the detection of bacterial contamination in liquids . The complex cellular composition of bacteria produces FT-NIR vibrational transitions (overtone and combination bands), forming the basis for identification and subtyping . A database including strains of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus cereus, and Bacillus thuringiensis was built, with special care taken to optimize sample preparation . The bacterial cells were treated with 70% (vol/vol) ethanolto enhance safe handling of pathogenic strains and then concentrated on an aluminum oxide membrane to obtain a thin bacterial film . This simple membrane filtration procedure generated reproducible FT-NIR spectra that allowed for the rapid discrimination among closely related strains . Principal component analysis and soft independent modeling of class analogy of transformed spectra in the region 5,100 to 4,400 cm(-1) were able to discriminate between bacterial species . Spectroscopic analysis of apple juices inoculated with different strains of E . coli at approximately 10(5) CFU/ml showed that FT-NIR spectralfeatures are consistent with bacterial contamination and soft independent modeling of class analogy correctly predicted the identity of the contaminant as strains of E . coli . FT-NIR in conjunction with multivariate techniques can be used for the rapid and accurate evaluation of potential bacterial contamination in liquids with minimal sample manipulation, and hence limited exposure of the laboratory worker to the agents.

Rinsho Ketsueki, 2004 Oct, 45(10), 1138 - 40
{Pseudomonas sepsis with ecthyma gangrenosum in an acute myeloid leukemia patient}; Obara Y et al.; A 56-year-old woman with acute myeloid leukemia had two rapidly growing necrotizing nodules with ulcer formation on her head after the first course of consolidation therapy . Clinical features corresponding to sepsis (e.g., fever) appeared following the development of the skin lesion . Pseudomonas aeruginosa was isolated from the blood as well as pus of the lesion . Based on these findings, a diagnosis of ecthyma gangrenosum was made . Treatment with ciprofloxacin and gamma-globulin dramatically improved the patient's clinical features . Since Pseudomonas sepsis with ecthyma gangrenosum is associated with a high mortality rate, it is important to start immediate treatment with appropriate antibiotics.

Ann Ig, 2004 Sep-Oct, 16(5), 693 - 7
{Cluster of Pseudomonas aeruginosa in a neurosurgical unit}; Torre I et al.; Hospital infections caused by Pseudomonas aeruginosa and particulary meningitis appear to be very common in neurosurgical wards and in patients with brain traumas . This study was carried out during the period between 25th October and the 11th December 2000 in a neurosurgical ward of the teaching hospital of the University "Federico II" of Naples . During this period, five patients contracted an infection caused by P . aeruginosa . The microorganisim was found to be responsible for three cases of meningitis and two cases of surgical site infections . The P . aeruginosa isolates responsible for the infections and the "environmental" isolates were subjected to genotypic typing through the analysis of macrorestriction patterns of genomic DNA after XbaI digestion of Pulsed Field Gel Electrophoresis (PFGE) . Four clinical isolates and an environmental isolate recovered from an hand washing basin located in the sub-intensive care area showed an identical PFGE pattern, as well as the some multiresistant antibiotype . The results of this study allows us to point out that the surveillance programs of infections in hospitals should include the molecular typing of micro-organisms singled out in clinical samples and, in case of outbreaks, also the typing of microorganisms originating from the environment and the hospital staff.

Am J Physiol Gastrointest Liver Physiol . 2004 Dec 9; {Epub ahead of print}
COMPONENTS OF INTESTINAL EPITHELIAL HYPOXIA ACTIVATE THE VIRULENCE CIRCUITRY OF PSEUDOMONAS; Kohler JE et al.; We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice . The aim of this study was to determine if intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression . A fusion construct was generated to express green fluorescent protein (GFP) downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P . aeruginosa . Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1-0.3% O2) for 1 hour, with or without a recovery period of normoxia (21% O2) for 2 hours, and then inoculated with P . aeruginosa containing the PA-I reporter construct . Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 hour; P < 0.001) . Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia . We conclude that the virulence circuitry of P . aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.

Cell Mol Life Sci, 2004 Nov, 61(21), 2753 - 9
Identification and characterization of a highly thermostable bacteriophage lysozyme; Lavigne R et al.; Pseudomonas aeruginosa bacteriophage phiKMV is a T7-like lytic phage . Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the phiKMV phage particle . The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P . aeruginosa PAO1 and Escherichia coli WK6 cells . The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg) . Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100 degrees C and 21% after autoclaving . This thermostability could prove an interesting characteristic for food conservation technology.

Mol Cell, 2004 Nov 19, 16(4), 497 - 8
The genetic basis for the commitment to chronic versus acute infection in Pseudomonas aeruginosa; Yahr TL et al.; The opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic airway infections . In a recent issue of Developmental Cell, Goodman et al . (2004) show that the RetS two-component gene regulatory module inversely controls expression of genes associated with acute and chronic infection.

J Appl Microbiol, 2004, 97(6), 1149 - 60
Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis; Messager S et al.; AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis . METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C . Concurrently variation in skin pH was evaluated at different time intervals during this period . In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM . All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E . coli and S . aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time . Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers . The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells . All micro-organisms had an acidifying effect on skin samples . CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin . In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis . SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.

J Biol Chem . 2004 Nov 15; {Epub ahead of print}
Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2; Zaas D et al.; Pseudomonas aeruginosa (Pa) is a major cause of pneumonia in patients with cystic fibrosis and other immuncompromising conditions . Here we show that Pa invades type I pneumocytes via a lipid-raft mediated mechanism . Pa invasion of rat primary type-I-like pneumocytes as well as a murine lung epithelial cell line (MLE-12) is inhibited by drugs that remove membrane cholesterol and disrupt lipid rafts . Confocal microscopy demonstrates co-localization of intracellular Pa with lipid raft components including caveolin-1 and 2 . We generated caveolin-1 and 2 knockdowns in MLE-12 cells using RNA interference techniques . Decreased expression of caveolin-2 significantly impairs the ability of Pa to invade MLE-12 cells . In addition, the lipid raft dependent tyrosine phosphorylation of caveolin-2 appears to be a critical regulator of Pa invasion.

Curr Pharm Des, 2004, 10(25), 3055 - 65
Regulatory effects of macrolides on bacterial virulence: potential role as quorum-sensing inhibitors; Tateda K et al.; Pseudomonas aeruginosa is an opportunistic pathogen, and this organism is a major cause of pulmonary damage and mortality in patients with cystic fibrosis (CF), diffuse panbronchiolitis (DPB) and other forms of bronchiectasis . A break-through in the treatment of DPB and associated chronic P . aeruginosa pulmonary infection was realized when a patient with DPB improved dramatically after treatment with erythromycin for years . Now, long-term macrolide therapy has become a first line of treatment in DPB patients, and the immunomodulatory properties have now been extended to other clinical settings, including CF . An important factor in the pathogenesis of chronic P . aeruginosa infection is a bacterial cell-to-cell signaling mechanism, referred to as "quorum sensing", which enables bacteria to coordinately turn on and off specific virulence genes through the production of autoinducer molecules . Interference or blocking of quorum-sensing systems has been considered an attractive therapeutic strategy . Clinical and basic science data suggests the potential of macrolides as relevant inhibitors of the Pseudomonas quorum-sensing system . In fact, certain macrolides strongly suppressed quorum-sensing associated genes and autoinducer production, in addition to inhibition of a variety of virulence factors . In this review, clinical efficacy of macrolides on DPB and CF patients will be briefly summarized . Additionally, the mechanisms of action of macrolides will be discussed from the standpoint of sub-MIC macrolide effects on P . aeruginosa, particularly the ability of this antibiotic to suppress quorum-sensing systems, which may be crucial in the pathogenesis of chronic P . aeruginosa infection.

Am J Respir Crit Care Med . 2004 Nov 12; {Epub ahead of print}
Prospective Surveillance for Pseudomonas aeruginosa Cross-infection at a Cystic Fibrosis Centre; Jones AM et al.; We have performed a 4-year prospective surveillance for Pseudomonas aeruginosa cross-infection at a large regional adult cystic fibrosis centre . Despite purpose built facilities in a new building and the practise of strict hygiene, P . aeruginosa cross-infection has continued . In contrast, individuals segregated from the cohort of patients with chronic P . aeruginosa infection but who attend the same centre have not acquired infection with transmissible P . aeruginosa strains . Simple infection control measures alone do not prevent the spread of transmissible P . aeruginosa strains between individuals with cystic fibrosis . However, in our clinic patient segregation effectively controlled spread of such strains.

Vaccine, 2004 Dec 16, 23(5), 656 - 63
Safety and immunogenicity of a booster dose of Staphylococcus aureus types 5 and 8 capsular polysaccharide conjugate vaccine (StaphVAX) in hemodialysis patients; Fattom A et al.; StaphVAX, an unadjuvanted, bivalent vaccine composed of Staphylococcus aureus (S . aureus) capsular polysaccharides (CPS) types 5 and 8 bound to the mutant non-toxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA) conferred approximately 60% protection for 10 months against bacteremia caused by this pathogen in hemodialysis patients . A protective level of 80 microg/ml was estimated based upon geometric mean (GM) antibody levels at the end of the efficacy period . To extend the duration of protection conferred by StaphVAX in hemodialysis patients, recipients of the vaccine were reinjected in a randomized double-blinded, placebo-controlled study . Vaccinees received StaphVAX and a saline placebo injection 14 days apart according to the randomization schedule . The booster dose of StaphVAX was administered an average of 958 days (753-1167 days) after the first injection . There were no serious adverse reactions . Antibody levels at day 14, 28, 92, and 182 post-injection were measured by ELISA . Maximal levels of IgG anti-CPS were observed at the 28-day interval . For type 5, GM antibody levels increased from 73 microg/ml at day 0 to 162 microg/ml (P < 0.001) and for type 8 from 59 microg/ml to 133 microg/ml (P < 0.001) . Anti-CPS antibody levels of approximately 80 microg/ml to type 5 and type 8 were achieved in 72.4 and 74.3% of vaccinees, respectively . There was excellent correlation between the level of anti-CPS and opsonic titer (r = 0.93) . Moreover, the decline of anti-CPS antibody levels at six months was significantly less rapid than that observed from the first immunization (P < 0.001) . We conclude that a booster immunization to maintain protective levels of specific antibodies for an extended period of time is feasible for patients at continuous risk for S . aureus bacteremia.

Chest, 2004 Nov, 126(5), 1575 - 82
Colonization of dental plaques: a reservoir of respiratory pathogens for hospital-acquired pneumonia in institutionalized elders; El-Solh AA et al.; STUDY OBJECTIVES: Poor dental hygiene has been linked to respiratory pathogen colonization in residents of long-term care facilities . We sought to investigate the association between dental plaque (DP) colonization and lower respiratory tract infection in hospitalized institutionalized elders using molecular genotyping . METHODS: We assessed the dental status of 49 critically ill residents of long-term care facilities requiring intensive care treatment . Plaque index scores and quantitative cultures of DPs were obtained on ICU admission . Protected BAL (PBAL) was performed on 14 patients who developed hospital-acquired pneumonia (HAP) . Respiratory pathogens recovered from the PBAL fluid were compared genetically to those isolated from DPs by pulsed-field gel electrophoresis . MEASUREMENTS AND RESULTS: Twenty-eight subjects (57%) had colonization of their DPs with aerobic pathogens . Staphylococcus aureus (45%) accounted for the majority of the isolates, followed by enteric Gram-negative bacilli (42%) and Pseudomonas aeruginosa (13%) . The etiology of HAP was documented in 10 patients . Of the 13 isolates recovered from PBAL fluid, nine respiratory pathogens matched genetically those recovered from the corresponding DPs of eight patients . CONCLUSIONS: These findings suggest that aerobic respiratory pathogens colonizing DPs may be an important reservoir for HAP in institutionalized elders . Future studies are needed to delineate whether daily oral hygiene in hospitalized elderly would reduce the risk of nosocomial pneumonia in this frail population.

Biotechnol Bioeng, 2004 Dec 30, 88(7), 861 - 8
Stimulating in-soil rhamnolipid production in a bioslurry reactor by limiting nitrogen; Hudak AJ et al.; A soil with aged contamination from lubricating oil (LO) and polychlorinated biphenyls (PCBs) was treated in a bioslurry reactor to investigate in-soil biosurfactant production by Pseudomonas aeruginosa, the most abundant indigenous, culturable, hydrocarbon-degrading microorganism . After 2 days of growth on LO, a depletion of nitrogen stimulated the production and accumulation of rhamnolipids to levels roughly 20 times the critical micelle concentration . Surface tensions and concentrations of monorhamnolipid and dirhamnolipid, PCBs, and total petroleum hydrocarbons (TPH) were measured in a slurry filtrate . Soil-bound PCBs and TPH were also quantified . Rhamnolipid production was observed within 1 to 2 days after nitrogen depletion in each of the 10 batches tested . By day 6, total rhamnolipid concentrations increased from below detection to average values over 1,000 mg/L, which caused over 98% of soil-bound PCBs and over 99% of TPH to be emulsified and recovered in the filtrate . After 70 days, rhamnolipid concentrations were only reduced by 15%, because of nitrogen-limited rates of rhamnolipid biodegradation . The results show that in-soil biosurfactant production can be stimulated in a controlled way with nutrient limitation and can be used to achieve soil washing . 2004 Wiley Periodicals, Inc.

J Eukaryot Microbiol, 2004 Sep-Oct, 51(5), 522 - 8
Modulation of a "CD59-like" protein in Naegleria fowleri amebae by bacteria; Fritzinger AE et al.; Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis . In the natural environment, amebae feed on bacteria . In the infected host, the amebae lyse and ingest nerve tissue . Recently, we have established that N . fowleri expresses a "CD59-like" surface protein, but the function of this protein in the ameba has not been elucidated . In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein . In the present study, expression of the "CD59-like" protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis . Co-culture of N . fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the "CD59-like" protein . Co-cultures of amebae and bacteria were examined by electron microscopy . The results of our study implicate a possible protective role of the "CD59-like" protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.

Dermatol Online J . 2004 Oct 15;10(2):20.
Facial cellulitis associated with Pseudomonas aeruginosa complicating ophthalmic herpes zoster; Atzori L et al.; Cellulitis is a rare and severe soft-tissue infection characterized by acute, diffuse, spreading inflammation, often associated with systemic symptoms such as malaise and fever . Surgery of the head and neck, dental infections, sinusitis, upper respiratory tract infections, and trauma are the most common portal of entry for pathogens in facial cellulitis . A very unusual case complicating an ophthalmic herpes zoster in a 74-year-old woman was observed at the department of dermatology, Cagliari University (Italy) . Culture of skin swabs showed growth of numerous Gram-negative bacilli, further identified as Pseudomonas aeruginosa . Therapy with intravenous ciprofloxacin was promptly instituted on the basis of the culture and sensitivity report . She was initially treated with daily drainage and twice-daily topical fusidic acid . The lesion completely resolved in 4 weeks, and no general complications or recurrence have been observed for 6 months . Early recognition and management of facial cellulitis is mandatory to avoid serious and generalized complications . Pseudomonas aeruginosa is rarely reported in facial cellulitis; there are apparently no reports of this infection occurring as a complication of ophthalmic herpes zoster . Herpetic damage of the anatomic barrier as well as impairment of defense mechanisms because of decompensated diabetes mellitus may have facilitated the colonization and proliferation of this opportunistic pathogen in our patient.

Biomacromolecules, 2004 Nov-Dec, 5(6), 2469 - 78
Microbial synthesis of poly(3-hydroxyalkanoates) by Pseudomonas aeruginosa from fatty acids: identification of higher monomer units and structural characterization; Barbuzzi T et al.; Pseudomonas aeruginosa ATCC 27853 accumulated poly(3-hydroxyalkanoates) (PHAs) after growth on saturated fatty acids with an odd number of carbon atoms . No nutrient limitation was required to induce PHA synthesis, although better yields were obtained when the medium was magnesium deprived . A comparative study was carried out between PHAs obtained from C-odd and those from C-even carbon sources . Repeating units identification was performed by gas chromatography (GC) and capillary liquid chromatography-electrospray mass spectrometry (LC-ESI MS) of methanolyzed samples . When C-odd n-alkanoic acids from nonanoic to pentadecanoic were used the lowest hydroxyalkanoate unit found was 3-hydroxyvalerate and the highest 3-hydroxypentadecanoate, whereas when C-even acids from octanoic to eicosanoic were used these were 3-hydroxycaproate and 3-hydroxyeicosanoate, respectively . Weight average molecular weights were in the range 187 000-596 000 . DSC traces showed Tm and DeltaHm which varied from 43 to 58 degrees C and from 5.9 to 24.8 J/g, with the PHAs generated from C-odd carbon sources having lower values . ESI MS of partially pyrolyzed samples allowed the identification of oligomers up to heptamers, and statistical analysis of the ions intensity in the mass spectra showed that these PHAs are random copolyesters.

J Environ Biol, 2004 Apr, 25(2), 197 - 200
Effect of Pseudomonas aeruginosa (MTCC 1688) on the tissue phosphatases activity on Macrobrachium rosenbergii (De Man); Ramalingam K et al.; A time course study on the endotoxin toxicity of the gram negative bacteria, Pseudomonas aeruginosa MTCC 1688 on the tissue phosphatases activity on the giant freshwater prawn, Macrobrachium rosenbergii was conducted . The results revealed marked elevation of both acid and alkaline phosphatase activity in the haemolymph and body muscle . The hepatopancreas showed reduced phosphatase activity compared to control . The enzymes, being non-specific in action and particularly the acid phosphatase being of lysosomal origin, their increase in muscle and haemolymph has pathogenic significance in the inoculum treated prawns.

J Clin Microbiol, 2004 Nov, 42(11), 5378 - 80
Clonal spread of IMP-1-producing Pseudomonas aeruginosa in two hospitals in Singapore; Koh TH et al.; Thirty-six isolates of carbapenem-resistant Pseudomonas aeruginosa were studied . Pulsed-field gel electrophoresis revealed the presence of two clones . One clone carried a bla(IMP-1) gene identical to that first described in Japan . The other clone carried a bla(IMP-1) variant containing four silent mutations . One isolate with a unique pulsed-field gel electrophoresis pattern contained bla(IMP-7).

J Clin Microbiol, 2004 Nov, 42(11), 5229 - 37
Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis; Jain M et al.; Pseudomonas aeruginosa is a frequent cause of respiratory exacerbations in individuals with cystic fibrosis . An important virulence determinant of this pathogen is its type III protein secretion system . In this study, the type III secretion properties of 435 P . aeruginosa respiratory isolates from 56 chronically infected individuals with cystic fibrosis were investigated . Although it had been previously reported that 75 to 90% of P . aeruginosa isolates from patients with hospital-acquired pneumonia secreted type III proteins, only 12% of isolates from cystic fibrosis patients did so, with nearly all of these isolates secreting ExoS and ExoT but not ExoU . Despite the low overall prevalence of type III protein-secreting isolates, at least one secreting isolate was cultured from one-third of cystic fibrosis patients . Interestingly, the fraction of cystic fibrosis patient isolates capable of secreting type III proteins decreased with duration of infection . Although 90% of isolates from the environment, the presumed reservoir for the majority of P . aeruginosa strains that infect patients with cystic fibrosis, secreted type III proteins, only 49% of isolates from newly infected children, 18% of isolates from chronically infected children, and 4% of isolates from chronically infected adults with cystic fibrosis secreted these proteins . Within individual patients, isolates of clonal origin differed in their secretion phenotypes, indicating that as strains persisted in cystic fibrosis patient airways, their type III protein secretion properties changed . Together, these findings indicate that following infection of cystic fibrosis patient airways, P . aeruginosa strains gradually change from a type III protein secretion-positive phenotype to a secretion-negative phenotype.

