Pseudomonas aeruginosa

Clin Infect Dis, 2004 Aug 15, 39(4), 497 - 503 Epub 2004 Aug 02.
Predicting hospital rates of fluoroquinolone-resistant Pseudomonas aeruginosa from fluoroquinolone use in US hospitals and their surrounding communities; Polk RE et al.; Rates of fluoroquinolone resistance among Pseudomonas aeruginosa in hospitals are increasing, but interhospital variability is great . We sought to determine whether this variability correlated to fluoroquinolone use in hospitals and in the surrounding community . Hospital quinolone use in 1999 (24 hospitals) through 2001 (35 hospitals) was determined from billing records . The number of fluoroquinolone prescriptions within a 10-mile (approximately 16-km) radius of each hospital was determined for 1999 and 2000 . Hospital fluoroquinolone use increased from 1999 through 2001, from 137 to 163 defined daily doses (DDD)/1000 patient-days (P=.01) . The rate of community fluoroquinolone use also increased, from 2.3 to 2.8 DDD/1000 inhabitant-days (P<.001) . Rates of fluoroquinolone-resistant P . aeruginosa increased from 29% in 1999 to 36% in 2001 (P=.003) . Both community and hospital fluoroquinolone use were predictive of rates of fluoroquinolone-resistant P . aeruginosa . Levofloxacin was associated with resistance, but ciprofloxacin was not . Most of the variability in resistance rates is explained by volume of fluoroquinolone use, both in the hospital and the surrounding community.

J Antimicrob Chemother, 2004 Oct, 54(4), 772 - 9 Epub 2004 Sep 08.
Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo are eradicated by a synthetic cationic polymer; Werthen M et al.; OBJECTIVES: Antimicrobial peptides are important effectors of innate immunity . Bacteria display multiple defence mechanisms against these peptides . For example, Pseudomonas aeruginosa releases potent proteinases that inactivate the human cathelicidin LL-37 . Hence, in conditions characterized by persistent bacterial colonization, such as in P . aeruginosa-infected skin wounds, there is a need for efficient means of reducing bacterial load . Here, the effect of the cationic molecule polyhexamethylenebiguanide (PHMB) was evaluated . METHODS: Infection models in human wound fluid and human skin were established . Radial diffusion methods, bacterial growth and bactericidal assays were used for determination of effects of PHMB on bacteria in the presence of plasma, wound fluid or human skin . At the protein and tissue levels, SDS-PAGE, light microscopy and scanning electron microscopy were used to study the effects of P . aeruginosa infection before and after addition of PHMB . RESULTS: PHMB killed common ulcer-derived bacteria in the presence of human wound fluid . Furthermore, elastase-expressing P . aeruginosa completely degraded wound fluid proteins as well as human skin during infection ex vivo . The infection, and consequent protein degradation, was reversed by PHMB . CONCLUSIONS: The ex vivo infection models presented here should be helpful in the screening of novel antimicrobials and constitute a prerequisite for future clinical studies.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Sep, 16(9), 540 - 3
{Characteristics and therapeutic strategies of nosocomial pneumonia in postoperative patients with acute hypertensive intracerebral hemorrhage}; Tong Y et al.; Objective: To investigate the characteristics of postoperative nosocomial pneumonia in patients with acute hypertensive intracerebral hemorrhage after evacuation of hematoma and the strategies of prevention and treatment . Methods: Retrospective analysis was performed on the clinical data of surgically treated patients with acute hypertensive intracerebral hemorrhage in the Department of Neurosurgery from 2000 to 2003 . The patients were divided into a group who developed nosocomial pneumonia to compare with a group who did not . Sputum was cultured B and drug sensitivity of isolated bacteria was performed . Appropriate treatment measures were given according to clinical manifestations . Results: In 112 post operative patients with acute hypertensive intracerebral hemorrhage, nosocomial pneumonia developed in 29 patients (25.89%), and 65.99% of microorganisms isolated from respiratory secretion was Gram-negative bacteria . The predominant bacteria were Pseudomonas aeruginosa and Staphylococcus aureus . The mortality was 34.48% (10/29 cases) . The prognosis of patients with nosocomial pneumonia was worse than the controls (7.23%, 6/83 cases, P<0.01) . Conclusion: The preventive and therapeutic strategies for nosocomial pneumonia in postoperative patients with acute hypertensive intracerebral hemorrhage include reduction of risk-factors, surveillance of pathogens, rational use of antibiotics, respiratory and nutrition support, and so on.

Biomaterials, 2005 Mar, 26(8), 917 - 24
Nitric oxide-releasing sol-gels as antibacterial coatings for orthopedic implants; Nablo BJ et al.; To assess the benefits of nitric oxide (NO)-releasing sol-gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol-gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane . Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol-gel-coated stainless steel is evaluated at both ambient and physiological temperature . Sol-gel films incubated at 25 degrees C have a lower NO flux over the first 24 h compared to those at 37 degrees C, but release more than five times longer . The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol-gel, and NO-releasing sol-gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25 degrees C and 37 degrees C . Cell adhesion to bare and sol-gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.