J Clin Microbiol, 2004 Nov, 42(11), 5176 - 83
characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling; Rogers GB et al.; Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients . A greater understanding of these bacterial infections is needed to improve lung disease management . As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients . Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients . Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth . The species richness and evenness were determined from the T-RF length and volume for the 71 profiles . Species richness was on average 13.3 +/- 7.9 per sample and 13.4 +/- 6.7 per patient . On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively . The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively . On average, the remaining T-RF bands represented 10.2% of the total profile volume . The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples . However, 18 other T-RF band lengths were dominant in at least one sample . In conclusion, this reveals the enormous complexity of bacteria within the CF lung . Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

J Clin Microbiol, 2004 Nov, 42(11), 5094 - 101
Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia; Crespo MP et al.; The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003 . Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants . Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis . Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR . PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A . Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained . The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA . They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2) . Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution . Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA . No bla(IMP) alleles were detected . During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.

Microbiology, 2004 Nov, 150(Pt 11), 3797 - 806
The Pseudomonas aeruginosa global regulator MvaT specifically binds to the ptxS upstream region and enhances ptxS expression; Westfall LW et al.; Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes . Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously . While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P . aeruginosa is not known . The presence of a P . aeruginosa protein complex that specifically binds to a segment within this region was determined . In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P . aeruginosa HU and MvaT . Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region . A PAO1 isogenic mutant defective in mvaT, PAODeltamvaT, was constructed and characterized . The lysate of PAODeltamvaT failed to bind to the 150 bp probe . The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments . The expression of ptxS was lower in PAODeltamvaT than in PAO1, but no difference was detected in ptxR expression . These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.

Appl Environ Microbiol, 2004 Nov, 70(11), 6914 - 9
Complete type III secretion system of a mesophilic Aeromonas hydrophila strain; Vilches S et al.; We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3 . We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa . Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A . hydrophila, A . veronii, and A . caviae . By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain . Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.

Appl Environ Microbiol, 2004 Nov, 70(11), 6435 - 43
Survival of Shewanella oneidensis MR-1 after UV radiation exposure; Qiu X et al.; We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure . MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of Escherichia coli K-12 values . Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose . UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage . A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of UVA m(-2), and 558 J of solar UVB m(-2), respectively . Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively . A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10(-5) to 10(-6) . Unlike in E . coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1 . Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude . Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents.

Am J Respir Cell Mol Biol . 2004 Nov 4; {Epub ahead of print}
Pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro; Beatty AL et al.; Although it is known that surfactant lipids and proteins are altered in patients with Pseudomonas aeruginosa infections, the mechanisms and implications of these alterations are not clear . In this study, the effects of P . aeruginosa on the surfactant large aggregate fraction were examined using an in vitro surface area cycling model . Large aggregates were isolated from porcine bronchoalveolar lavage fluid and incubated with supernatants from P . aeruginosa cultures (PAO1, parent strain; PAO1-A1, lasA-negative mutant; PAO1-B1, elastase-negative mutant) or purified elastase . Amounts of surfactant protein (SP)-A and SP-B, phospholipid content, and large aggregate conversion were assessed . Additionally, lipid degradation was assessed by incubating a mixture of radiolabeled phospholipids with P . aeruginosa supernatants . The results demonstrated that SP-A was degraded by PAO1 and PAO1-A1 supernatants, and by purified elastase . SP-B was degraded by PAO1 and PAO1-B1 supernatants, but not by elastase . P . aeruginosa supernatants degraded phospholipids, a process inhibited by ZnCl2 . P . aeruginosa supernatants and elastase increased conversion . The data suggest that protein degradation facilitates increased conversion, and that phospholipid degradation and conversion enhance degradation of surfactant proteins . In conclusion, P . aeruginosa secretes multiple virulence factors that cooperate to result in degradation of surfactant components and alteration of large aggregate conversion.

Biochem Biophys Res Commun, 2004 Dec 3, 325(1), 85 - 90
Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa; Cipollone R et al.; Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans . P . aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide . Here, the biochemical characterization of recombinant P . aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported . Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue . Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule . Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C . However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent . Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay) . Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle . As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P . aeruginosa from endogenous and environmental cyanide.

Mol Microbiol, 2004 Nov, 54(4), 1090 - 103
A novel sensor kinase-response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa; Laskowski MA et al.; The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media . We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P . aeruginosa . RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains . Deletion of rtsM resulted in a defect in production and secretion of the type III effectors . Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the DeltartsM strain under TTSS-inducing conditions . Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact . The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in deltartsM than in the wild-type parent, PA103 . The defect in TTSS effector production and secretion of deltartsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation . DeltartsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo . We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals.

Folia Biol (Praha), 2004, 52(1-2), 91 - 6
Effect of Pseudomonas aeruginosa crude proteolytic fraction on antibacterial activity of Galleria mellonella haemolymph; Andrejko M; The antibacterial activity of immune haemolymph Galleria mellonella directed against Escherichia coli D31 was destroyed by Pseudomonas aeruginosa crude proteolytic fraction . This was demonstrated by diffusion well assay and acid gel electrophoresis and subsequent bioautography . On the contrary, lysozyme activity appeared to be insensitive to extracellular proteases of P . aeruginosa when activity was determined using the bioautography method . In addition, no change in lysozyme protein level was observed by immunoblotting with specific antibodies directed against G . mellonella lysozyme, which confirmed that lysozyme was not degraded by the crude proteolytic fraction of P . aeruginosa . However, a significant decrease of lysozyme activity in naive and immune haemolymph exposed to the action of P . aeruginosa proteins determined by using diffusion well assay was observed . Mechanisms of the observed inhibition require further studies.

Acta Biotheor, 2004, 52(4), 379 - 90
Epigenesis and dynamic similarity in two regulatory networks in pseudomonas aeruginosa; Guespin-Michel JF et al.; Mucoidy and cytotoxicity arise from two independent modifications of the phenotype of the bacterium Pseudomonas aeruginosa that contribute to the mortality and morbidity of cystic fibrosis . We show that, even though the transcriptional regulatory networks controlling both processes are quite different from a molecular or mechanistic point of view, they may be identical from a dynamic point of view: epigenesis may in both cases be the cause of the acquisition of these new phenotypes . This was highlighted by the identity of formal graphs modelling these networks . A mathematical framework based on formal methods from computer science was defined and implemented with a software environment . It allows an easy and rigorous validation and certification of these models and of the experimental methods that can be proposed to falsify or validate the underlying hypothesis.

J Biol Chem, 2005 Jan 7, 280(1), 418 - 27 Epub 2004 Oct 31.
A Primer-dependent Polymerase Function of Pseudomonas aeruginosa ATP-dependent DNA Ligase (LigD); Zhu H et al.; Pseudomonas aeruginosa encodes two putative DNA ligases: a classical NAD(+)-dependent DNA ligase (LigA) plus an ATP-dependent DNA ligase (LigD) . LigD exemplifies a family of bacterial proteins that consist of a ligase domain fused to flanking domains that resemble nucleases and/or polymerases . Here we purify LigD and show that it possesses an intrinsic polymerase function resident within an autonomous C-terminal polymerase domain, LigD-(533-840), that flanks an autonomous DNA ligase domain, LigD-(188-527) . Native LigD and the polymerase domain are both monomeric proteins . The polymerase activity is manifest in three ways: (i) non-templated nucleotide addition to a blunt-ended duplex DNA primer; (ii) non-templated addition to a single-stranded DNA primer; and (iii) templated extension of a 5'-tailed duplex DNA primer-template . The divalent cation cofactor requirement for non-templated and templated polymerase activity is satisfied by manganese or cobalt . rNTPs are preferred over dNTPs as substrates for non-templated blunt-end addition, which typically entails the incorporation of only 1 or 2 nucleotides at the primer terminus . Templated dNMP addition to a 5'-tailed substrate is efficient with respect to dNTP utilization; the primer is elongated to the end of the template strand and is then further extended with a non-templated nucleotide . The polymerase activity is abolished by alanine substitution for two aspartates (Asp-669 and Asp-671) within the putative metal-binding site . We speculate that polymerase activity is relevant to LigD function in nonhomologous end-joining.

Int J Antimicrob Agents, 2004 Nov, 24(5), 515 - 8
Antimicrobial susceptibility profiles of Pseudomonas aeruginosa and Staphylococcus aureus isolated in Italy from patients with hospital-acquired infections; Blandino G et al.; Here we report the results of the Sentinel Project 2000 and give the susceptibility to selected antibiotics of 108 Pseudomonas aeruginosa and 108 Staphylococcus aureus strains isolated from patients with hospital-acquired lower respiratory tract infections . In P . aeruginosa, susceptibility to aztreonam and ciprofloxacin was lower than 50% . The resistance rate to beta-lactams was up to 25% and to amikacin 15.7% . Blood isolates showed 80-90% susceptibility to all antibiotics tested except for aztreonam and tobramycin . Overall, oxacillin resistance in S . aureus was 45%, reaching 64.3% among the bronchoalveolar lavage isolates, and 42.9% among the blood isolates . These worrying results confirm the need for continuous monitoring of bacterial resistance trends in the hospitals, mainly in ICUs.

Infect Control Hosp Epidemiol, 2004 Oct, 25(10), 856 - 9
Multidrug-resistant Pseudomonas aeruginosa cholangitis after endoscopic retrograde cholangiopancreatography: failure of routine endoscope cultures to prevent an outbreak; Fraser TG et al.; BACKGROUND: Nosocomial infections due to medical devices are of increasing concern to infection control practitioners . Attempts to prevent such infections have included surveillance cultures of endoscopes and bronchoscopes . In July 2002, the infectious disease consultation service was asked to see three patients with sepsis due to multidrug-resistant Pseudomonas aeruginosa after endoscopic retrograde cholangiopancreatography (ERCP) . OBJECTIVE: To describe an outbreak of multidrug-resistant P . aeruginosa sepsis after ERCP at an institution that performs routine surveillance cultures of endoscopes . DESIGN: A traditional outbreak investigation supplemented by pulsed-field gel electrophoresis (PFGE) was undertaken, including a case-control analysis based on the hypothesis that all infected individuals had their ERCP performed with the same endoscope . SETTING: A tertiary-care academic medical center . RESULTS: The case-control analysis confirmed the hypothesis that undergoing ERCP with the implicated endoscope was associated with a culture positive for Pseudomonas (P = .01) . The available strains were identical by PFGE . This outbreak occurred despite a negative surveillance culture of the implicated endoscope 1 month earlier . CONCLUSIONS: Infectious morbidity can occur after endoscopy despite negative surveillance cultures . The practice of routine endoscope cultures does not prevent device-related infectious morbidity.

Infect Control Hosp Epidemiol, 2004 Oct, 25(10), 825 - 31
Endemic multidrug-resistant Pseudomonas aeruginosa in critically ill patients; Ortega B et al.; OBJECTIVE: To describe the epidemiology of endemic multidrug-resistant Pseudomonas aeruginosa colonizations and infections in critically ill patients . DESIGN: Prospective study on bacterial strain typing and retrospective cohort study of charts of patients in the intensive care unit (ICU) . PATIENTS: Fifty-three patients with P . aeruginosa isolated from clinical cultures in 2001 were selected, divided into those with P . aeruginosa in vitro resistant to at least two classes of antibiotics (multidrug-resistant, n = 18) and those susceptible to all or resistant to only one antibiotic (susceptible, n = 35) . RESULTS: Risk factors for multidrug-resistant P . aeruginosa included maxillary sinusitis, long-dwelling central venous catheters, prolonged use of certain antibiotics, a high lung injury score, and prolonged mechanical ventilation and duration of stay . The frequency of colonization (approximately 50%) versus infection (ie, ventilator-associated pneumonia) did not differ between the groups . On amplified fragment-length polymorphism analysis, 64% of the multidrug-resistant strains had been potentially transmitted via cross-colonization and 36% had probably originated endogenously . ICU mortality was 22% in the multidrug-resistant group and 23% in the susceptible group, although the duration of mechanical ventilation was longer in the former . CONCLUSIONS: Patients with sinusitis who stayed in the ICU longer, were ventilated longer because of acute lung injury, received antibiotics for longer durations, and had long-dwelling central venous catheters ran an elevated risk of acquiring multidrug-resistant P . aeruginosa . These patients did not have a higher mortality than patients with susceptible P . aeruginosa . Prevention of the emergence of multidrug-resistant strains requires changes in infection control measures and antibiotic policies in our ICU.

J Bacteriol, 2004 Nov, 186(22), 7575 - 85
MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa; Wu W et al.; The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor . The T3SS of P . aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells . The exact pathway of signal sensing and T3SS activation is not clear . By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions . Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype . Alginate production responds to various environmental stresses and plays a protective role for P . aeruginosa . Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis . Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression . An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes . It is likely that P . aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

Am J Respir Crit Care Med . 2004 Oct 29; {Epub ahead of print}
Multiple-breath Washout is a Marker of Lung Disease in Preschool Children with Cystic Fibrosis; Aurora P et al.; Rationale: Sensitive measures of lung function applicable to young subjects are needed to detect early cystic fibrosis lung disease . Methods: Forty children with cystic fibrosis aged 2-5 years and 37 age-matched healthy controls performed multiple-breath inert gas washout, plethysmography, and spirometry . Results: Thirty children in each group successfully completed all measures, with success on first visit being between 68% and 86% for all three measures . Children with cystic fibrosis had significantly higher lung clearance index (mean {95% CI} difference for CF-control 2.7 {1.9,3.6}, p<0.001), and specific airway resistance (1.65 z-scores {0.96,2.33}, p<0.001), and significantly lower forced expired volume in 0.5 seconds (-0.49 z-scores {-0.95,-0.03}, p<0.05) . Abnormal lung function results were identified in 22/30 (73%) children with cystic fibrosis by multiple-breath washout, compared with 14/30 (47%) by plethysmography, and 4/30 (13%) by spirometry . Children with cystic fibrosis who were infected with Pseudomonas aeruginosa had significantly higher lung clearance index, but no significant difference in other lung function measures, when compared with non-infected children . Conclusion: Most preschool children can perform multiple-breath washout, plethysmography, and spirometry at first attempt . Multiple-breath washout detects abnormal lung function in children with cystic fibrosis more readily than plethysmography or spirometry.

Am J Physiol Lung Cell Mol Physiol . 2004 Oct 29; {Epub ahead of print}
NF-{kappa}B activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa; Joseph T et al.; The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection . Recent studies have demonstrated up-regulation of nuclear factor-kappa B (NF-kappaB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8 . Using human airway epithelial cells grown in primary culture we examined in vitro activation of NF-kappaB in cells isolated from five CF (DeltaF508/DeltaF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa . Immunofluorescence, gel-shift and immunoblot assays demonstrated a rapid translocation of NFkappa-B subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures . However, nuclear extracts from CF cells both before and following P . aeruginosa stimulation revealed elevated NF-kappaB activation compared to NCF cells . Additionally, elevated nuclear levels of the NF-kappaB inhibitor IkappaBalpha were detected in nuclei of CF cells after P . aeruginosa stimulation but this increase was transient . There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times . Our results also demonstrated increased baseline translocation of NF-kappaB to nuclei of primary CF epithelial cell cultures, but intranuclear IkappaBalpha may initially block its effects following P . aeruginosa stimulation . Thus, IL-8 mRNA expression was prolonged after P . aeruginosa stimulation in CF epithelial cells and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

Am J Physiol Lung Cell Mol Physiol, 2005 Feb, 288(2), L409 - 18 Epub 2004 Oct 29.
Pseudomonas aeruginosa protease IV degrades surfactant proteins and inhibits surfactant host defense and biophysical functions; Malloy JL et al.; Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense . Both functions are dependent on surfactant-associated proteins . Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia . P . aeruginosa secretes a number of proteases that contribute to its virulence . We hypothesized that P . aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions . Protease IV was isolated from cultured supernatant of P . aeruginosa by gel chromatography . Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B . SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-l-lysine-chloromethyl ketone (TLCK) . Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages . Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK . We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P . aeruginosa via loss of surfactant function within the lung.

APMIS, 2004 Jun, 112(6), 369 - 73
Ginseng modulates the immune response by induction of interleukin-12 production; Larsen MW et al.; In infections with intracellular microorganisms such as mycobacteria and Leishmania parasites as well as certain extracellular chronic infections such as Pseudomonas aeruginosa a Th1 response with activation of macrophages is desirable . Several studies indicate that such a response is associated with better recovery from infection, improved course of the chronic infection, and higher survival rate . In Th1 responses there is increased interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) production, whereas that of interleukin-10 (IL-10) is decreased . The present study indicated that Ginseng modulation of stimulated peripheral blood mononuclear cells (PBMC) results in a higher IL-12 production . The enhanced IL-12 production could induce a stronger Th1 response, resulting in better protection against infection with a variety of pathogens.

An Med Interna, 2004 Oct, 21(10), 483 - 7
{Antimicrobial susceptibility of the bloodstream infections: a study in a nonteaching hospital}; Pazos Anon R et al.; BACKGROUND: To try established antimicrobial susceptibility patterns and frequency of both nosocomial and community-acquired bloodstream infections and and to try identified the prognostic factors that can be modified . MATERIAL AND METHODS: A prospective study of 310 bloodstream infections with clinical significance detected in a non teaching hospital over period from October 2000-2001 . A blood culture were identified by Bact-Alert system and the confirmation was performed by MicroScan system; an antimicrobial susceptibility test was performed by reference microdilution methods as described by NCCLS . We studied sentinel antimicrobial/organism combinations with potential clinical importance . Data were computerized using SPSS . Qualitative variables were compared using the X2 test or the Fisher exact test, and quantitative variables with t Student or ANOVA . RESULTS: Gram positive and Candida were frequently recovered in nosocomial bloodstreams . The proportion of oxacillin-resistant S . aureus isolates was 24% and the penicillin resistant pneumococci was 14% . Vancomycin was universal active against gram positive . Gram negatives were often recovered in community bloodstream . The proportion of EBSL E . coli isolates was < 2% and the proportion of multiresistance Pseudomonas aeruginosa was higher among UCI isolates . An independent risk factors for death identified after multivariate analysis was the inappropriate antimicrobial therapy OR 2.6 . CONCLUSIONS: Ongoing surveillance of microbial pathogens and their resistance profiles is essential on local scale and permit the selection of appropriate antibiotic therapy which would be reduce the mortality.

Laryngoscope, 2004 Nov, 114(11), 2038 - 43
Albumin-coated tympanostomy tubes: prospective, double-blind clinical study; Kinnari TJ et al.; OBJECTIVES: Coating an implant with albumin prevents adhesion of proteins, bacteria, and platelets and thus may lead to its improved and prolonged function . Previously, we have demonstrated the inhibition of binding of fibronectin, one of the most adhesive glycoproteins, on human serum albumin (HSA)-coated tympanostomy tubes and the durability of this binding inhibition in a 8-month trial . We have also demonstrated that the HSA coating inhibits the binding of Staphylococcus aureus and Pseudomonas aeruginosa to titanium plates . This prospective study evaluated the effect of albumin coating on tympanostomy tube sequelae and on the outcome of tympanostomized patients . STUDY DESIGN: Double-blind, prospective, randomized clinical trial . METHODS: Two otolaryngological centers in southern Finland enrolled 179 pediatric patients . Number of tube occlusions and otorrhea and tube ventilation time in the ears with HSA-coated titanium tympanostomy tubes were compared with the contralateral ear with its uncoated, otherwise identical titanium tube during a 9-month follow-up period . RESULTS: In HSA-coated tubes, average ventilation time was slightly longer and the number of early tube occlusions significantly less (P < .05) . Moreover, in patients with perioperative bleeding, the coating prolonged average ventilation time of tympanostomy tubes significantly (P < .05) . CONCLUSIONS: HSA coating reduces early tube occlusions by preventing adherence of blood and secretion.

Laryngoscope, 2004 Nov, 114(11), 2021 - 4
Xylitol enhances bacterial killing in the rabbit maxillary sinus; Brown CL et al.; OBJECTIVES: Factors that alter airway surface liquid (ASL) ionic concentrations may influence the course of sinusitis . Xylitol has been shown to effect ASL ionic composition in vitro and to reduce nasal bacterial carriage, otitis media, and dental caries in vivo . We examined the effect of xylitol on experimental sinusitis in the rabbit model . STUDY DESIGN: Prospective randomized controlled study of xylitol, saline, and Pseudomonas aeruginosa administration to the rabbit maxillary sinus . METHODS: P . aeruginosa was administered to the sinuses of 26 New Zealand white rabbits . Saline was placed in the left maxillary sinus and xylitol in the right . The rabbits were randomly assigned to one of three groups: one, simultaneous administration of bacteria and solutions with bacterial analysis at 20 minutes, 11 rabbits; two, preadministration of solutions 1 hour before bacterial infection with analysis at 20 minutes, 11 rabbits; three, established sinusitis, 4 rabbits had daily injections of solutions for 5 days starting 7 days after P . aeruginosa administration . RESULTS: In group 1, 6.96% of injected bacteria were retrieved on the left (saline), whereas 0.095% were retrieved on the right (xylitol) (P = .034) . In group 2, 5.64% of inoculum was recovered from the left and 2.89% from the right (P = .188) . Group 3 demonstrated evidence of sinusitis with recovery of noninoculate bacteria . with no difference between right and left . CONCLUSIONS: Xylitol reduces experimental sinusitis when administered simultaneously with bacteria . Its effect in established sinusitis is less clear . A role may exist for xylitol in nasal irrigation fluid in human disease.

Kansenshogaku Zasshi, 2004 Sep, 78(9), 829 - 34
{In vitro indirect pathogenesis of Pseudomonas aeruginosa against anti MRSA chemotherapy}; Satoh N et al.; In the patient with a chronic respiratory disease, both Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) are frequently detected from expectoration . Vancomycin (VCM) and arbekacin (ABK) are both recommended for the chemotherapy of MRSA infection in Japan . Minocycline (MINO) is also selected for the treatment of MRSA infection . While rifampicin (RFP) and a trimetoprim-sulfamethoxazole combination (ST) are also recommended in Europe and USA but not recommended in Japan for the chemotherapy of MRSA infection . It is pointed out that coexistence bacteria affect chemotherapy as an indirect pathogen . Not only an antibacterial action but the immunological action or the metabolic effect against chronic P . aeruginosa infection such as DPB is known by the administration of 14-membered ring macrolides including erythromycin (EM) . We considered the influence of P . aeruginosa isolated with MRSA on the activity against anti-MRSA agents by the disk diffusion method with bilayer flat agar in vitro . Moreover, we also examined the influence of EM against the activity of the anti-MRSA agents when P . aeruginosa was coexistence . One strain of MRSA as an indicator strain and 100 strains of P . aeruginosa as test strains, which were obtained from clinical materials, were used for the following experiment . P . aeruginosa was streaked on to the Mueller-Hinton agar culture medium (MHA), and they incubated at 35 degrees C for 24 hours . Then, the blood agar plate was piled up, MRSA was streaked on the blood agar surface, the susceptibility test disks (VCM, ABK, MINO, RFP, ST) were put on it, and incubated at 35 degrees C for a further 24 hours . The diameter of the zone of inhibition around the susceptibility disks against MRSA was measured and compared with P . aeruginosa free experiments . The anti-MRSA activity of MINO, ST and ABK was reduced by coexistence of P . aeruginosa . In RFP and VCM, the anti-MRSA activity was reinforced by coexistence of P . aeruginosa . Although the anti-MRSA activity of ST and ABK has improved by EM addition in the MHA plates, the anti-MRSA activity has not improved in MINO . These results are suggesting that in a MRSA infection, the chemotherapy by anti-MRSA agents were affected by coexistence of P . aeruginosa as an indirect pathogen . The macrolides such as EM may be useful as a modulator for chemotherapy by ST or ABK when MRSA and P . aeruginosa are isolated at the same time from the patient.

Kansenshogaku Zasshi, 2004 Sep, 78(9), 823 - 8
{Bacterial interaction and indirect pathogenesis of Pseudomonas aeruginosa at the growth of MRSA}; Kondo S et al.; Bacterial interactions such as methicillin-resistant Staphylococcus aureus (MRSA) growth inhibition or inactivation of anti-MRSA antibiotics by Pseudomonas aeruginosa as an indirect pathogen were tested by in vitro assay . Paired strains, P . aeruginosa and MRSA, used in this experiment were isolated from 63 respiratory samples at Juntendo University Hospital from 2002 to 2003 . Growth inhibitory activities against MRSA by P . aeruginosa were tested with reversed agar plate method . Inactivation of anti-MRSA antibiotics by P . aeruginosa were assayed with disk diffusion method using agar over lay technique . Fifty-six (88.9%) out of 63 samples showed the significant MRSA growth inhibitory activity by co-existed P . aeruginosa . Anti-MRSA antibiotics such as trimetoprim-sulfamethoxazole combination (ST), arbekacin (ABK) and minocycline (MINO) except Vancomycin (VCM) and Teicoplanin (TEIC) were inactivated by the co-existed P . aeruginosa . Our data suggests that P . aeruginosa may play not only as a chronic respiratory pathogen but also as an indirect pathogen . Further, the most P . aeruginosa with anti-MRSA activity isolated respiratory sample may play as a modulator of MRSA infection.

J Virol, 2004 Nov, 78(22), 12147 - 56
New class of orthopoxvirus antiviral drugs that block viral maturation; Byrd CM et al.; By using a homology-based bioinformatics approach, a structural model of the vaccinia virus (VV) I7L proteinase was developed . A unique chemical library of approximately 51,000 compounds was computationally queried to identify potential active site inhibitors . The resulting biased subset of compounds was assayed for both toxicity and the ability to inhibit the growth of VV in tissue culture cells . A family of chemotypically related compounds was found which exhibits selective activity against orthopoxviruses, inhibiting VV with 50% inhibitory concentrations of 3 to 12 microM . These compounds exhibited no significant cytotoxicity in the four cell lines tested and did not inhibit the growth of other organisms such as Saccharomyces cerevisiae, Pseudomonas aeruginosa, adenovirus, or encephalomyocarditis virus . Phenotypic analyses of virus-infected cells were conducted in the presence of active compounds to verify that the correct biochemical step (I7L-mediated core protein processing) was being inhibited . Electron microscopy of compound-treated VV-infected cells indicated a block in morphogenesis . Compound-resistant viruses were generated and resistance was mapped to the I7L open reading frame . Transient expression with the mutant I7L gene rescued the ability of wild-type virus to replicate in the presence of compound, indicating that this is the only gene necessary for resistance . This novel class of inhibitors has potential for development as an efficient antiviral drug against pathogenic orthopoxviruses, including smallpox.

J Biol Chem, 2004 Dec 17, 279(51), 52816 - 9 Epub 2004 Oct 26.
Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end; Akama H et al.; The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane . The channel structure must differ from that of the porin-type open pore because the protein facilitates the exit of antibiotics but not the entry . For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-angstroms resolution . The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring beta-barrel and the cavity-forming alpha-barrel . OprM anchors the outer membrane by two modes of membrane insertions . One is via the covalently attached NH(2)-terminal fatty acids and the other is the beta-barrel structure consensus on the outer membrane-spanning proteins . The beta-barrel had a pore opening with a diameter of about 6-8 angstroms, which is not large enough to accommodate the exit of any antibiotics . The periplasmic alpha-barrel was about 100 angstroms long formed mainly by a bundle of alpha-helices that formed a solvent-filled cavity of about 25,000 angstroms(3) . The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule . The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics . However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends . The crystal structure offers possible mechanisms of pore opening and pump assembly.

Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 15833 - 9 Epub 2004 Oct 25.
Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR; Schuster M et al.; Along with their cognate acyl-homoserine lactone signals, the quorum sensing regulators LasR and RhlR control the expression of hundreds of genes in the opportunistic human pathogen Pseudomonas aeruginosa . This extensive, overlapping regulatory network affords the opportunity to systematically investigate the sequence requirements and specificity determinants of large families of target promoters . Many of the P . aeruginosa quorum-controlled genes possess conserved palindromic promoter elements predicted to be binding sites for either one or both transcriptional regulators, but biochemical proof has not been reported . We have purified native LasR and characterized binding to various quorum-controlled promoters in vitro . Purified LasR was a dimer in solution that irreversibly bound two molecules of 3-oxo-C12-homoserine lactone . LasR bound several las-responsive promoters specifically and with high affinity, interacting cooperatively with some promoters and noncooperatively with others . LasR recognized some, but not all, of the predicted binding sites, and also bound to several unexpected sites . In contrast to predictions from genetic data, we found that the recognition sequences of las-specific promoters showed little overall sequence conservation and did not require dyad symmetry . We found distinct differences in sequence composition between las-specific noncooperative, las-specific cooperative, and rhl-responsive promoters . These results provide the basis for defining promoter specificity elements in P . aeruginosa quorum sensing . Insights into the molecular mechanism of LasR function have implications for the development of quorum-sensing targeted antivirulence compounds.

Invest Ophthalmol Vis Sci, 2004 Nov, 45(11), 4066 - 74
Hypoxia increases corneal cell expression of CFTR leading to increased Pseudomonas aeruginosa binding, internalization, and initiation of inflammation; Zaidi T et al.; PURPOSE: To investigate the effect of hypoxia-induced molecular responses of corneal epithelial cells on the surface of rabbit and human corneas and corneal cells in culture on interactions with Pseudomonas aeruginosa that may underlie increased susceptibility to keratitis . METHODS: Organ cultures of rabbit and human corneal tissue, primary rabbit and human corneal cells, and transformed human corneal cells from a patient with cystic fibrosis and the same cell line corrected for expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), the cellular receptor for P . aeruginosa, were exposed to hypoxic conditions for 24 to 72 hours . Changes in binding and internalization of P . aeruginosa were measured using cellular association and gentamicin-exclusion assays, and expression of CFTR and activation of NF-kappaB in response to hypoxia were determined by confocal laser microscopy and quantitative measurements of NF-kappaB activation . RESULTS: Hypoxia induced in a time- and oxygen-concentration-dependent manner increased association and internalization of clinical isolates of P . aeruginosa in all cells tested . Hypoxia increased CFTR expression and NF-kappaB nuclear translocation in rabbit and human cells with wild-type CFTR . Corneal cells lacking CFTR had reduced NF-kappaB activation in response to hypoxia . Hypoxia did not affect the increase in corneal cell CFTR levels or NF-kappaB activation after P . aeruginosa infection . CONCLUSIONS: Hypoxic conditions on the cornea exacerbate the binding and internalization of P . aeruginosa due to increased levels of CFTR expression and also induce basal NF-kappaB activation . Both of these responses probably exacerbate the effects of P . aeruginosa infection by allowing lower infectious doses of bacteria to induce disease and promote destructive inflammatory responses.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4315 - 21
Novel approach to characterization of combined pharmacodynamic effects of antimicrobial agents; Tam VH et al.; There is considerable need for new modeling approaches in the study of combined antimicrobial effects . Current methods based on the Loewe additivity and Bliss independence models are associated with implicit assumptions about the interacting system . To circumvent these limitations, we propose an alternative approach to the quantification of pharmacodynamic drug interaction (PDI) . Pilot time-kill studies were performed with 10(8) CFU of Pseudomonas aeruginosa/ml at baseline with meropenem or tobramycin alone . The studies were repeated with 25 concentration combinations of meropenem (0 to 64 mg/liter) and tobramycin (0 to 32 mg/liter) in a five-by-five array . The data were modeled with a three-dimensional response surface using effect summation as the basis of null interaction . The interaction index (Ii) is defined as the ratio of the volumes under the planes (VUP) of the observed and expected surfaces: VUP(observed)/VUP(expected) . Synergy and antagonism are defined as Ii values of <1 and >1, respectively . In all combinations, an enhanced killing effect was seen compared to that of either drug at the same concentration . The most significant synergism was observed between 1 and 5 mg/liter of meropenem and between 1 and 4 mg/liter of tobramycin; seven out of nine combinations had a >2-log drop compared to the more potent agent . The Ii was found to be 0.76 (95% confidence interval, 0.65 to 0.91) for the concentration ranges of the agents . The results corroborate previous data indicating that meropenem is synergistic with an aminoglycoside when used in combination against P . aeruginosa . Our parametric approach to quantifying PDI appears robust and warrants further investigations.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4226 - 33
Hypermutation and the preexistence of antibiotic-resistant Pseudomonas aeruginosa mutants: implications for susceptibility testing and treatment of chronic infections; Oliver A et al.; Whether or not resistant mutants will be present before the start of antibiotic treatment of an initially susceptible population of bacteria depends on the size of the infecting population, the rate of mutation to resistance, and the amount of time that the population has been maintained . In the present investigation, we argue that for the treatment of chronic infections caused by hypermutable Pseudomonas aeruginosa of the sort frequently found in cystic fibrosis patients, mutants resistant to all single antipseudomonal drugs will almost invariably be present in a high proportion at the onset of treatment, and consequently, these strains should be considered resistant to all agents when they are used as monotherapy . Using a construct of P . aeruginosa strain PAO1 with a mutS deletion (strain PAODeltamutS), we show that when in vitro populations of less than 5 x 10(4) seemingly susceptible hypermutable bacteria are confronted with any of 11 antipseudomonal agents, mutants for which the MICs and the minimum bactericidal concentrations are in the range of clinical resistance will almost invariably ascend to dominance within 24 to 36 h . This does not occur for PAO1 without the mutS deletion . The results of our detailed analysis of this evolution of acquired resistance to two of these antibiotics, imipenem and ciprofloxacin, indicate that although the rates of mutation to resistance in PAODeltamutS are on the order of 1 x 10(-6) per generation, resistant mutants are very likely to either be present in cultures of between 2 x 10(4) and 4 x 10(4) bacteria or arise after the bacterial populations are confronted with antibiotics . We also demonstrate with in vitro experiments that the problem of acquired resistance to treatment with single antibiotics can be thwarted by combination therapy with pairs of antibiotics of different classes with synergistic activities . We discuss the clinical implications of our analysis of these observations.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4084 - 8
In vitro testing of antimicrobial activity of bone cement; Alt V et al.; The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples . Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin . After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts . The medium was monitored in real time by recording a time proliferation curve for each well . Microplate testing revealed no antibacterial effect of plain bone cement . The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains . The effect of high-porosity silver was dose dependent . Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver . The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility . In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver.

Biochemistry, 2004 Nov 2, 43(43), 13666 - 73
Phosphonoformate: a minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA; Rigsby RE et al.; Fosfomycin {(1R,2S)-epoxypropylphosphonic acid} is a simple phosphonate found to have antibacterial activity against both Gram-positive and Gram-negative microorganisms . Early resistance to the clinical use of the antibiotic was linked to a plasmid-encoded resistance protein, FosA, that catalyzes the addition of glutathione to the oxirane ring, rendering the antibiotic inactive . Subsequent studies led to the discovery of a genomically encoded homologue in the pathogen Pseudomonas aeruginosa . The proteins are Mn(II)-dependent enzymes where the metal is proposed to act as a Lewis acid stabilizing the negative charge that develops on the oxirane oxygen in the transition state . Several simple phosphonates, including the antiviral compound phosphonoformate (K(i) = 0.4 +/- 0.1 microM, K(d) approximately 0.2 microM), are shown to be inhibitors of FosA . The crystal structure of FosA from P . aeruginosa with phosphonoformate bound in the active site has been determined at 0.95 A resolution and reveals that the inhibitor forms a five-coordinate complex with the Mn(II) center with a geometry similar to that proposed for the transition state of the reaction . Binding studies show that phosphonoformate has a near-diffusion-controlled on rate (k(on) approximately 10(7)-10(8) M(-1) s(-1)) and an off rate (k(off) = 5 s(-1)) that is slower than that for fosfomycin (k(off) = 30 s(-1)) . Taken together, these data suggest that the FosA-catalyzed reaction has a very early transition state and phosphonoformate acts as a minimal transition state analogue inhibitor.

Infect Immun, 2004 Nov, 72(11), 6463 - 70
Differential immune modulatory activity of Pseudomonas aeruginosa quorum-sensing signal molecules; Hooi DS et al.; Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM) . We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity . One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity . In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro . 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule) . 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release . Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation . Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K . Tateda et al., Infect . Immun . 64:37-43, 1996), whereas PQS did not inhibit in this assay . These data highlight the presence of two differentially active immune modulatory QSSM from P . aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P . aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.

FEMS Microbiol Lett, 2004 Nov 1, 240(1), 111 - 6
Galangin expresses bactericidal activity against multiple-resistant bacteria: MRSA, Enterococcus spp . and Pseudomonas aeruginosa; Pepeljnjak S et al.; The antimicrobial activity of three propolis ethanol extracts (EEP) was examined for various Gram-negative and Gram-positive bacterial species, including multiple-resistant Staphylococcus aureus, Enterococcus spp . and Pseudomonas aeruginosa strains . EEP had a good bactericidal activity against Gram-positive species, and all multiple-resistant bacterial strains tested were sensitive to EEP . Minimal inhibitory concentrations (MICs) were lower in samples of higher flavonoid content (from 0.65 to 7.81 mg mL(-1)), indicating the influence of the concentration of some potent bactericidal compound(s) in propolis or synergism among some bactericidal compounds . Antimicrobial-guided separation of flavonoid aglycones (bioassay in situ on thin-layer chromatogram) showed that galangin (3,5,7-trihydroxyflavone) is one compound in EEP with bactericidal activity . Galangin was isolated by preparative chromatography . After determining the quantity present, the MIC against multiple-resistant bacteria was determined . The MIC of galangin against multiple-resistant bacterial strains was significantly lower (from 0.16 to 0.44 mg mL(-1), p < 0.05) than that of EEP . The bactericidal activity of galangin against P . aeruginosa strains was present at 0.17+/-0.05 mg mL(-1).

J Vet Pharmacol Ther, 2004 Oct, 27(5), 293 - 8
Cefotaxime kinetics in plasma and synovial fluid following intravenous administration in horses; Orsini JA et al.; Cefotaxime powder was diluted with sterile water to a concentration of 100 mg/mL . The volume of solution was adjusted for each experimental horse to provide a total dose of 15, 20, and 25 mg/kg and was administered by infusion through a jugular vein catheter over a 10-min period . All three doses were administered to each of the six experimental horses at three different times . Cefotaxime concentrations in plasma and synovial fluid samples were measured by high-performance liquid chromatography (HPLC) . Standard compartmental analysis techniques and the WinSAAM modeling program were used to determine standard pharmacokinetic parameters for cefotaxime . The plasma and synovial fluid data from the five horses administered the 25 mg/kg dose was analyzed . Plasma cefotaxime concentrations appeared to be linearly related to dose infused and declined in parallel, suggesting linear drug kinetics . Moreover, cefotaxime concentrations declined monotonically suggesting that its disposition kinetics could essentially be described by a one-compartment model rather than the fact that sampling occurred after the infusion was discontinued . Maximum concentration of cefotaxime in plasma occurred immediately after cessation of the infusion . Minimum inhibitory concentrations were determined for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, common isolates from septic arthritis in horses . Based on our pharmacokinetic data, a regimen of 25 mg/kg administered i.v . every 6 h appears appropriate for susceptible joint infections in adult horses.