Arch Pediatr, 2004 Sep, 11(9), 1070 - 2
{Acute bartholinitis caused by Pseudomonas aeruginosa in an 18-month-old infant}; Touzot F et al.; Acute bartholinitis is a disease usually seen in women in the period of genital activity . Its occurence in a prepubertal child is an extremely rare event . CASE REPORT: We describe the case of a 18-month-old infant presenting a Bartholin's gland abces caused by Pseudomonas Aeruginosa with a resolutive evolution after antibiotherapy and surgical drainage . CONCLUSION: Diagnosis of Bartholinitis should be considered in any female infant with a labial enlargement.

Biochemistry, 2004 Sep 14, 43(36), 11427 - 35
Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture; Audette GF et al.; Adherence of pathogens to host cells is critical for the initiation of infection and is thus an attractive target for anti-infective therapeutics and vaccines . In the opportunistic human pathogen Pseudomonas aeruginosa, host-cell adherence is achieved predominantly by type IV pili . Analysis of several clinical strains of P . aeruginosa reveals poor sequence conservation between pilin genes, including the residues in the receptor-binding site . Interestingly, the receptor-binding sites appear to retain a conserved surface epitope because all Pseudomonas type IV pili recognize the same receptor on the host cell and cross-reactive antibodies specific for the receptor-binding site exist . Here, we present the crystallographic analysis of two crystal forms of truncated pilin from P . aeruginosa strain K122-4 (DeltaK122-4) at 1.54 and 1.8 A resolution, respectively . The DeltaK122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of DeltaK122-4 pilin . A comparison with the structure of the highly divergent P . aeruginosa strain K (DeltaPAK) pilin indicates that the receptor-binding loop in both pilins forms a shallow depression with a surface that is formed by main-chain atoms . Conservation of this putative binding site is independent of the sequence as long as the main-chain conformation is conserved and could therefore explain the shared receptor specificity and antibody cross reactivity of highly divergent Pseudomonas type IV pilins.

J Mater Sci Mater Med, 1998, 9(1), 17 - 22
The influence of substratum topography on bacterial adhesion to polymethyl methacrylate; Taylor RL et al.; The effect of substratum roughness on the adhesion of Pseudomonas aeruginosa and Staphylococcus epidermidis was investigated using PMMA . A small increase in Ra values (0.04-1.24 microm) resulted in a significant increase (P<0.05) in bacterial attachment . Subsequent increases in surface roughness (Ra=1.86-7.89 microm) resulted in a decrease in adhesion, although adhesion was still higher than to the smooth surface . When the PMMA surfaces were coated with protein (bovine serum albumin), no difference (P<0.05) could be determined in the amount of protein adsorbed, irrespective of surface topography . However, the influence of the underlying topography on adhesion was still evident . Substratum topography is an important parameter affecting bacterial adhesion to surfaces.

J Mater Sci Mater Med, 2000 Dec, 11(12), 817 - 823
beta-Chitin-based wound dressing containing silver sulfurdiazine; Lee YM et al.; Physical and biological properties of some wound dressing materials based on beta-chitin were studied . Water vapor transmission rates (WVTR), oxygen permeabilities and biodegradation kinetics were examined for film-type samples . WVTR of samples was in the range 2400-2800 g/m^2/day . However, oxygen permeabilities of the samples were relatively low . To improve oxygen permeabilities, porous sponge-type wound dressing materials were prepared . In addition, these sponge-type samples contained antimicrobial agents, silver sulfadiazine (AgSD), in order to prevent bacteria infection on a wound surface . Anti-microbacterial tests on agar plate were carried out to confirm the bactericidal capacity of present materials . These materials impregnating AgSD had the complete bactericidal capacity against pseudomonas aeruginosa up to 7 days . Finally, a wound healing effect of beta-chitin-based semi-interpenetrating polymer networks was evaluated from the animal test using the wistar rat in vivo . Histological studies confirm the proliferation of fibroblasts in the wound bed and a distinct reduction in infectious cells .