Eye Contact Lens, 2004 Jul, 30(3), 173 - 8
Pseudomonas aeruginosa corneal binding after 24-hour orthokeratology lens wear; Ladage PM et al.; PURPOSE: To examine the effect of short-term 24-hr orthokeratology lens (OKL) wear on Pseudomonas aeruginosa binding, epithelial surface cell morphology, epithelial sheet thickness, and stromal thickness in a rabbit model . METHODS: Seventeen New Zealand white rabbits were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research . Partial membranectomy was performed on all rabbits 1 week before the experiments . Baseline values for epithelial and stromal thickness and epithelial surface cell size were determined by in vivo confocal microscopy in one randomly chosen eye (n = 6) . One week later, rabbits were fitted in the same eye with a hyper oxygen-transmissible OKL . Twenty-four hours later, confocal microscopy was repeated . The second group of rabbits (n = 6) was fitted with an OKL in one randomly chosen eye for 24 hr . P . aeruginosa binding to the corneal epithelium was assessed for the control corneas and those exposed to the test lens . Scanning electron microscopy was performed on a third group of rabbits to assess epithelial surface damage (n = 5) . RESULTS: There was a statistically significant difference (P<0.001) in P . aeruginosa binding between the control (1.11 +/- 0.74 x 10(5) colony-forming units per cornea) and the OKL-wearing eyes (2.74 +/- 0.69 x 10(5) colony-forming units per cornea) . The central epithelium thinned by 6.5% after lens wear (48.2 +/- 1.9 microm to 45 +/- 1.7 microm, P=0.005); however, central stromal thickness increased by 7.3% (322 +/- 22 microm to 345 +/- 29 microm, P=0.006) . Compared with the baseline value, central epithelial cell size increased significantly from 1,253 +/- 140 mm(2) to 1,627 +/- 393 mm(2) (29.4%, P=0.02) . Scanning electron microscopy showed increased surface epithelial damage associated with OKL wear . CONCLUSIONS: This prospective, masked, pilot study showed that 24-hr hyper oxygen-transmissible OKL wear induced a statistically significant increase in P . aeruginosa binding to the epithelium of the rabbit cornea, accompanied by central epithelial thinning, stromal thickening, and surface cell damage assessed by scanning electron microscopy . Collectively, the data suggest that despite adequate lens oxygen transmissibility, the mechanical pressure inherent in the OKL design exerted on the corneal surface appears to be associated with increased adherence of P . aeruginosa to surface corneal epithelial cells, which may pose an increased risk for lens-related microbial keratitis, especially in overnight (i.e., closed-eye) wearing conditions . Future studies are needed to determine whether these results are similar in human wear and how P . aeruginosa binding during OKL wear compares with other lens-wearing modalities, such as daily or continuous soft lens wear.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Sep, 27(9), 608 - 10
{Diffuse panbronchiolitis complicated by thymoma: a case report and a review of literature}; Zhang D et al.; OBJECTIVE: To highlight the characteristics of diffuse panbronchiolitis (DPB) . METHOD: One patient with DPB confirmed by thorocoscopic biopsy was described and relevant literatures were reviewed . RESULTS: DPB is a chronic lower respiratory tract disease common in Japanese, rare in China, characterized by infiltration of inflammatory cells around bronchioles . Although the etiology and precise mechanisms are under investigation, it is commonly hypothesized that heredity and immunity have a major role in DPB . Symptoms include cough, expectoration, and dyspnea after exercises . Pseudomonas aeruginosa is isolated from sputum in some cases . If left untreated, DPB progresses rapidly and has a poor prognosis if respiratory failure occurs . CONCLUSIONS: DPB should be included in the differential diagnosis of bilateral multiple pulmonary nodular shadows . Long-term, low-dose macrolide therapy may improve the prognosis through an anti-inflammatory effect.

J Med Microbiol, 2004 Nov, 53(Pt 11), 1151 - 4
Predictive value of isolating Pseudomonas aeruginosa from aerobic and anaerobic blood culture bottles; Enoch DA et al.; Pseudomonas aeruginosa is a particularly virulent pathogen when it causes bacteraemia and early diagnosis is essential to reduce morbidity and mortality . It is an aerobe and is thought by many to be almost exclusively isolated from the aerobic blood culture bottle in cases of bacteraemia . This study analysed 277 Gram-negative bacteraemic episodes over 1 year at a single institution in order to assess the predictive value of this finding . In 39 of 44 episodes of P . aeruginosa bacteraemia, the organism was isolated from the aerobic bottle only, which gave a sensitivity of 88.6 % for this 'test' and a specificity of 73.8 % . However, for all episodes of Gram-negative bacteraemia, the likelihood of a Gram-negative bacillus occurring in the aerobic bottle first being P . aeruginosa was only 39 % . The converse finding of a Gram-negative bacillus isolated first in the anaerobic bottle or from both bottles together was clinically helpful, having a negative predictive value of 97.2 % (i.e . that the organism was not P . aeruginosa).

Ann Clin Microbiol Antimicrob . 2004 Oct 20;3(1):21.
Early detection of Pseudomonas aeruginosa - comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF); Xu J et al.; BACKGROUND: Pseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients . Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection . The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum . METHODS: Adult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise . Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar) . In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus . RESULTS: By sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients . In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci . The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative . Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4-17 months later, with a mean lag time of 4.5 months . CONCLUSIONS: This study indicates that molecular detection of PA in sputum employing the oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture . It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.

J Immunol, 2004 Nov 1, 173(9), 5712 - 20
Polymorphonuclear cell transmigration induced by Pseudomonas aeruginosa requires the eicosanoid hepoxilin A3; Hurley BP et al.; Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology . A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site . It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen . An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers . A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers . This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface . Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8 . However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration . Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner . These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.

J Immunol, 2004 Nov 1, 173(9), 5671 - 8
Human monoclonal antibodies to Pseudomonas aeruginosa alginate that protect against infection by both mucoid and nonmucoid strains; Pier GB et al.; Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia . The specificity of the mAbs for alginate was determined by ELISA and killing assays . Some strains of P . aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung . No protection against strains genetically unable to make alginate was achieved . These mAbs have potential to be passive therapeutic reagents for all strains of P . aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.

J Immunol, 2004 Nov 1, 173(9), 5659 - 70
Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease; Cobb LM et al.; Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern . Two P . aeruginosa phenotypes relevant in human disease include motility and mucoidy . Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients . Indeed, chronic infection of the lung by mucoid P . aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients . We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P . aeruginosa . The response of airway epithelial cells to exposure to P . aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense . By contrast, the response of airway epithelia to the stimuli presented by mucoid P . aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen . The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines . By contrast, infection with alginate-producing P . aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.

Biosens Bioelectron, 2004 Oct 15, 20(3), 538 - 44
Detection of Mycobacterium tuberculosis (TB) in vitro and in situ using an electronic nose in combination with a neural network system; Pavlou AK et al.; The use of volatile production patterns produced by Mycobacterium tuberculosis and associated bacterial infections from sputum samples were examined in vitro and in situ using an electronic nose based on a 14 sensor conducting polymer array . In vitro, it was possible to successfully discriminate between M . tuberculosis (TB) and control media, and between M . tuberculosis and M . avium, M . scrofulaceum and Pseudomonas aeruginosa cultures in the stationary phase after 5-6h incubation at 37 degrees C based on 35 samples . Using neural network (NN) analysis and cross-validation it was possible to successfully identify 100% of the TB cultures from others . A second in vitro study with 61 samples all four groups were successfully discriminated with 14 of 15 unknowns within each of the four groups successfully identified using cross-validation and discriminant function analysis . Subsequently, lipase enzymes were added to 46 sputum samples directly obtained from patients and the head space analysed . Parallel measurements of bacterial contamination were also carried out for confirmation using agar media . NN analysis was carried out using some of the samples as a training set . Based on the NN and genetic algorithms of up to 10 generations it was possible to successfully cross-validate 9 of 10 unknown samples . PCA was able to discriminate between TB infection alone, the controls, M . avium, P . aeruginosa and a mixed infection . These findings will have significant implications for the development of rapid qualitative systems for screening of patient samples and clinical diagnosis of tuberculosis.

Bioresour Technol, 2005 Mar, 96(4), 429 - 36
Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K; Rahman RN et al.; The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated . Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P . aeruginosa . Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum . Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v) . Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0 . However, the highest yield of protease was observed at pH 7.0 . Neutral media increased the protease production compared to acidic or alkaline media.

Mol Microbiol, 2004 Nov, 54(3), 620 - 31
Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli; Gerken H et al.; Genetic data have suggested that TolC, AcrA and AcrB constitute a major antibiotic efflux system in Escherichia coli . Through reversion analysis of an unstable and antibiotic-sensitive TolC mutant (TolCP246R,S350C), we isolated extragenic suppressors that mapped within the acrRAB loci . DNA sequence analysis revealed that 18 isolates contained 10 different missense mutations within the acrA gene, whereas a single isolate had a missense mutation within the acrR gene, which codes for the acrAB repressor . Besides reversing the hypersensitivity phenotype of TolCP246R,S350C, AcrA and AcrR alterations elevated the mutant TolC protein level, thus indicating that the mechanism of suppression involves the stabilization of an unstable mutant TolC protein . Eight of the 10 AcrA alterations were clustered in the 202-265 region of the mature protein, whereas the other two suppressors affected residues 30 and 146 . Based on the recently solved crystal structure of MexA, an AcrA counterpart from Pseudomonas aeruginosa, the regions encompassing residues 30 and 202-265 constitute the alpha+beta-domain of AcrA (MexA), whereas that of 146 form the alpha-domain . The data suggest that residues of these two AcrA domains either directly or indirectly influence interactions with TolC . Curiously, the stability of three mutant AcrA proteins, bearing an L222Q, L222R or P265R substitution, became dependent on the presence of either wild-type or mutant TolC . This dependence of the mutant AcrA proteins on TolC further supported the notion of a direct physical interaction between these two proteins . Because a mutation in acrR or acrAB expression from a multicopy plasmid also suppressed the TolCP246R,S350C defects, it indicated that wild-type AcrA when produced in high levels presumably establishes similar interactions with the mutant TolC protein as do the suppressor forms of AcrA produced from the chromosomal copy . The AcrA-mediated suppression of mutant TolC phenotypes and the stabilization of mutant TolC protein were dependent on AcrB, reflecting the existence of a functional complex between TolC and AcrAB in vivo.

Biochemistry, 2004 Oct 26, 43(42), 13478 - 86
Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa; Kim SK et al.; APS reductase from Pseudomonas aeruginosa has been shown to contain a {4Fe-4S} cluster . Thiol determinations and site-directed mutagenesis studies indicate that the single {4Fe-4S} cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines . Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert {4Fe-4S}(2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species . This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a {3Fe-4S}(+) form by treatment with a 3-fold excess of ferricyanide . Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster . The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.

Perit Dial Int, 2004 Sep-Oct, 24(5), 447 - 53
Pharmacokinetics of oral ciprofloxacin in continuous cycling peritoneal dialysis; Yeung SM et al.; BACKGROUND: In order to avoid aminoglycosides, the International Society for Peritoneal Dialysis recommends cefazolin and ceftazidime for empirical treatment of peritonitis . Ciprofloxacin covers relevant gram-negative pathogens without the resistance associated with ceftazidime . However, ciprofloxacin pharmacokinetic data in patients on continuous cycling peritoneal dialysis (CCPD) are lacking . OBJECTIVES: (1) To determine the pharmacokinetics of oral ciprofloxacin in CCPD patients, (2) to compare serum and dialysate ciprofloxacin concentrations with minimum inhibitory concentrations (MIC) of the gram-negative bacteria associated with peritonitis, and (3) to establish oral ciprofloxacin dosing guidelines for the empirical treatment of peritonitis in patients receiving CCPD . METHODS: Eligible CCPD patients received 2 doses of ciprofloxacin: 750 mg orally every 12 hours . Serial blood and end-of-dwell dialysate samples were collected during the first 12-hour interval; an end-of-dwell dialysate sample from the overnight dwell and a final blood sample were collected at the end of the second 12-hour interval . Ciprofloxacin concentrations were determined using a liquid chromatographic (HPLC)-fluorescence method . Pharmacokinetic calculations were completed assuming a one-compartment model . RESULTS: Eight patients completed the study . The pharmacokinetic parameters determined for ciprofloxacin were (mean +/- SEM) serum half-life 10.1 +/- 1.2 hours, maximum serum concentration 2.7 +/- 0.5 mg/L, time to maximum serum concentration 1.6 +/- 0.1 hours after the first dose, and peritoneal clearance 1.2% +/- 0.1% of the mean calculated total body clearance . While all patients achieved serum area under the concentration-time curve:MIC > 125 for Escherichia coli and Klebsiella species after the first dose, only 2 patients achieved this goal for Pseudomonas aeruginosa . End-of-dwell dialysate concentrations were above the MIC for E . coli, Klebsiella spp, and P . aeruginosa after the second dose . CONCLUSION: Ciprofloxacin 750 mg orally every 12 hours in CCPD patients may be useful for empirical gram-negative coverage of CCPD peritonitis and for treatment of documented peritonitis caused by sensitive E . coli or Klebsiella species . While ceftazidime may be required for documented pseudomonal peritonitis, the oral ciprofloxacin regimen achieved adequate serum concentrations to treat systemic gram-negative infections caused by sensitive E . coli or Klebsiella species.

Mikrobiyol Bul, 2004 Jul, 38(3), 273 - 84
{Bacterial communication: quorum-sensing}; Cakar A; The interaction between the host and a pathogenic bacterium is mainly controlled by the bacterial population size . An individual bacterial cell is able to sense other members of the same species and in response, differentially expresses specific genes . Such cell to cell communication is called quorum sensing (QS) and involves the direct or indirect activation of a response regulator by a signal molecule . The major QS signal molecules are N-acyl homoserine lactones in Gram negative bacteria and post-translationally modified peptides in Gram positive bacteria . QS system is used by a wide variety of bacteria including human pathogens . QS genes are important for the pathogenic potential of Pseudomonas aeruginosa and Staphylococcus aureus, as well as other invasive bacteria . Thus QS interfering molecules promise new therapeutic strategies or prophylactic measures in infectious diseases . In this review article, the role of QS system on bacterial virulence, its effects on the host immune response and QS inhibitors for prophylaxis and therapy are discussed.

J Bacteriol, 2004 Nov, 186(21), 7369 - 77
AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa; Robles-Price A et al.; Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients . Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P . aeruginosa chromosome . By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm . Consistent with these results is the presence of a 26-amino-acid signal sequence . To examine the requirement for AlgX in alginate biosynthesis, part of algX in P . aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette . The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing) . The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P . aeruginosa algX provided on a plasmid . The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths . Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid . These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.

Genome Res, 2004 Oct, 14(10B), 2190 - 200
The Pseudomonas aeruginosa PA01 gene collection; Labaer J et al.; Pseudomonas aeruginosa, a common inhabitant of soil and water, is an opportunistic pathogen of growing clinical relevance . Its genome, one of the largest among bacteria {5570 open reading frames (ORFs)} approaches that of simple eukaryotes . We have constructed a comprehensive gene collection for this organism utilizing the annotated genome of P . aeruginosa PA01 and a highly automated and laboratory information management system (LIMS)-supported production line . All the individual ORFs have been successfully PCR-amplified and cloned into a recombination-based cloning system . We have isolated and archived four independent isolates of each individual ORF . Full sequence analysis of the first isolate for one-third of the ORFs in the collection has been completed . We used two sets of genes from this repository for high-throughput expression and purification of recombinant proteins in different systems . The purified proteins have been used to set up biochemical and immunological assays directed towards characterization of histidine kinases and identification of bacterial proteins involved in the immune response of cystic fibrosis patients . This gene repository provides a powerful tool for proteome- and genome-scale research of this organism, and the strategies adopted to generate this repository serve as a model for building clone sets for other bacteria.

Biochem Biophys Res Commun, 2004 Nov 19, 324(3), 1116 - 23
Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates; Riester D et al.; Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells . Deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone-DNA and histone-non-histone protein interactions . However, surprisingly little is known about the substrate specificities of different HDACs . Here, we use the epsilon-acyl moieties of epsilon-modified l-lysine in peptidic substrates as a probe to examine the active site cavity of HDACs and HDAC-like enzymes . Measurements were based on a fluorogenic assay with small synthetic substrates . Four different enzyme preparations were used derived from rat, human, and bacterial sources . None of the enzymes was able to utilize substrates with epsilon-acyl moieties larger than acetyl, except rat liver HDAC, which was the only enzyme to convert a substrate containing epsilon-propionyl-l-lysine . All enzymes exhibited a distinct enantioselectivity toward l-lysine-containing substrates except FB188 HDAH which also deacetylated Boc-d-Lys(epsilon-acetyl)-MCA . Moreover, all enzymes also exhibited a distinct specificity for the length of the lysine side chain; acetylated ornithine, which comprises one CH(2) unit less in the side chain, was not a substrate . In line with these results, only acetylcadaverin the metabolic degradation product of lysine but neither acetylputrescine (degradation product of ornithine) nor acetylspermidine strongly inhibited enzyme activity . Boc-l-Lys(epsilon-trifluoroacetyl)-MCA was observed to be a superior substrate for FB188 HDAH, Pseudomonas aeruginosa HDAH (PA3774), and particularly HDAC 8 compared to rat liver HDAC, and is the first suitable (synthetic) substrate for (human-derived) HDAC 8 reported to date . Altogether, the results reveal clear differences in substrate specificity between different HDACs as analyzed in the fluorogenic HDAC assay . Finally, we present the first candidates for HDAC-type-selective substrates that may be useful as biochemical tools to establish the function of particular pathways and to elucidate the role of distinct HDAC subtypes in cellular differentiation and cancer.

Biochem Biophys Res Commun, 2004 Nov 19, 324(3), 1065 - 8
In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux; Babayan A et al.; Xu et al . {Biochem . Biophys . Res . Commun . 305 (2003) 941} reported that, when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 microM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually . The authors concluded that the intracellular ethidium bromide "induced" either its degradation or its efflux through the assembly of unknown efflux pumps . We found, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under these conditions, indicating the absence of its degradation . Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.

Infect Control Hosp Epidemiol, 2004 Sep, 25(9), 753 - 8
Ventilator-associated pneumonia in a pediatric intensive care unit in Saudi Arabia: a 30-month prospective surveillance; Almuneef M et al.; OBJECTIVE: To describe the rate, risk factors, and outcome of ventilator-associated pneumonia (VAP) in pediatric patients . METHODS: This prospective surveillance study of VAP among all patients receiving mechanical ventilation for 48 hours or more admitted to a pediatric intensive care unit (PICU) in Saudi Arabia from May 2000 to November 2002 used National Nosocomial Infections Surveillance (NNIS) System definitions . RESULTS: Three hundred sixty-one eligible patients were enrolled . Most were Saudi with a mean age of 28.6 months . Thirty-seven developed VAP . The mean VAP rate was 8.87 per 1,000 ventilation-days with a ventilation utilization rate of 47% . The mean duration of mechanical ventilation was 21 days for VAP patients and 10 days for non-VAP patients . The mean PICU stay was 34 days for VAP patients and 15 days for non-VAP patients . Among VAP patients, Pseudomonas aeruginosa was the most common organism, followed by Staphylococcus aureus . Other gram-negative organisms were also encountered . There was no significant difference between VAP and non-VAP patients regarding mortality rate . Witnessed aspiration, reintubation, prior antibiotic therapy, continuous enteral feeding, and bronchoscopy were associated with VAP . On multiple logistic regression analysis, only prior antibiotic therapy, continuous enteral feeding, and bronchoscopy were independent predictors of VAP . CONCLUSIONS: The mean VAP rate in this hospital was higher than that reported by NNIS System surveillance of PICUs . This study has established a benchmark for future studies of VAP in the pediatric intensive care population in Saudi Arabia . Additional studies from the region are necessary for comparison and development of preventive measures.

Crit Care Med, 2004 Oct, 32(10), 1997 - 2001
Acute pancreatitis in mice impairs bacterial clearance from the lungs, whereas concurrent pneumonia prolongs the course of pancreatitis; van Westerloo DJ et al.; OBJECTIVE: Nosocomial pneumonia is a feared complication in the critically ill patient . Serious acute pancreatitis is frequently complicated by infections . The objectives of this study were to determine the influence of acute pancreatitis on host defense against Pseudomonas pneumonia and to determine the influence of Pseudomonas pneumonia on the severity of concurrent pancreatitis . DESIGN: A controlled, in vivo laboratory study . SETTING: Research laboratory of a health sciences university . SUBJECTS: Female C57Bl/6 mice . INTERVENTIONS: Pancreatitis was induced by 12 hourly intraperitoneal injections of cerulein (pancreatitis) or saline (sham) immediately followed by intranasal administration of Pseudomonas aeruginosa (to induce pneumonia) or saline (controls) . Mice were killed 24 hrs later . Hence, four groups were studied: sham/control, pancreatitis/control, sham/pneumonia, and pancreatitis/pneumonia mice . MEASUREMENTS AND MAIN RESULTS: When compared with sham/pneumonia mice, pancreatitis/pneumonia mice demonstrated exaggerated lung inflammation, higher bacterial counts in lungs and pancreas, and enhanced dissemination of the infection . Concurrently, pneumonia prolonged the course of pancreatitis, as reflected by histopathology and higher plasma amylase and relative pancreas weights (all p < .05 for the difference between pancreatitis/pneumonia and pancreatitis/control mice), which was associated with the localization of Pseudomonas in the pancreas . CONCLUSIONS: Acute pancreatitis impairs host defense against Pseudomonas pneumonia, whereas pneumonia prolongs the course of pancreatitis.

Toxicol Sci, 2005 Jan, 83(1), 155 - 65 Epub 2004 Oct 13.
Inhaled Diesel Engine Emissions Reduce Bacterial Clearance and Exacerbate Lung Disease to Pseudomonas aeruginosa Infection In Vivo; Harrod KS et al.; Despite experimental evidence supporting an adverse role for air pollution in models of human disease, little has been done in the way of assessing the health effects of inhalation of whole mixtures from defined sources at exposure levels relevant to ambient environmental exposures . The current study assessed the impact of inhaled diesel engine emissions (DEE) in modulating clearance of Pseudomonas aeruginosa (P.a.) and the adverse effects of infection to the pulmonary epithelium . At DEE concentrations representing from high ambient to high occupational exposures, mice were exposed to DEE continuously for one week or six months (6 h/day), and subsequently infected with P.a . by intratracheal instillation . At 18 h following P.a . infection, prior exposure to DEE impaired bacterial clearance and exacerbated lung histopathology during infection . To assess the airway epithelial cell changes indicative of lung pathogenesis, markers of specific lung epithelial cell populations were analyzed by immunohistochemistry . Both ciliated and non-ciliated airway epithelial cell numbers were decreased during P.a . infection by DEE exposure in a concentration-dependent manner . Furthermore, the lung transcription regulator, thyroid transcription factor 1 (TTF-1), was also decreased during P.a . infection by prior exposure to DEE concordant with changes in airway populations . These findings are consistent with the notion that environmental levels of DEE can decrease the clearance of P.a . and increase lung pathogenesis during pulmonary bacterial infection.

Protein Expr Purif, 2004 Nov, 38(1), 79 - 83
In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA; Folschweiller N et al.; The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years . We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation . To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation . Selenomethionine incorporation greater than 95% was achieved using P . aeruginosa as an overexpression system.

Burns, 2004 Nov, 30(7), 660 - 4
The time-related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients; Altoparlak U et al.; To examine the bacterial isolates from the burn patients and to compare the antibiograms of the predominant bacteria isolated from 51 patients who were hospitalized at least 3 weeks or more over a period of 7 months, a prospective study was undertaken . Periodic swabs were taken from burn wound, nasal, axillary, inguinal, and umbilical region of the patients on admission and on 7th, 14th, and 21st days of hospitalization . Mean hospital stay was 36.5 days . A total of 1098 microbial isolates were detected during the study period . Coagulase-negative staphylococci (CNS, 63.0%) and Staphylococcus aureus (19.7%) were the most prevalent isolates in admission cultures . During the next weeks, these bacteria were superceded by mainly Pseudomonas aeruginosa . Between admission and 21st day, the rates of methicillin resistance of staphylococci strains increased steadily . There was no vancomycin resistance in any staphylococci strains, although nine of the S . aureus isolates (2.7%) were resistant to teicoplanin . There were no strains producing inducible beta lactamase (IBL) among P . aeruginosa strains . One extended-spectrum beta-lactamase (ESBL)-producing strain was recovered on admission, although strains producing IBL and ESBL were detected at rates of 79.6 and 57.1%, respectively, on the 21st day . The nature of microbial wound colonization, flora changes, and antimicrobial sensitivity profiles should be taken into consideration in using empirical antimicrobial therapy of burned patients.

Burns, 2004 Nov, 30(7), 619 - 27
Host defense peptides in burns; Steinstraesser L et al.; Overuse of antibiotics and failure to apply basic infection control policies and procedures have contributed to the increasing multi-drug resistance of many nosocomial pathogens . The alarming increase of multi-drug-resistant bacteria (e.g . Pseudomonas aeruginosa, methicilin-resistant Staphylococci, vancomycin-resistant Enterococci) causes infected wounds associated with high mortality and morbidity in burned patients and focuses attention on the need for better treatment and prevention of wound infections . The review points out and discusses some emerging alternatives to antibiotics used in clinical practice, with special emphasis on the role of the innate immune response and potential application of human host defense peptides in thermal injury.

Biodegradation, 2004 Aug, 15(4), 249 - 60
Enhanced biodegradation of Casablanca crude oil by a microbial consortium in presence of a rhamnolipid produced by Pseudomonas aeruginosa AT10; Abalos A et al.; The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate . Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium . The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures . The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation . Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction . The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.

J Biochem Mol Biol, 2004 Jul 31, 37(4), 387 - 93
Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation; El-Bessoumy AA et al.; The L-asparaginase (E . C . 3 . 5 . 1 . 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation . Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation . The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa . A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU . The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min . The amino acid composition of the purified enzyme was also determined.

Mol Microbiol, 2004 Oct, 54(2), 307 - 20
An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system; Ha UH et al.; We have previously reported on the isolation of in vivo inducible genes of Pseudomonas aeruginosa using IVET system . One of such genes isolated from burn mouse infection model encodes a short open reading frame with unknown function . In this study, we demonstrate that this gene product specifically suppresses the expression of type III secretion genes in P . aeruginosa, thus named PtrA (Pseudomonas type III repressor A) . A direct interaction between the PtrA and type III transcriptional activator ExsA was demonstrated, suggesting that its repressor function is probably realized through inhibition of the ExsA protein function . Indeed, an elevated expression of the exsA compensates the repressor effect of the PtrA . Interestingly, expression of the ptrA is highly and specifically induced by copper cation . A copper- responsive two-component regulatory system, copR-copS, has also been identified and shown to be essential for the copper resistance in P . aeruginosa as well as the activation of ptrA in response to the copper signal . Elevated expression of the ptrA during the infection of mouse burn wound suggests that P . aeruginosa has evolved tight regulatory systems to shut down energy-expensive type III secretion apparatus in response to specific environmental signals, such as copper stress.

Protein Pept Lett, 2004 Aug, 11(4), 291 - 300
Design, synthesis and antibacterial activity studies of model peptidess of proline/arginine-rich region in bactenecin7; Abiraj K et al.; Bactenecin 7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue) . To investigate the structure and property of a Pro/Arg-rich region, e synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized . The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism . The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions . Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and gram-positive bacteria such as Staphylococcus aureus were measured at various peptide concentrations . Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed with gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 microg/mL.

J Bacteriol, 2004 Oct, 186(20), 7015 - 8
Evidence for polyadenylated mRNA in Pseudomonas aeruginosa; Saravanamuthu SS et al.; In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers . Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P . aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.

J Bacteriol, 2004 Oct, 186(20), 6837 - 44
The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa; Carterson AJ et al.; Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients . One characteristic of P . aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR . Transcriptional profiling analyses comparing mucoid P . aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants . S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P . aeruginosa . Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains . In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates . Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P . aeruginosa CF isolates from 24 patients . Our data indicate that (i) mucoid P . aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P . aeruginosa, and (iii) P . aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains . Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

Pathol Biol (Paris), 2004 Oct, 52(8), 455 - 61
{Two efflux systems expressed simultaneously in clinical Pseudomonas aeruginosa.}; Hocquet D et al.; Active efflux systems MexAB-OprM and MexXY were found to be overexpressed simultaneously in 12 multiresistant clinical isolates of Pseudomonas aeruginosa . Nine of these strains (agrZ mutants) harbored mutations in gene mexZ, the product of which down-regulates expression of operon mexXY . Eight of the 12 strains exhibited mutations in genes known to control transcription of operon mexAB-oprM, such as mexR (four nalB mutants) or PA3721 (three nalC mutants) . One strain was a nalB/nalC double mutant . For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the over-expression levels of genes mexA or mexX, and (iii) the resistance levels to effluxed antibiotics . Finally, three and four isolates overproduced MexXY (agrW mutants) or MexAB-OprM (nalD mutants), respectively, without any mutation in the known regulator genes . These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.

J Cyst Fibros, 2004 Aug, 3 Suppl 2, 197 - 201
Airway epithelial cell-pathogen interactions; Bragonzi A et al.; Cystic fibrosis (CF) airway becomes colonized with only a limited number of bacterial pathogens . It is of paramount importance to establish in vitro and in vivo models to better understand bacterial-host interactions under CF-like conditions . In this article, in vitro methods suitable to study Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) adherence to and uptake by airway epithelial cells are described . Acute and chronic respiratory infection models, which have been used in CF transgenic mice and mimic human CF lung pathology, are also taken into consideration.

J Cyst Fibros, 2004 Aug, 3(3), 179 - 83
Anti-neutrophil cytoplasmatic antibodies and lung disease in cystic fibrosis; Dorlochter L et al.; Background: Bactericidal-permeability-increasing protein (BPI) is a potent anti-microbial protein produced by neutrophil granulocytes . Anti-neutrophil cytoplasmatic antibodies (ANCA) directed against BPI have been detected in up to 91% in patients with cystic fibrosis (CF) . We aimed to evaluate the prevalence of BPI-ANCA in our CF patients and to determine whether presence of BPI-ANCA is correlated with organ damage . Methods: Twenty-four patients performed respiratory function testing and pulmonary high-resolution computed tomography (HRCT) . HRCT was scored by using a modified Bhalla method . Serum samples were analysed by direct binding enzyme-linked immunosorbent assay for BPI-ANCA . Results: The prevalence of anti-BPI-IgG was 71% and anti-BPI-IgA 33% . Twenty-nine percent of our patients were positive for both BPI-ANCA isotypes . Mean HRCT score was 8.0 ranging from 0 to 22, bronchiectasis presented the most common finding (79%) . There was a significant correlation between BPI-ANCA and both HRCT score and FEV(1) (p<0.01) . High levels of BPI-ANCA were correlated to chronic Pseudomonas aeruginosa lung infection (p<0.01) . Conclusions: BPI-ANCA was common in our study group . Highly significant correlations between BPI-ANCA and parameters to evaluate lung disease in CF may be a consequence of the inflammation process, or it may indicate a pathogenic role of BPI-ANCA levels in the development of lung disease . More research is needed and the clinical significance of our findings needs further evaluation.

J Cyst Fibros, 2004 Mar, 3(1), 37 - 44
Genotyping of Pseudomonas aeruginosa in cystic fibrosis suggests need for segregation; Edenborough FP et al.; BACKGROUND: Emerging resistance of Pseudomonas aeruginosa within cystic fibrosis (CF) populations is attributed to antibiotic pressure and spread of transmissible strains . We describe increasing resistance of P . aeruginosa isolates, resulting in the identification of two multiresistant strains and their impact on morbidity . METHODS: Susceptibility reports of all P . aeruginosa isolates since 1998 in our unit were reviewed . Isolates were submitted for genomic finger-printing by pulsed-field gel electrophoresis . Clinical measures and the consumption of treatment resources were compared between those harbouring resistant organisms and those with sensitive strains . RESULTS: Analysis of 407 reports from 43 patients revealed isolation of multiresistant (MR) organisms increased during 1999 . Those harbouring MR strains consumed more resources than non-MR . Strain typing showed a new 'Sheffield' strain in seven patients (100% MR), and the 'Liverpool' strain in 10 patients (40% MR) . Individuals in these groups consumed significantly more resources than 23 patients with unique, susceptible strains (4% MR) . DISCUSSION: Increasing resistance in isolates of P . aeruginosa may herald the arrival of a transmissible strain in CF Units which though sometimes sensitive, may become multiply resistant and require more intensive treatment . We now segregate those with transmissible strains from each other and from those with unique strains.

J Cyst Fibros, 2004 Mar, 3(1), 23 - 8
Effect of nebulized colistin sulphate and colistin sulphomethate on lung function in patients with cystic fibrosis: a pilot study; Westerman EM et al.; BACKGROUND: Pulmonary administration of colistin is one of the antimicrobial treatments used in Cystic Fibrosis (CF) patients chronically infected with Pseudomonas aeruginosa . Dry powder inhalation of colistin may be an attractive alternative to nebulization of colistin . However, nebulized colistin can cause bronchoconstriction in CF patients . Therefore, in the progress of developing a dry powder formula, the choice of the inhaler and its contents should be guided by optimal efficacy and the least possible side effects . To investigate the side effects, a study was initiated to compare the tolerability of colistin sulphate to colistin sulphomethate per nebulization in CF-patients . METHODS: Nine CF-patients chronically infected with P . aeruginosa participated in a double blind, randomized cross over study . On two visits to the outpatient clinic, patients were submitted to either nebulized colistin sulphate or colistin sulphomethate solution . Lung function tests were performed immediately before and 15 and 30 min after nebulization . RESULTS: Nebulization of colistin sulphate caused a significant larger mean decrease in lung function compared to nebulized colistin sulphomethate . A significant decrease in mean changes (SD) in FEV1 at 30 min and FVC at 15 and 30 min after nebulization compared to baseline of -7.3% (8.6%), -5.7% (7.3%) and -8.4% (7.5%) respectively was seen after colistin sulphate nebulization compared to colistin sulphomethate (P < 0.05) . Seven patients were not able to complete the nebulization of colistin sulphate because of throat irritation and severe cough . CONCLUSION: Based on these results it was concluded that inhalation with nebulized colistin sulphate is not suitable for treatment of CF patients chronically infected with P . aeruginosa . Colistin sulphomethate is the drug of choice for pulmonary administration of colistin.

J Cyst Fibros, 2003 Dec, 2(4), 199 - 205
Different opinions of physicians on the importance of measures to prevent acquisition of Pseudomonas aeruginosa from the environment; Steinkamp G et al.; BACKGROUND: Since chronic infection with mucoid Pseudomonas aeruginosa (PA) is associated with deteriorating lung function, many parents of young children with cystic fibrosis (CF) fear the first PA positive throat swab as a milestone in the progression of the disease . To reduce the risk of PA acquisition from the environment, they perform preventive measures at home or outdoors . METHODS: In an attempt to evaluate the attitude of CF physicians towards these measures and the respective consulting practice, we mailed a questionnaire to all 65 certified paediatric CF centres in Germany . RESULTS: Physicians from 54 (83%) CF clinics replied . They expressed widely different ideas about the impact of the environment for the acquisition of P . aeruginosa, and recommended a large spectrum of preventive measures . Some physicians proposed only few precautions, which focussed on the prevention of cross-infection between patients, whereas others suggested prevention of any contact with moist or wet places, e.g . use different toothbrushes for mornings and evenings, or do without air-conditioning in the car . CONCLUSIONS: CF physicians have different opinions on the risk of PA acquisition from the environment . Doctors who recommend strict precautions could engender a parental fear of a ubiquous threat from invisible bacteria . The resulting extended safety measures might impair the family's quality of life .

J Cyst Fibros, 2003 Sep, 2(3), 143 - 7
Exhaled nitric oxide increases following admission for intravenous antibiotics in children with cystic fibrosis; Jaffe A et al.; BACKGROUND: Lack of standardisation for the measurement of exhaled nitric oxide (NO) (FENO) has resulted in conflicting data in cystic fibrosis (CF) . The aim of this study was to assess whether FENO is a useful non-invasive marker of lung disease in CF by assessing the effect of intravenous (IV) antibiotics on FENO . METHODS: FENO was measured on line, according to recently published ERS/ATS guidelines, using a chemiluminescence analyser together with pulmonary function in 14 CF children prior to and following a course of IV antibiotics . RESULTS: There was a significant improvement in mean (S.E.M.) % FEV1 from 60.0 (6.3) to 68.0 (5.4) (P < 0.05) and mean (S.E.M.) % FVC from 66.3 (5.5) to 75.1 (4.9) (P < 0.01) . FENO increased significantly from median (range) 5.8 (2.0-14.3) to 9.2 ppb (0.8-25.1) (P < 0.05) . There was no correlation between FE(NO) and lung function . Subgroup analysis on those with chronic Pseudomonas aeruginosa infection (n = 6) demonstrated no significant change in FENO . CONCLUSIONS: Using a flow of 50 ml/s, FENO increases following admission for IV antibiotic treatment in children with CF but does not correlate with lung function . It is not a useful marker of lung diseases in CF, which has implications for clinical practice .

J Cyst Fibros, 2003 Sep, 2(3), 120 - 8
Economic evaluation of Tobramycin nebuliser solution in cystic fibrosis; Iles R et al.; Background: The cost effectiveness of inhaled TOBIR tobramycin nebuliser solution (TNS) in CF and chronic pulmonary Pseudomonas aeruginosa infection has been shown in US but not in European studies . Methods: An economic evaluation of TNS was undertaken in children and adults . Lung function and resource utilisation were recorded for 24 months before and during TNS therapy . Interventions were costed . Results: Forty-one patients received TNS; 30 of them matched with a paired control on usual therapy . TNS cases received more inhaled and IV antibiotics in the year before TNS than controls, and were hospitalised more . In the TNS treated group mean days in hospital before and after (change) were 32.0, 24.2 (-7.8); days on IV antibiotics 55.4, 38.9 (-16.4); total cost 22,102 pounds, 28,394 pounds (+ 6292 pounds), composed of cost of TNS 0 pounds, 10,010 pounds (+ 10,010 pounds), cost of hospitalisation 10,897 pounds, 8552 pounds (- 2345 pounds), cost of drugs 11,205 pounds, 9832 pounds (- 1374 pounds) . In 19 patients aged < 18 the change in days hospitalised was -10.7 and days on IVs -20.2 . Incremental cost was 3830 pounds . Conclusions: TNS was associated with clinically and socially important reductions of hospital attendances and parenteral antibiotics . This would improve patients' quality of life and reduce interference with work and schooling . Its maximal acquisition cost of 10,010 pounds may be reduced by delays in prescribing and dispensing, and was offset by savings of approximately 3500-6200 pounds .

J Cyst Fibros, 2003 Sep, 2(3), 112 - 9
Cytokine modulating effect of ginseng treatment in a mouse model of Pseudomonas aeruginosa lung infection; Song Z et al.; The major cause of morbidity and mortality in cystic fibrosis (CF) patients is chronic Pseudomonas aeruginosa lung infection . In a mouse model of P . aeruginosa lung infection mimicking that in CF patients, the effects of ginseng treatment on cytokine responses and the correlation between the changes in cytokine production and the lung pathology were studied . Mice were challenged with alginate beads containing P . aeruginosa (10(9) CFU/ml) . A saline extract of ginseng was injected subcutaneously at a dosage of 250 mg/kg of body weight/day for 7 days . Saline was used as a placebo control . One week after challenge, a significantly lower mortality was found in the ginseng treated group (P < 0.005) . The lung cells from the ginseng treated group produced more interferon-gamma (IFN-gamma) (P < 0.04) and tumor necrosis factor-alpha (TNF-alpha) (P < 0.03) but less interleukin-4 (IL-4) (P < 0 .02) with a higher ratio of IFN-gamma/IL-4 (P < 0.004) after 6 and/or 24 h of incubation with specific and non-specific antigens as compared to the control group . The ginseng treated splenocytes produced more TNF-alpha (P < 0.03) and IFN-gamma (P0.05) than the control spleen cells . Furthermore, a significantly milder lung pathology (P < 0.025) and a faster bacterial clearance (P < 0.038) from the lungs were also found in the ginseng treated group compared to the control group . These results indicate a Th1-like immune response in the mice with P . aeruginosa lung infection after 7 days of ginseng treatment, which is an important mechanism accounting for ginseng's favorable action . We therefore believe that Th1 response might benefit the host with P . aeruginosa lung infection and ginseng treatment might be a promising alternative measure for the treatment of chronic P . aeruginosa lung infection in CF patients .

J Cyst Fibros, 2003 Jun, 2(2), 84 - 90
Cost of care and clinical condition in paediatric cystic fibrosis patients; Baumann U et al.; BACKGROUND: The clinical course of cystic fibrosis (CF) shows considerable variation resulting in differences in health care utilisation . We investigated important clinical parameters and their relation to costs . METHODS: We collected clinical parameters together with health care utilisation of a representative paediatric CF population (n=138 patients) attending Hanover Medical School over a period of 1 year . 49% of the patients were chronically infected with Pseudomonas aeruginosa . Costs were calculated on the basis of the annual individual health care utilisation from the perspective of health insurance . RESULTS: Total annual expenditure per patient amounted to 23,989 euro (S.D . 18,026), with home drug treatment representing the most important single cost factor (47% of total costs) . While costs rose with age and doubled in the first 18 years, they correlated foremost with P . aeruginosa airway colonisation status and lung function expressed as FEV(1) . Costs of patients with chronic P . aeruginosa infection were more than three times higher than of uninfected patients . CONCLUSIONS: Health care expenditures for patients with CF vary with the clinical course . The variation can be explained to a large extend by clinical parameters.

J Cyst Fibros, 2003 Jun, 2(2), 69 - 71
Improved lung function and body mass index associated with long-term use of Macrolide antibiotics; Pirzada OM et al.; BACKGROUND: A number of studies have suggested that the non-antimicrobial actions of macrolide antibiotics may be valuable in treating patients with cystic fibrosis . The use of long-term macrolide antibiotics for the management of CF patients colonised by Pseudomonas aeruginosa and progressive pulmonary disease was introduced into our clinic in 1997 . A retrospective study was undertaken to assess of the impact of this therapy . METHODS: Twenty patients with progressive pulmonary disease (>10% fall in FEV(1) over 12 months despite optimising conventional therapy) were commenced on Azithromycin, 250 mg daily during a 21-month period . At the time of assessment they had remained on therapy for a mean of 0.9 years . Changes in lung function, weight, body mass index (BMI) and frequency of pulmonary exacerbations were assessed . A group of 20 patients with stable lung function and matched as far as possible for age and sex was identified for comparison . RESULTS: Pulmonary function increased significantly in the Azithromycin group with FEV1% predicted increasing from a mean of 50.2-59.1% (P=0.001) while FVC% predicted increase from 64.5 to 76.1% (P=0.002) . There was small but non-significant fall in lung function in the comparison group . Body mass index increased by a mean of 1.1 in the Azithromycin group but remained unchanged in the comparison group . The number of pulmonary exacerbations requiring intravenous antibiotics declined by 48.3% in macrolide treated subjects compared to the pre-treatment period (P<0.025); frequency of exacerbations in the control group was unchanged . CONCLUSION: Long-term Azithromycin treatment in patients with progressive deterioration in lung function appears to have led to an improvement in pulmonary function, increased body mass index and decreased the frequency of pulmonary exacerbations requiring intravenous antibiotics.

J Cyst Fibros, 2003 Mar, 2(1), 29 - 34
Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients; Lee TW et al.; BACKGROUND: Patients were defined each successive month as either 'chronic' when more than 50% of the preceding 12 months were PA culture positive, 'intermittent' when < or =50% of the preceding 12 months were PA culture positive, 'free of PA', with no growth of PA for the previous 12 months, having previously been PA culture positive, or 'never infected', when PA had never been cultured . METHODS: Cross-sectional analysis of 146 children attending the Leeds Regional Cystic Fibrosis Centre was performed to assess relationship between the new definition and clinical scores and investigations . The response variable was regressed on age and sex and the residuals analysed using the Kruskal-Wallis test . RESULTS: The 'chronic' group (18% of patients) had significantly worse Shwachman-Kulczycki (SK) and Northern chest X-ray scores, and % predicted FEV(1) values than the 'free' (28%) or 'never' (20%) categories (P<0.004) . The 'intermittent' group (34%) had a significantly higher SK score than the 'chronic' group (P<0.0001), and a significantly lower % predicted FEV(1) value than the 'free' or 'never' groups (P<0.0003) . 'Chronic' patients were significantly associated with a positive, and 'never' patients with a negative, PA antibody result (P<0.001) . CONCLUSIONS: The validity and importance of identifying these four subgroups is demonstrated . Previous definitions may over-estimate the prevalence of chronic infection.

J Cyst Fibros, 2003 Mar, 2(1), 19 - 24
Fosfomycin therapy for multiresistant Pseudomonas aeruginosa in cystic fibrosis; Mirakhur A et al.; BACKGROUND: Increasing resistance to standard antibiotics has been demonstrated in CF patients colonised by Pseudomonas aeruginosa . The antibiotic Fosfomycin has a unique mode of action against this organism, and may protect against aminoglycoside mediated renal and ototoxic effects . However, there is little published experience of this drug in IV form, and it is not licensed for use in the UK . METHODS: In combination with other antibiotics, we used Fosfomycin to treat 30 pulmonary exacerbations in 15 adult CF patients colonised by P . aeruginosa, mainly multiresistant strains . All patients gave informed consent . We cultured sputum prior to treatment and measured spirometry, renal function, and symptoms before and after treatment, and recorded any side effects . RESULTS: One patient developed nausea and Fosfomycin treatment was withdrawn . The remaining patients showed clinical resolution of their chest exacerbations (mean FEV1% predicted: pre 41.1 vs . post 49.4, P<0.001) . Although there was a statistical increase in plasma urea (pre 3.9 mmol/l vs . post 4.3, P<0.03), this was still within the normal range . Plasma creatinine was unchanged . CONCLUSIONS: This study shows that IV Fosfomycin is well tolerated by adult patients with CF and can be useful in the treatment of those colonised with multiresistant P . aeruginosa.

J Cyst Fibros, 2002 Dec, 1(Suppl 2), 199 - 202
New clinical evidence from the European tobramycin trial in cystic fibrosis; Hodson ME et al.; The major cause of morbidity and mortality in patients with cystic fibrosis (CF) is respiratory disease (Penketh et al., Thorax 1987; 42: 526-532) . Recent studies in the USA have shown that intermittent administration of inhaled tobramycin is beneficial to patients with CF who are chronically infected with Pseudomonas aeruginosa (Ramsey et al., N Engl J Med 1999; 340: 23-30; Ramsey et al., Proceedings of the 12th Annual North American Cystic Fibrosis Conference, 1998, Montreal, Canada; Ramsey et al., Abstract from 23rd European Cystic Fibrosis Conference, 1999, the Hague, Netherlands) . In Europe, the use of nebulised colistin in patients chronically infected with P . aeruginosa is widespread . A recently published study compared the efficacy and safety of tobramycin nebuliser solution (TNS) and nebulised colistin in CF patients . One hundred and fifteen patients were randomised to receive either TNS or colistin in a multi-centre open-labelled study that assessed change from baseline in FEV(1) and sputum P . aeruginosa density . TNS produced a mean 6.7% improvement in lung function (P=0.006), whilst there was no significant improvement in the colistin-treated patients . The TNS-treated patients had a significantly greater improvement in lung function than those treated with colistin (P=0.008) . The safety profile of both treatments was good . We conclude that patients treated with TNS for 1 month experience improved lung function compared with patients treated with colistin.

J Cyst Fibros, 2002 Dec, 1(Suppl 2), 189 - 93
Inhaled antibiotic therapy: What drug? What dose? What regimen? What formulation?
Smith AL.
Early studies of the use of antibiotics in patients with cystic fibrosis suggested that they would be of benefit in preventing or reducing infection by Pseudomonas aeruginosa . In seeking to optimize treatment, factors such as the drug used, the dose, the regimen and the formulation must be considered . Aminoglycosides are ideal for aerosolization because they have a long post-antibiotic effect and have an acceptable taste . Tobramycin is one of the aminoglycosides with the lowest systemic toxicity, which enables the aerosol delivery of doses high enough to overcome the antagonistic effects of the sputum . The most dramatic benefits from inhaled tobramycin have been shown to occur in the first 2-4 weeks of administration . Continual administration for longer periods can result in the development of resistance and loss of the improvement in lung function . However, this resistance is transient, and susceptibility to tobramycin returns after a short drug holiday . Optimal drug administration therefore consists of a 4-week on, 4-week off cycle . Such a cycle also helps to maintain patient compliance . Successful drug delivery also depends upon a formulation that does not provoke bronchoconstriction, which demands a formulation that is both preservative free, and osmotically and pH balanced . This research has enabled the development of a novel formulation of tobramycin optimized for use as an inhalation therapy in cystic fibrosis.

J Cyst Fibros, 2002 Sep, 1(3), 122 - 30
Parental fears of Pseudomonas infection and measures to prevent its acquisition; Ullrich G et al.; BACKGROUND AND AIM OF THE STUDY: Chronic infection with Pseudomonas aeruginosa (PA) is associated with accelerated worsening of lung disease in patients with cystic fibrosis (CF) . Fears of PA are widespread among parents of CF children, and many parents take precautions at home to prevent acquisition of the bacterium from the environment . The present study was undertaken to describe the type and intensity of these activities . METHODS: Parents of 21 CF children (7 without prior PA infection, 10 with intermittent and 4 with chronic PA infection) were investigated using semistructured interviews . These were analyzed descriptively and with respect to predominant themes . Additionally, a German personality test was used to evaluate the influence of psychological factors . RESULTS: The clinical impression of widespread parental anxieties of PA infection was confirmed . Misunderstandings concerning PA infections were related to a simplistic concept of the underlying biological mechanisms . Some parents which we classified as 'bacterium-focussed' thought that each contact with PA would lead to bacterial infection . These parents used a large variety of measures, which concerned both domiciliary and outdoor surroundings and activities . At the other end of the spectrum were parents which we classified as 'child-focussed' who mostly supported (and relied on) the child's defense mechanism instead of hygienic measures . CONCLUSIONS: Recommendations by physicians on how to prevent PA acquisition from the environment should take into account possible non-intended side effects, since some parents will exaggerate daily precautions to the detriment of the child's (and the parent's) quality of life.

J Cyst Fibros, 2002 Jun, 1(2), 99 - 101
Chronic mucoid Pseudomonas aeruginosa cholangitis complicating ERCP in a CF patient; Wallick K et al.; We report a case of P . aeruginosa cholangitis in an adult with cystic fibrosis (CF) . The patient had a past history of cholecystectomy and a new finding of intrahepatic biliary duct stricture . Evaluation and treatment with endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous biliary tract drainage was complicated by post-procedure pain and fever . The only organism recovered from biliary drainage was P . aeruginosa . Southern blot analysis of respiratory and biliary cultures confirmed that the isolates were identical . Despite aggressive antibiotic therapy and drainage, persistent cholangitis and infection have not been eradicated after 6 months . The most likely mechanism of infection of the biliary tract was direct introduction of the upper respiratory tract pathogen during the diagnostic procedure.

Orv Hetil, 2004 Aug 15, 145(33), 1719 - 21
{Metastatic endophthalmitis following lung transplantation}; Csiszer E et al.; Intraocular septic--bacterial, viral or fungal--metastasis originating from an infectious focus is very rare but serious complication . The authors performed one enucleation and one evisceration on two patients because of endophthalmitis secondary to pulmonary infection following lung transplantation . The pathogens were Pseudomonas aeruginosa and Mycobacterium xenopi respectively . With this case report the authors aim to draw attention to a rare, but serious ophthalmic complication of organ transplantation: endophthalmitis; its early care, to help prevent the need for enucleation.

New Microbiol, 2004 Jul, 27(3), 263 - 72
E-test method for detecting antibiotic synergy against Pseudomonas aeruginosa from neutropenic patients: a cost-effective approach; Di Bonaventura G et al.; The aim of this study was to evaluate the accuracy of E-test for the detection of synergy or antagonism of antibiotic combinations against Pseudomonas aeruginosa isolates from neutropenic patients . The activity of levofloxacin or grepafloxacin combined with ceftriaxone or cefotaxime against 20 P . aeruginosa clinical strains was assessed by checkerboard technique in comparison with results performed by E-test . The combination grepafloxacin + ceftriaxone appeared to be most effective (synergy, 55%) by checkerboard technique . The agreement between checkerboard and E-test results was 71.2% . Synergy was detected by checkerboard and E-test methods in 35 (43.8%) and 23 (31.3%) of 80 possible combinations, respectively . Antagonism was detected once (1.2%) by checkerboard method only . No major errors were recorded . E-test was preferable to checkerboard method for the total cost (reagent cost + cost of technologist time) (8,60 vs 21,80 euros/test, respectively) . E-test appeared a promising alternative for testing antibiotic combinations although further testing should be performed to better refine this metodology.

Biochem J, 2005 Feb 1, 385(Pt 3), 667 - 75
Structure-function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa; Yates SP et al.; The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD(+) to its protein substrate . A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD(+) was investigated for their inhibition of the catalytic domain of ETA . The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC(50) values . Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible . One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant K(i) of 140 nM . We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor . The 2.1 A (1 A=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop . Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs {poly(ADP-ribose) polymerases} shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP . The present study provides insight into the important features required for inhibitors that mimic NAD(+) and their binding to the mono-ADPRT family of toxins.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1325 - 31
Modeling rhl quorum-sensing regulation on rhamnolipid production by Pseudomonas aeruginosa; Chen F et al.; The effect of autoinducer PAI2 on rhamnolipid (RL) production by Pseudomonas aeruginosa was evaluated using an rhlI null mutant of PAO1 added with PAI2 at various concentrations . A model has also been developed to describe the production kinetics regulated by the rhl quorum-sensing system in three steps: First, PAI2 combines with RhlR protein . Second, the activated complex RhlR:PAI2 triggers the transcription (and expression) of the rhlAB operon that encodes for rhamnosyltransferase . Finally, the enzyme catalyzes the RL synthesis . The model describes fairly well the experimental results/profiles from three different studies (this and two others reported in the literature) . The overall picture predicted by the model is as follows: The induced enzyme synthesis proceeds at the highest rate following PAI2 addition . The rate decreases with time as the autoinducer is degraded . The enzyme concentration nonetheless continues to increase until reaching the plateau at the exhaustion of autoinducer . Higher added PAI2 concentrations thus give not only higher initial enzyme synthesis rates but also longer induced synthesis . As the enzyme concentration increases with time, the RL production rate also increases, resulting in an accelerated rise in RL concentrations initially . The increase in RL concentrations becomes linear at the exhaustion of PAI2 . The best-fit model parameters obtained also provided important insights . To complex half of the intracellular RhlR proteins would require 1.61 microM PAI2, about half of the PAI2 concentration obtained in the stationary-phase culture of wild-type PAO1 . On the other hand, to activate the rhamnosyltransferase synthesis at half of its maximum rate would require the binding of 39% of RhlR with PAI2 . The maximum RL production rate of the culture was found to be 0.042 g/L.h, and the fully induced culture would require at least 1.61 h to synthesize the enzyme to the necessary level for producing RL at half of the maximum rate.

Arch Pathol Lab Med, 1996 Dec, 120(12), 1123 - 8
Botryomycosis ('bacterial ball') of the sinonasal tract caused by Pseudomonas aeruginosa; Wenig BM et al.; BACKGROUND: Botryomycosis is a chronic bacterial infection that typically presents as a cutaneous lesion . Visceral involvement may occur, but mucosal disease is uncommon . We report two cases of sinonasal tract botryomycosis that clinically simulated a neoplasm . METHODS: Two cases of sinonasal tract botryomycosis were identified from the Otolaryngic Tumor Registry at the Armed Forces Institute of Pathology, Washington, DC . The clinical records, slides, and paraffin blocks were available for both cases . Histochemical stains, including Brown and Hopps, Gomori's methenamine-silver, acid-fast bacilli, mucicarmine, periodic acid-Schiff, and Warthin-Starry, were performed . RESULTS: The patients were an 81-year-old man and a 43-year-old woman . The man presented with acute ethmoiditis and a bulging eye . Radiographic studies showed a soft tissue mass in his left maxillary antrum with osseous erosion of adjacent anatomic sites . The woman presented with persistent headaches of more than 1 year's duration, with increasing severity in the months prior to presentation . An expansile soft tissue mass was identified in her right maxillary and ethmoid sinuses . Surgery was performed on both patients . The histology included amorphous, acellular material and separate, rounded eosinophilic granules associated with a neutrophilic infiltrate . A Splendore-Hoeppli phenomenon was seen . Filamentous gram-negative bacilli, identifiable only by histochemical staining, were morphologically compatible with Pseudomonas aeruginosa . Cultures of samples taken from both patients intraoperatively confirmed the organisms as P . aeruginosa . CONCLUSIONS: Sinonasal botryomycosis is a rare localized disease that may be mistaken clinically for an aggressive neoplasm . Complete surgical evacuation is curative.

Infect Genet Evol, 2004 Sep, 4(3), 193 - 7
The use of molecular typing for epidemiological surveillance and investigation of endemic nosocomial infections; Blanc DS; While molecular typing methods are widely used to help the epidemiologist in the investigation of outbreaks, their use for the investigation of endemic infections has been limited . For the investigation of a micro-epidemiological setting such as the understanding of endemic nosocomial infections, discriminant methods are needed, such as pulsed field gel electrophoresis (PFGE), or PCR related typing methods . However, for long-term surveillance, preference should be given to typing methods that give definite results . The identification of the source or reservoir is only possible with comprehensive screening of the environment and the endogenous flora of patients and staff, and therefore, requires significant resources . However, cross-contamination or the existence of a common exogenous source might be investigated by typing clinical isolates during non-epidemic periods . Cross-contamination is suspected when isolates from different patients belong to the same type and when an epidemiological relation can be established . Thus, molecular typing makes it possible to track the dissemination of specific clones, it may facilitate the breaking down of endemic transmission to the level of micro-epidemics . This is illustrated by the example of one investigation of Pseudomonas aeruginosa colonization/infection in patients hospitalized in intensive care units.

Biochemistry, 2004 Oct 5, 43(39), 12563 - 74
Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin; Tigerstrom A et al.; Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications . This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa . Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures . The atomic displacement parameters describe the molecular movement at higher temperatures . The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry . Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein . Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R . Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin . Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R . The mutants involving electrostatic interactions had a minor effect on the thermostability . The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution . In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution . These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin.

Ann Biomed Eng, 2004 Aug, 32(8), 1108 - 19
Quantitative dynamics of in vivo bone marrow neutrophil production and egress in response to injury and infection; Rosinski M et al.; Production rates of blood cells from the bone marrow (BM) can be determined from pool size and residence time in the circulation only during steady state . We describe a method to evaluate changes in BM neutrophil production following severe injury . Male CD-1 mice underwent nonlethal cutaneous burn injury, a lethal burn injury with Pseudomonas aeruginosa infection, or sham treatment, and received bromodeoxyuridine (BrdU) to label proliferative cells . Rates of BM neutrophil production and release into the circulation were determined using a mathematical model that integrates BM neutrophil pool size and fraction of BrdU labeled cells as a function of time . Absolute rates could not be quantified without BrdU data for the neutrophil progenitor pool; however, relative rates could be determined . BM neutrophil production and release significantly increased after injury . After nonlethal burn, release transiently exceeded production, causing a temporary decrease in BM neutrophil stores followed by reestablishment of a steady-state BM neutrophil pool similar to sham controls . After lethal burn infection, release always exceeded production, causing complete depletion of BM neutrophils and suppression of BM neutrophil production . This method is generally applicable to estimating production rates of nonproliferating, terminally differentiated cells, arising from a stem cell pool in vivo.

Acta Crystallogr D Biol Crystallogr, 2004 Oct, 60(Pt 10), 1919 - 21 Epub 2004 Sep 23.
Crystallization and X-ray diffraction analyses of the outer membrane pyochelin receptor FptA from Pseudomonas aeruginosa; Cobessi D et al.; FptA, the pyochelin outer membrane receptor from Pseudomonas aeruginosa, is a siderophore receptor involved in iron uptake when the bacterium grows under iron limitation . Two crystal forms of the FptA-pyochelin complex were obtained under different crystallization conditions . They belong to space groups P1 and P2(1)2(1)2(1) and data sets were collected for both crystal forms . The triclinic crystals diffract to 3.2 A resolution and the orthorhombic crystals show a 1.9 A resolution limit . A data set at the peak of the iron K edge was also collected at 3.1 A resolution.

Protein Sci, 2004 Oct, 13(10), 2628 - 38
Apo-azurin folds via an intermediate that resembles the molten-globule; Sandberg A et al.; The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates . Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A) . The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1) . The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure . Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O) . This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state . In both mutants we also noted a burst-phase intermediate . Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status . However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1531 - 5
Streptomyces africanus sp . nov., a novel streptomycete with blue aerial mycelium; Meyers PR et al.; An actinomycete with blue aerial mycelium and yellow substrate mycelium was isolated from a suburban soil sample collected in Cape Town, South Africa and named strain CPJVR-HT . The colour of the substrate mycelium was not sensitive to changes in pH . The organism produced spiny spores in Spirales spore chains . Chemical taxonomy indicated that it is a member of the genus Streptomyces . Strain CPJVR-HT grew at 45 degrees C and did not produce melanin or any diffusible pigments . It exhibited weak antibacterial activity against a clinical isolate of Enterococcus faecium, but no antibacterial activity against Escherichia coli ATCC 25922 or Pseudomonas aeruginosa ATCC 27853 . Analysis of its 16S rRNA gene sequence, DNA-DNA hybridization studies and the results of physiological tests showed that this strain represents a novel species of Streptomyces, for which the name Streptomyces africanus sp . nov . is proposed . The type strain is CPJVR-HT (= NRRL B-24143T = DSM 41829T).

Antimicrob Agents Chemother, 2004 Oct, 48(10), 4059 - 62
Rapid detection and sequence-specific differentiation of extended-spectrum beta-lactamase GES-2 from Pseudomonas aeruginosa by use of a real-time PCR assay; Weldhagen GF; The LightCycler was compared to nested PCR for the detection of bla(GES/IBC) genes from 100 Pseudomonas aeruginosa clinical isolates . The real-time PCR assay detected a bla(GES/IBC) gene product from 83 isolates, exhibiting a sensitivity and specificity of 94.3 and 100% respectively, compared to nested PCR and DNA sequencing.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3817 - 22
Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage; Hagens S et al.; Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens . A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia . Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection . An export protein gene of the P . aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene . This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell . The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum . Treatment of P . aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3 . However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5 . This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage . Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release . Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.

Mol Microbiol, 2004 Sep, 53(5), 1423 - 36
MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719; Cao L et al.; MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon . While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains . Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified . Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC . PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721 . Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants . The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype . Introduction of PA3719 into wild-type P . aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain . Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression . Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity . The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur .

Mol Microbiol, 2004 Sep, 53(5), 1279 - 90
ExoU is a potent intracellular phospholipase; Sato H et al.; The combination of a large genome encoding metabolic versatility and conserved secreted virulence determinants makes Pseudomonas aeruginosa a model pathogen that can be used to study host-parasite interactions in many eukaryotic hosts . One of the virulence regulons that likely plays a role in the ability of P . aeruginosa to avoid innate immune clearance in mammals is a type III secretion system (TTSS) . Upon cellular contact, the P . aeruginosa TTSS is capable of delivering a combination of at least four different effector proteins, exoenzyme S (ExoS), ExoT, ExoU, and ExoY . Two of the four translocated proteins, ExoS and ExoU, are cytotoxic to cells during infection and transfection . The mechanism of cytotoxicity of ExoS is unclear . ExoU, however, has recently been characterized as a member of the phospholipase A family of enzymes, possessing at least phospholipase A2 activity . Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryotic-specific modification or cofactor for its activity in vitro . The biologic effects of minimal expression of ExoU in yeast can be visualized by membrane damage to different organelles and fragmentation of the vacuole . In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death . ExoU likely represents a unique enzyme and is the first identified phopholipase virulence factor that is translocated into the cytosol by TTSS .

Ann Saudi Med, 2004 Jul-Aug, 24(4), 284 - 7
Effect of obstructive airway disease in patients with non-cystic fibrosis bronchiectasis; Khalid M et al.; BACKGROUND: Extensive research has been devoted to cystic fibrosis-related brochiectasis, compared with non-cystic fibrosis bronchiectasis but the latter is more common and results in significant morbidity and mortality . We assessed the relationship between pulmonary function test (PFT) findings and sputum bacteriology, blood gases, number of hospital admissions and mortality in patients with non-cystic fibrosis bonchiectasis (NCFB) . METHODS: We conducted a retrospective review of 88 consecutive patients admitted with exacerbation of bronchiectasis over 5 years from 1996 to 2001 . Demographic and clinical data collected included gender, age, pulmonary functions, arterial blood gases, sputum bacteriology during stable and exacerbation periods, and number of hospital admissions due to exacerbation of bronchiectasis . A comparison was made between patients having obstructive airway disease (OAD group) and patients with normal or restrictive pulmonary functions (non-OAD group) . RESULTS: OAD in patients with NCFB adversely affected clinical outcome . There was a significant increase in Pseudomonas colonization (60.3% vs . 16%; P<0.0003), hypercapnic respiratory failure (63.4% vs . 20%; P<0.0003), and mean number of admissions due to exacerbation (6 vs . 2; P<0.0001) in the OAD group as compared with the non-OAD group . Although mortality was increased in the OAD group, the difference was not statistically significant . CONCLUSION: Patients with NCFB who have OAD have a significantly higher rate of colonization with Pseudomonas aeruginosa (PSA), hypercapnic respiratory failure, a greater number of hospital admissions due to exacerbation of bronchiectasis, and a higher mortality compared with patients with restrictive or normal pulmonary functions.

J Biomed Mater Res, 2004 Nov 15, 71B(2), 336 - 42
Physicochemical factors influencing bacterial transfer from contact lenses to surfaces with different roughness and wettability; Vermeltfoort PB et al.; The aim of this study was to determine the transfer of Pseudomonas aeruginosa No . 3 and Staphylococcus aureus 835 from contact lenses to surfaces with different hydrophobicity and roughness . Bacteria were allowed to adhere to contact lenses (Surevue, PureVision, or Focus Night & Day) by incubating the lenses in a bacterial suspension for 30 min . The contaminated lenses were put on a glass, poly(methylmethacrylate), or silicone rubber substratum, shaped to mimic the eye . After 2 and 16 h, lenses were separated from the substrata and bacteria were swabbed off from the respective surfaces and resuspended in saline . Appropriate serial dilutions of these suspensions were made, from which aliquots were plated on agar for enumeration . Bacterial transfer varied between 4 and 60%, depending on the combination of strain, contact time, contact lens, and substratum surface . For P . aeruginosa No . 3, transfer was significantly higher after 16 h than after 2 h, whereas less increase with time was seen for S . aureus 835 . Bacterial transfer from all tested contact lenses was least to silicone rubber, the most hydrophobic and roughest substratum surface included . (c) 2004 Wiley Periodicals, Inc.

Curr Microbiol, 2004 Oct, 49(4), 261 - 6
Diversity of oleic acid, ricinoleic acid and linoleic acid conversions among Pseudomonas aeruginosa strains; Kuo TM et al.; Sixteen Pseudomonas aeruginosa strains, including patent strain NRRL B-18602, three recent isolates from composted materials amended with ricinoleic acid, and 12 randomly selected from the holdings of the ARS Culture Collection, were examined for their fatty acid converting abilities . The study examined the bioconversion of oleic acid to 7,10-dihydroxy-8( E)-octadecenoic acid (DOD) and ricinoleic acid to 7,10,12-trihydroxy-8( E)-octadecenoic acid (TOD) . A new DOD-like compound from linoleic acid was observed . All strains except NRRL B-247 exhibited varying levels of DOD production . NRRL B-1000, NRRL B-18602 and NRRL B-23258 with yields up to 84% were among the best DOD producers . TOD production generally paralleled DOD production at a relatively lower yield of up to 15% . Strains NRRL B-1000 and NRRL B-23260 were the best TOD producers . A DOD-like product in low yields was obtained from linoleic acid . The fatty acid bioconversion capability was related neither to growth rate nor to variation in the greenish pigmentation of the strains . Production of significant quantities of DOD and TOD from oleic and ricinoleic acids, respectively, appeared to be a characteristic trait of P . aeruginosa strains . A number of highly effective strains for DOD production were identified.

Infect Immun, 2004 Oct, 72(10), 6176 - 80
Limited role for interleukin-18 in the host protection response to pulmonary infection with Pseudomonas aeruginosa in mice; Nakasone C et al.; We report that clearance of Pseudomonas aeruginosa, accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice . Our results suggest a limited role for IL-18 in the host defense against P . aeruginosa.

Infect Immun, 2004 Oct, 72(10), 5638 - 45
Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster; Erickson DL et al.; The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme . We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection . Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems . The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation . We found that strains lacking relA exhibited reduced virulence in a D . melanogaster feeding assay . In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-{3-oxododecanoyl}-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response . In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase . Deletion of relA also resulted in reduced levels of the RpoS sigma factor . These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P . aeruginosa adaptation in pathogenic relationships.

J Mol Microbiol Biotechnol, 2004, 7(4), 212 - 23
Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03; Ogino H et al.; A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced . In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found . Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile . The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis . The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain . Addition of glycerol to a solution containing Lip8 stabilized this enzyme . By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids .

Diagn Microbiol Infect Dis, 2004 Sep, 50(1), 43 - 50
Multidrug resistance associated with mexXY expression in clinical isolates of Pseudomonas aeruginosa from a Texas hospital; Wolter DJ et al.; Fluoroquinolones and carbapenems are important drug classes used for the treatment of Pseudomonas aeruginosa infections . However, overexpression of the P . aeruginosa efflux pump, MexEF-OprN, can provide dual resistance to both fluoroquinolones and carbapenems . Recently, a hospital in Texas encountered several P . aeruginosa isolates resistant to both of these drug classes . The purpose of this study was to determine whether the mechanism responsible for this multidrug resistance involved the overexpression of MexEF-OprN . To test this hypothesis, 7 clinical isolates from the Texas hospital were analyzed for clonality, antimicrobial susceptibility, expression of the porin, oprD, and four multidrug-resistant efflux pumps (mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY), quinolone resistance-determining region mutations, and beta-lactamase production . Two groups of isolates possessed similar pulse field gel electrophoresis patterns indicating their genetic relatedness . In addition to fluoroquinolone and carbapenem resistance, each isolate also displayed varying degrees of susceptibility to additional beta-lactams tested and resistance to gentamicin . Interestingly, none of the 7 clinical isolates overexpressed mexEF-oprN as determined by semiquantitative reverse transcriptase polymerase chain reaction, but overexpression of mexXY was observed in 6 of the 7 isolates . All 7 isolates showed a decrease of OprD in the outer membrane and a reduction in transcriptional expression of oprD compared to wild-type strain, PAO1 . These results demonstrate that multidrug resistance to the fluoroquinolones and carbapenems in these clinical isolates was not a result of the overexpression of the mexEF-oprN pump . Instead, resistance to these two agents seemed to arise through independent mutational events.

Int J Antimicrob Agents, 2004 Oct, 24(4), 357 - 61
Experimental sepsis using Pseudomonas aeruginosa: the significance of multi-drug resistance; Giamarellos-Bourboulis EJ et al.; In order to clarify whether susceptible and multidrug-resistant Pseudomonas aeruginosa differ in the mechanism of induction of sepsis, three different isolates were used; one susceptible (isolate A) and two (isolates B and C) multidrug-resistant . Isolate B had moderately elevated MICs of antipseudomonal antimicrobials and isolate C highly elevated MICs . Each isolate was infused by a catheter inserted into the right jugular vein of six rabbits . Survival was recorded; blood was sampled at regular time intervals for estimation of bacterial blood counts, malondialdehyde (MDA) and tumour necrosis factor-alpha (TNFalpha) . Quantitative cultures of various organs were performed after death or sacrifice . Mean survival after challenge by isolates A, B and C was 0.73, 2.58 and 11.00 days, respectively (P of comparisons A versus B, 0.0048; A versus C, 0.0012; B versus C, 0.0005) . The number of viable organisms in the blood after challenge using isolates A and B was greater than the viable counts of C . Serum MDA was lower after challenge with B and C compared with A . Serum TNFalpha levels were higher after challenge by isolate A compared with isolate C . The bacterial loads of the liver, lower right lung lobe, spleen and mesenteric lymph nodes were greater after challenge by isolate A than the other isolates . It is concluded that infection by multidrug-resistant P . aeruginosa is accompanied by increased survival compared with infection by susceptible isolates; that finding might be explained by the different mechanisms leading to sepsis . Further studies must be done to clarify the significance of these observations for therapeutics.

Int J Antimicrob Agents, 2004 Oct, 24(4), 346 - 51
Associations between antibiotic use and changes in susceptibility patterns of Pseudomonas aeruginosa in a private, university-affiliated teaching hospital: an 8-year-experience: 1995-2002; Mohr JF et al.; Increasing resistance in Pseudomonas aeruginosa to multiple antibiotics has been observed and is posing therapeutic dilemmas . Antibiotic utilization is one factor that has been associated with the emergence of antimicrobial resistance . We examined the overall and specific antimicrobial use in relation to changes in susceptibility patterns in P . aeruginosa . Regression analysis was performed to explore the relationships between annual antibiotic use and the incidence of resistant P . aeruginosa . There were statistically significant relationships between increasing anti-pseudomonal cephalosporin and levofloxacin use and the increasing incidence of ciprofloxacin resistant P . aeruginosa . However, there was not an association between other fluoroquinolone or overall fluoroquinolone use and this change . In addition, there was no association between increasing anti-pseudomonal cephalosporin use and cefepime resistant P . aeruginosa . No statistical relationship was seen with overall antibiotic use and the development of resistance in P . aeruginosa, suggesting that the development of resistance is associated with the use of individual agents, rather than overall antibiotic consumption.

Int J Antimicrob Agents, 2004 Oct, 24(4), 339 - 45
The evolution of Pseudomonas aeruginosa during antibiotic rotation in a medical intensive care unit: the RADAR-trial; Tsukayama DT et al.; Bacterial spread between patients may contribute to the high prevalence of antibiotic-resistant pathogens within ICUs . The aim of this study was to evaluate the fate of Pseudomonas aeruginosa during the different antibiotic regimens . Susceptibility patterns and genotyping were performed to determine whether there was a predominant clone and to track the spread of resistant strains within the unit . Twenty-eight different ribotypes were found among 82 Pseudomonas isolates . Four ribotypes accounted for 42 (51%) isolates and were designated the "major clones" occurring throughout multiple cycles . The ribotypes with multiple occurrences were more resistant to antibiotics than ribotypes that appeared only once . The correlation of antibiotic use with antibiotic resistance and the finding of a large number of ribotypes suggested that de novo development of antibiotic resistance is a likely event in P . aeruginosa . In addition, ribotypes associated with antibiotic resistance appeared to have a survival advantage and can become frequent colonizers in the ICU.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2004 Aug, 26(4), 359 - 63
{Drug screening model acting on out-membrane protein OprM in pseudomonas aeruginosa efflux pump system}; Tian R et al.; OBJECTIVE: To establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point . METHODS: Efflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain . Expression of OprM protein was induced in E . coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector . In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly . RESULTS: OprM protein was highly expressed . Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM . A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5% . CONCLUSION: OprM plays an important role in drug efflux . The established model has good specificity and maneuverability.

Am J Physiol Lung Cell Mol Physiol, 2005 Jan, 288(1), L150 - 8 Epub 2004 Sep 17.
SP-A1 and SP-A2 variants differentially enhance association of Pseudomonas aeruginosa with rat alveolar macrophages; Mikerov AN et al.; Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF) . Surfactant protein A (SP-A) enhances phagocytosis of P . aeruginosa . Two genes, SP-A1 and SP-A2, encode human SP-A . We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist . To test this, we studied association of a nonmucoid P . aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants . We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants . We tested the 6A(2) and 6A(4) alleles (for SP-A1), the 1A(0) and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products . After incubation of alveolar macrophages with P . aeruginosa in the presence of the SP-A variants at 37 degrees C for 1 h, the cell association of bacteria was assessed by light microscopy analysis . We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was approximately 52-95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was approximately 18-41% of the human SP-A from bronchoalveolar lavage . We conclude that human SP-A variants in vitro enhance association of P . aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.

Jpn J Antibiot, 2004 Jun, 57(3), 288 - 93
{Combined effects of arbekacin with biapenem against in vitro and in vivo model of a mixture of MRSA and Pseudomonas aeruginosa}; Niida M et al.; Combined effects of arbekacin (ABK) with biapenem (BIPM) were examined on both in vitro and in vivo model of a mixture of MRSA and Pseudomonas aeruginosa . As a result, significant effect in vitro was observed in combined use of ABK (1/2 MIC) with BIPM (1/4 and 1/2 MIC) against MRSA as compared with ABK or BIPM alone . Against P . aeruginosa combined effect was also observed, showing reduction of viable cells to the limitation of detection within 2 hours . Moreover, with respect to the protective effect on mixed systemic infection of MRSA and P . aeruginosa, the combined treatment with ABK and BIPM showed more excellent efficacy as compared with the single use of each drug.

Am J Ind Med, 2004 Oct, 46(4), 375 - 7
Solubility of endotoxins from Escherichia coli and Pseudomonas aeruginosa; Eduard W et al.; BACKGROUND: The Limulus amebocyte lysate (LAL) assay may underestimate endotoxins because only soluble endotoxins are determined . The solubility of endotoxins was, therefore, studied in two species of Gram-negative bacteria . METHODS: Cultures were grown in serum broth, cells were harvested by centrifugation and washed in physiological saline . Bacterial suspensions were either filtered through PTFE filters and air dried at room temperature, dried in polypropylene tubes at room temperature in a desiccator, or freeze dried . Samples were extracted with aqueous Tween 20 . The soluble and insoluble fractions were dried, methanolysed, and hydroxy-fatty acid methyl esters were determined by gas chromatography (GC) as markers of endotoxins . RESULTS: The solubility of the endotoxins from Escherichia coli and Pseudomonas aeruginosa ranged from 9% to 83% . Species and drying conditions had substantial influence on the solubility . CONCLUSIONS: Endotoxin exposure may be underestimated in environmental samples by the LAL test because a substantial fraction can be non-soluble and is not detected.

Rev Physiol Biochem Pharmacol, 2004, 152, 79 - 92 Epub 2004.
Pseudomonas aeruginosa ExoS and ExoT; Barbieri JT et al.; ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains . The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42 . Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis . Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity . Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates . While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins . Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.

J Biol Chem, 2004 Nov 19, 279(47), 49315 - 22 Epub 2004 Sep 16.
The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice; Power MR et al.; Toll-like receptors (TLR) induce distinct patterns of host responses through myeloid differentiation factor 88 (MyD88)-dependent and/or -independent pathways, depending on the nature of the pathogen . Pseudomonas aeruginosa is a cause of serious lung infection in immunocompromised individuals and cystic fibrosis patients . The role of the TLR-MyD88 pathway in P . aeruginosa-induced lung infection in vivo was examined in this study . MyD88-/- mice demonstrated an impaired clearance of P . aeruginosa from the lung . Little or no neutrophil recruitment was observed in the airways of MyD88-/- mice following P . aeruginosa lung infection . This observation was associated with a reduced production of inflammatory mediators that affect neutrophil recruitment, including macrophage-inflammatory protein-2, tumor necrosis factor, and interleukin-1beta in the airways of MyD88-/- mice . Similarly, MyD88-/- mice showed inhibited NF-kappaB activation in the lung following P . aeruginosa infection . Interestingly, P . aeruginosa infection induced a 7.5-fold increase of TLR2 mRNA expression in the lungs of MyD88+/+ mice . Furthermore, host responses to P . aeruginosa lung infection in TLR2-/- and TLR4 mutant mice were partially inhibited compared with the responses of respective control mice . Taken together, our results indicate that the MyD88-dependent pathway is essential for the development of early host responses to P . aeruginosa infection, leading to the clearance of this bacterium, and that TLR2 and TLR4 are involved in this process.

J Bacteriol, 2004 Oct, 186(19), 6560 - 74
Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa; Braid MD et al.; Bacteriophage B3 is a transposable phage of Pseudomonas aeruginosa . In this report, we present the complete DNA sequence and annotation of the B3 genome . DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3% . The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons . Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins . Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes . Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions . Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster . These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.

J Bacteriol, 2004 Oct, 186(19), 6367 - 73
PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa; Reimmann C et al.; In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF . A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF . In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins . In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type . Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not . These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

J Bacteriol, 2004 Oct, 186(19), 6341 - 50
The complex flagellar torque generator of Pseudomonas aeruginosa; Doyle TB et al.; Flagella act as semirigid helical propellers that are powered by reversible rotary motors . Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation . The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria . Here, we show that these five genes contribute to motility . Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments . However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions . Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility . Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together . motY appears to be required for motAB function . The combination of motY and motCD mutations rendered the cells nonmotile . Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to approximately 85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded . Thus, the flagellar motor of P . aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.

Clin Microbiol Infect, 2004 Oct, 10(10), 877 - 83
Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa; Islam S et al.; The MexZ-MexX-MexY multidrug efflux system in Pseudomonas aeruginosa was studied to determine its contribution to aminoglycoside resistance . Amikacin-resistant (AR) mutants were generated from P . aeruginosa strain PAO1, and clinical isolates of P . aeruginosa were collected from cystic fibrosis patients . The regulatory gene mexZ and the intergenic region (mexOZ) between mexZ and mexX were investigated for mutation by PCR and DNA sequence analysis . The results showed that 14 of 15 AR clinical isolates and one of ten laboratory mutants had at least one mutation in mexZ and/or mexOZ . To study the effect of mexZ and mexOZ mutations, the production of MexY mRNA was investigated quantitatively by real-time PCR . Seven of ten AR mutants (MIC 4-8 mg/L) produced 8-21-fold more MexY mRNA than PAO1 . These isolates were sensitive to fluoroquinolones, carbapenems and ceftazidime . One AR mutant (MIC 64 mg/L) that produced > 200-fold more MexY mRNA than PAO1 was also resistant to fluoroquinolones, carbapenems and ceftazidime . Thirteen of 15 AR clinical isolates produced 3.4-727-fold more MexY mRNA . No evidence was found for the aminoglycoside-modifying enzymes 6'-N-acetyltransferase type Ib, 4'-O-nucleotidyltransferase type IIb or aminoglycoside 3'-phosphotransferase IIps in these strains . Nine AR mutants overproduced MexY without mutations in mexZ or mexOZ, suggesting that MexXY efflux is also regulated by gene(s) other than mexZ.

Isr Med Assoc J, 2004 Sep, 6(9), 531 - 4
Lack of evidence of transmission of Pseudomonas aeruginosa among cystic fibrosis patients attending health camps at the Dead Sea, Israel; Greenberg D et al.; BACKGROUND: Transmission of Pseudomonas aeruginosa among cystic fibrosis patients attending health camps has been reported previously . OBJECTIVES: To determine the transmission of P . aeruginosa among CF patients during three winter camps in the Dead Sea region in southern Israel . METHODS: Three consecutive CF patient groups were studied, each of which spent 3 weeks at the camp . The patients were segragated prior to camp attendance: patients who were not colonized with P . aeruginosa constituted the first group, and colonized patients made up the two additional groups . Sputum cultures were obtained upon arrival, at mid-camp and on the last day . Environmental cultures were also obtained . Patients were separated during social activities and were requested to avoid social mingling . Isolates were analyzed by antibiotic susceptibility profile and by pulsed field gel electrophoresis . RESULTS: Ninety isolates from 19 patients produced 28 different fingerprint patterns by PFGE . Isolates from two siblings and two patients from the same clinic displayed the same fingerprint pattern . These patients were already colonized with these organisms upon arrival . Two couples were formed during the camp, but PFGE showed no transmission of organisms . All other patients' isolates displayed unique fingerprint patterns and were distinguishable from those of other attendees, and none of the P . aeruginosa-negative patients acquired P . aeruginosa during camp attendance . Environmental cultures were negative for P . aeruginosa . CONCLUSIONS: We were unable to demonstrate cross-infection of P . aeruginosa among CF patients participating in health camps at the Dead Sea who were meticulously segregated.

J Biol Chem, 2004 Dec 10, 279(50), 52593 - 602 Epub 2004 Dec 10.
The Structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa; Kimber MS et al.; Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery . The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase . There are two isoforms . FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system . FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis . We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution . PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface . Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization . Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

Eur J Hum Genet, 2005 Jan, 13(1), 96 - 101
Fcgamma receptor IIA genotype and susceptibility to P . aeruginosa infection in patients with cystic fibrosis; De Rose V et al.; It has been suggested that genes other than CFTR could modulate the severity of lung disease in cystic fibrosis (CF) . Neutrophil Fcgamma receptor II (FcgammaRII) is involved in host defense against microorganisms and in inflammatory response . We evaluated the association between genetic variability of this gene and both airway infection with Pseudomonas aeruginosa and severity of lung disease in patients with CF . We studied 167 Italian unrelated patients with CF and 50 control subjects . The distribution of FcgammaRIIA genotypes in CF patients was compared with that in control subjects and the different genotypes were related with the presence or absence of P . aeruginosa infection and markers of disease severity in CF patients . The distribution of FcgammaRIIA genotypes was not significantly different between CF patients and controls . We observed that in CF patients with the same CFTR genotype (DeltaF508/DeltaF508), those carrying the R allele of FcgammaRIIA had an increased risk of acquiring chronic P . aeruginosa infection (P=0.042, R.R.: 4.38; 95% CI: 1.17/22.4) . Moreover, the frequency of R/R genotype in patients with chronic P . aeruginosa infection seems to be higher than that of control subjects and patients without chronic infection . The observation that CF patients carrying the R allele of FcgammaRIIA are at higher risk of acquiring chronic P . aeruginosa infection suggests that the FcgammaRII loci genetic variation is contributing to this infection susceptibility.

Biochemistry, 2004 Sep 7, 43(35), 11275 - 82
Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI MS; Reid CW et al.; Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer, peptidoglycan, between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue . With 72% amino acid sequence identity between the enzymes, the theoretical structure of the membrane-bound lytic transglycosylase B (MltB) from Psuedomonas aeruginosa was modeled on the known crystal structure of Escherichia coli Slt35, the soluble derivative of its MltB . Of the twelve residues in Slt35 known to make contacts with peptidoglycan derivatives in Slt35, nine exist in the same position in the P . aeruginosa homologue, with two others only slightly displaced . To probe the binding properties of an engineered soluble form of the P . aeruginosa MltB, a SUPREX method involving hydrogen/deuterium exchange coupled with MALDI mass spectrometry detection was developed . Dissociation constants were calculated for a series of peptidoglycan components and compared to those obtained by difference UV absorption spectroscopy . These data indicated that GlcNAc alone does not bind to MltB with any measurable affinity but it does contribute to the binding of GlcNAc-MurNAc-dipeptide . With the MurNAc series of ligands, significant binding contributions are made through both the N-acetyl and C-3 lactyl moieties of the aminosugar with additional contributions to binding provided by associated peptides.

Transpl Int, 2004 Oct, 17(9), 545 - 8 Epub 2004 Sep 08.
Refractory pulmonary aspergillosis treated with caspofungin after heart-lung transplantation; Carby MR et al.; Invasive pulmonary aspergillosis (IPA) is a serious complication of lung transplantation . Pre-mortem diagnosis is difficult and is made according to defined criteria . Most patients with a post mortem diagnosis of IPA only reach the possible or probable levels of diagnostic certainty during life . Here, we report a case of probable IPA that was refractory to conventional treatment, including amphotericin, but which responded to therapy with caspofungin . A 23-year-old man underwent heart-lung transplantation for cystic fibrosis . Ten years after transplantation he developed IPA . His condition continued to deteriorate despite treatment with itraconazole, liposomal amphotericin and flucytosine together with treatment of a concomitant infection with Pseudomonas aeruginosa . Following treatment with caspofungin there was progressive and sustained clinical and radiological improvement . No adverse reaction occurred during treatment . Caspofungin should be considered as an alternative treatment for IPA in lung transplant recipients who fail to respond to other therapy.

Otolaryngol Head Neck Surg, 2004 Sep, 131(3), 248 - 52
Hearing loss with semicircular canal transection and Pseudomonas aeruginosa otitis media; Prevatt AR et al.; OBJECTIVE: To determine if Pseudomonas aeruginosa (PA) otitis media (OM) increases the risk of sensorineural hearing loss (SNHL) with semicircular canal transection in the guinea pig model . METHODS: PA was injected transtympanically into both middle ears of pigmented guinea pigs . Saline-injected animals served as controls . Two to 4 days after injection, one horizontal semicircular canal was transected and sealed . Hearing was tested using electrocochleography before and after transection . RESULTS: PA-OM was induced in all PA-injected ears . Five days after semicircular canal transection, click-evoked thresholds were significantly elevated in ears with PA-OM (P = 0.00009) but not in saline-injected ears (P = 0.398) . CONCLUSION: Violation of the inner ear in the presence of PA-OM is associated with increased risk of SNHL in the guinea pig . SIGNIFICANCE: Factors unique to PA infection may be responsible for SNHL with labyrinthine injury in humans with chronic otitis media.

J Mol Biol, 2004 Oct 1, 342(5), 1599 - 611
Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures; Zhuravleva AV et al.; Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein . Following a careful model-free analysis, several protein regions with different dynamic regimes are identified . Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117 . These findings are consistent with a gated electron transfer process according to the "dynamic docking" model . Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature . Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion . The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.

Am J Respir Crit Care Med, 2004 Dec 15, 170(12), 1331 - 9 Epub 2004 Sep 10.
Azithromycin blocks neutrophil recruitment in Pseudomonas endobronchial infection; Tsai WC et al.; Macrolides exert their effects on the host by modulation of immune responses . In this study, we assessed the therapeutic efficacy of azithromycin in a murine model of mucoid Pseudomonas aeruginosa endobronchial infection . The clearance of Pseudomonas from the airway of mice treated with the macrolide azithromycin was not different than untreated mice challenged with Pseudomonas beads . However, the azithromycin-treated mice showed a remarkable reduction in lung cellular infiltrate in response to Pseudomonas beads, as compared with untreated mice . This effect was associated with significant decreases in lung levels of tumor necrosis factor-alpha and keratinocyte-derived chemokine in azithromycin-treated mice compared with untreated mice . Furthermore, there was a significant reduction in the response of both mouse and human neutrophils to chemokine-dependent and -independent chemoattractants when studied in vitro . Inhibition of chemotaxis correlated with azithromycin-mediated inhibition of extracellular signal-regulated kinase-1 and -2 activation . This study indicates that the azithromycin treatment in vivo results in significant reduction in airway-specific inflammation, which occurs in part by inhibition of neutrophil recruitment to the lung through reduction in proinflammatory cytokine expression and inhibition of neutrophil migration via the extracellular signal-regulated kinase-1 and -2 signal transduction pathway.

Eur Respir J, 2004 Sep, 24(3), 449 - 52
Cystic fibrosis patients and families support cross-infection measures; Griffiths AL et al.; A clonal strain of Pseudomonas aeruginosa (PA) was isolated in 1999 at the Royal Children's Hospital, Melbourne, Australia, after five unrelated children with cystic fibrosis (CF) died from severe lung disease aged <5 yrs . Subsequently, more than half of the patients in the clinic with PA were found to harbour this strain, and segregation measures were instituted at the hospital to prevent further spread . The aim of this study was to assess CF parent and patient responses to the segregation measures to determine overall support . A questionnaire was sent out to the families of 291 CF children treated at the centre . A 65% response rate was obtained . The majority of parents (85%) and patients > or=12 yrs old (63%) were positive about the segregation measures instituted . A total of 11% of parents and 25% of patients were unsure, and 4% of parents and 12% of children gave negative responses . Those who were not happy listed reasons such as concerns about the emotional impact of not socialising with other CF children, inconclusive evidence about person-person spread of infection and feelings of alienation created in the clinic by the separation . In conclusion, the majority of responding cystic fibrosis patients and their families understand and are supportive of infection control measures instituted at the Royal Children's Hospital, Melbourne, Australia.

FEBS Lett, 2004 Sep 10, 574(1-3), 73 - 9
Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline; Reid CW et al.; The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue . The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis . Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase . The beta-hexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance . Using surface plasmon resonance and difference absorbance spectroscopy, Kd values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.

Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 109 - 15
Antibacterial synergism of novel antibiotic peptides with chloramphenicol; Park Y et al.; HP (2-20) is an antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 . We previously tested whether several analogues of HP (2-20), with amino acid substitutions that increased or decreased net hydrophobicity, could be useful as therapeutic agents . In the present study, we show that substituting Gln and Asp for Trp at positions 17 and 19, respectively, of HP (2-20) (peptide A3) had potent antibacterial activity in minimal inhibition concentration and minimal bactericidal concentration without having hemolytic activity . In contrast, when we decreased hydrophobicity by substituting Leu or Phe for Ser at positions 12 and 19, respectively, of HP (2-20) (Anal 4, Anal 5), there was no significant effect on antibacterial activity . We found that A3 acted synergistically with chloramphenicol against bacterial cells . Fluorescence activated flow cytometry showed that A3-treated cells had higher fluorescence intensity than untreated cells, similar to that of melittin-treated cells . Furthermore, A3 caused significant morphological alterations of Staphylococcus aureus and Pseudomonas aeruginosa, as shown by scanning electron microscopy . Our results suggest that peptide A3 may be useful for the design of novel antibiotic peptides that possess high bacterial cell selectively and synergistic effects with conventional antibiotic agents but lack hemolytic activity.

Biochem Biophys Res Commun, 2004 Aug 27, 321(3), 631 - 7
Antibiotic activity and synergistic effect of antimicrobial peptide against pathogens from a patient with gallstones; Park Y et al.; HP (2-20) is a peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1 that has been shown to have antimicrobial activity against various species of bacteria . When we tested the effects of HP (2-20), we found that this peptide displayed strong activity against pathogens from a patient with gallstones, but it did not have hemolytic activity against human erythrocytes . We also found that HP (2-20) had potent activity against cefazolin sodium-resistant bacterial cell lines, and that HP (2-20) and cefazolin sodium had synergistic effects against cell lines resistant to the latter . To investigate the mechanism of action of HP (2-20), we performed fluorescence activated flow cytometry using pathogens from the patient with gallstones . As determined by propidium iodide (PI) staining, pathogenic bacteria treated with HP (2-20) showed higher fluorescence intensity than untreated cells, similar to melittin-treated cells, and that HP (2-20) acted in an energy- and salt-dependent manner . Scanning electron microscopy showed that HP (2-20) caused significant morphological alterations in the cell surface of pathogens from the patient with gallstones . By determining their 16S rDNA sequences, we found that both the pathogens from the patient with gallstones and the cefazolin sodium-resistant cell lines showed 100% homology with sequences from Pseudomonas aeruginosa . Taken together, these results suggest that HP (2-20) has antibiotic activity and that it may be used as a lead drug for the treatment of acquired pathogens from patients with gallstones and antibiotic-resistant cell lines.

Rev Panam Salud Publica, 2004 Aug, 16(2), 125 - 30
Antibiotic resistance in clinical isolates of Pseudomonas aeruginosa in Jamaica; Brown PD et al.; OBJECTIVE: To assess antibiotic resistance in clinical isolates of Pseudomonas aeruginosa in Jamaica, and to obtain baseline information on the presence of this important pathogen . METHODS: A total of 51 isolates of Pseudomonas aeruginosa, obtained from 162 clinical specimens from major hospitals and laboratories in seven parishes in Jamaica, were analyzed between May and August 2002 . Isolates were tested against 18 different antibiotics by a disk diffusion method . RESULTS: Organisms were cultured from wound swabs (56%), high vaginal swabs (10.5%) and ear swabs (42.5%) . Overall, the highest percentage rates of resistance were found for cefaclor (100% of all isolates), nalidixic acid (82.4%), kanamycin (76.5%), and trimethoprim/sulfamethoxazole (56.9%) . Resistance rates were 25.5% or lower for tobramycin, gentamicin and polymyxin B, cefotaxime, ciprofloxacin and norfloxacin, piperacillin, carbapenems and amikacin . Forty-one isolates showed intermediate sensitivity to most of the antipseudomonal antibiotics, and the remaining 10 isolates were resistant to eight or more antibiotics . The multiresistant isolates, most of which were hospital isolates, were all resistant to tetracycline, nalidixic acid and trimethoprim/sulfamethoxazole, and highly (80%-90%) resistant to kanamycin, ciprofloxacin and norfloxacin . CONCLUSIONS: This study confirms that antibiotic resistance in this clinical pathogen is emerging in Jamaica, and suggests that due care must be taken in hospital settings to adequately diagnose pseudomonal infections and prescribe the antibiotic treatment most effective in preventing the increase in multidrug resistant organisms.

J Appl Microbiol, 2004, 97(4), 699 - 711
Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000; Lambert RJ; AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested . METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph . aureus {169 methicillin-sensitive Staph . aureus (MSSA), 87 methicillin-resistant Staph . aureus (MRSA)} and 111 clinical isolates of Ps . aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis . Comparisons suggest that alterations in the mean susceptibility of Staph . aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes . Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group . In both Staph . aureus and Ps . aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found . CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph . aureus or in Ps . aeruginosa . SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.

J Microbiol, 2004 Mar, 42(1), 47 - 50
Effect of titanium-ion on the growth of various bacterial species; Yu TS; There are a number of studies that explain the metabolism and roles of metallic titanium and titaniumion . One of the most intriguing results from these studies is the finding of metallic titanium having no bacteriostatic effects on oral bacterial species . In this research, the effects of titanium-ion on the growth of twenty-two bacterial species, some of which are commonly found in foods such as yoghurt, kimchi, and soy fermented products, were investigated . All but two bacteria, Escherichia coli and Pseudomonas aeruginosa appeared to be sensitive to titanium-ion . These two species were grown on 360 microg/ml of titanium-ions, and they were found to be resistant to the titanium-ion . Both the wild-type and plasmid-cured E . coli showed good growth in a medium with 200 microg/ml of titanium-ions . These results suggest that titanium-resistance was independent from the effects of the plasmid in E . coli.

Mayo Clin Proc, 2004 Sep, 79(9), 1185 - 91
Vestibular toxicity due to inhaled tobramycin in a patient with renal insufficiency; Edson RS et al.; Inhaled tobramycin is being used increasingly in patients with cystic fibrosis and other forms of bronchiectasis for treatment of bronchial colonization with Pseudomonas aeruginosa . The goal of inhaled antibiotics is to provide maximal concentrations at the site of infection without risking systemic toxicity . We report an unusual case of reversible vestibular toxicity due to inhaled tobramycin in a patient with renal failure who was undergoing hemodialysis . Although systemic absorption after inhaled tobramycin is reportedly negligible, no recommendations have been published regarding monitoring of serum concentrations in patients receiving this form of therapy . We suggest that clinicians consider monitoring serum concentrations of tobramycin in patients at risk of renal toxicity and/or ototoxicity, such as those with predisposing renal or otologic compromise . Further studies in at-risk patients are needed to determine the optimal frequency and timing of such monitoring.

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