J Mater Sci Mater Med, 1999 Dec, 10(12), 853 - 5
Initial adhesion and surface growth of Pseudomonas aeruginosa on negatively and positively charged poly(methacrylates); Gottenbos B et al.; The infection risk of biomaterial implants is determined by an interplay of bacterial adhesion and surface growth of the adhering organisms . In this study, we compared initial adhesion and surface growth of Pseudomonas aeruginosa AK1 (zeta potential -7 mV) on negatively charged (PMMA/MAA, zeta potential -18 mV) and positively charged (PMMA/TMAEMA-Cl, zeta-potential +12 mV) methacrylate copolymers in situ in a parallel plate flow chamber . Initial adhesion was measured using phosphate-buffered saline and subsequent surface growth of the adhering bacteria using nutrient broth as growth medium . Initial adhesion was twice as fast on the positively charged methacrylate than on the negatively charged copolymer . Surface growth, however, was absent on the positively charged copolymer, while on the negatively charged methacrylate the number of bacteria increased exponentially during surface growth with a generation time of 32 min . From the results of this study it can be concluded that positively charged biomaterial surfaces might show reduced risks of biomaterials-centred infections, despite being more adhesive .

Appl Environ Microbiol, 2004 Sep, 70(9), 5357 - 65
Localization and characterization of two novel genes encoding stereospecific dioxygenases catalyzing 2(2,4-dichlorophenoxy)propionate cleavage in Delftia acidovorans MC1; Schleinitz KM et al.; Two novel genes, rdpA and sdpA, encoding the enantiospecific alpha-ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified . Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other alpha-ketoglutarate dioxygenases . RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134 . The functionally important amino acid sequence motif HX(D/E)X(23-26)(T/S)X(114-183)HX(10-13)R/K, which is highly conserved in group II alpha-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes . Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene . Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes . Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon . Furthermore, two copies of a sequence similar to IS91-like elements were identified . Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements . Two other dichlorprop-degrading bacterial strains, Rhodoferax sp . strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected . This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains.

Appl Environ Microbiol, 2004 Sep, 70(9), 5102 - 10
The gnyRDBHAL cluster is involved in acyclic isoprenoid degradation in Pseudomonas aeruginosa; Diaz-Perez AL et al.; Pseudomonas aeruginosa PAO1 mutants affected in the ability to degrade acyclic isoprenoids were isolated with transposon mutagenesis . The gny cluster (for geranoyl), which encodes the enzymes involved in the lower pathway of acyclic isoprenoid degradation, was identified . The gny cluster is constituted by five probable structural genes, gnyDBHAL, and a possible regulatory gene, gnyR . Mutations in the gnyD, gnyB, gnyA, or gnyL gene caused inability to assimilate acyclic isoprenoids of the citronellol family of compounds . Transcriptional analysis showed that expression of the gnyB gene was induced by citronellol and repressed by glucose, whereas expression of the gnyR gene had the opposite behavior . Western blot analysis of citronellol-grown cultures showed induction of biotinylated proteins of 70 and 73 kDa, which probably correspond to 3-methylcrotonoyl-coenzyme A (CoA) carboxylase and geranoyl-CoA carboxylase (GCCase) alpha subunits, respectively . The 73-kDa biotinylated protein, identified as the alpha-GCCase subunit, is encoded by gnyA . Intermediary metabolites of the isoprenoid pathway, citronellic and geranic acids, were shown to accumulate in gnyB and gnyA mutants . Our data suggest that the protein products encoded in the gny cluster are the beta and alpha subunits of geranoyl-CoA carboxylase (GnyB and GnyA), the citronelloyl-CoA dehydrogenase (GnyD), the gamma-carboxygeranoyl-CoA hydratase (GnyH), and the 3-hydroxy-gamma-carboxygeranoyl-CoA lyase (GnyL) . We conclude that the gnyRDBHAL cluster is involved in isoprenoid catabolism.

Am J Chin Med, 2004, 32(3), 389 - 96
Shiunko promotes epithelization of wounded skin; Huang KF et al.; Shiunko is a traditional botanic formula (ointment) which is used clinically for the treatment of wounded skin caused by cuts, abrasions, frost or burn . The aim of this study was to evaluate the effect of Shiunko on epithelization of wounded skin . Experimental cutting wounds on the back skin of Sprague-Dawley rats were induced . Different bacterial inoculations (Pseudomonus aeruginosa and Staphylococcus aureus) and treatment (Shiunko, Povidone-iodine and saline) were arranged herein . The incidences of infection and the speed of epithelization were evaluated . We observed that the incidences of wound infection following Pseudomonas aeruginosa inoculation were lower on both the Shiunko-treated group (0%, p < 0.01) and Povidine-iodine-treated group (5%, p < 0.05), than the saline-treated group (40%) . The Shiunko-treated group reported higher percentages of complete epithelization not only on the sterilized wounds (100%) but also on the contaminated wounds (90%) when compared to the saline-treated group (60% sterilized wounds, 40% and 50% contaminated wounds) on day 7 (p < 0.01) . Povidone-iodine did not promote epithelization of wounded skin, whereas Shiunko did.

J Am Vet Med Assoc, 2004 Aug 15, 225(4), 548 - 53
Evaluation of outcome of otitis media after lavage of the tympanic bulla and long-term antimicrobial drug treatment in dogs: 44 cases (1998-2002); Palmeiro BS et al.; OBJECTIVE: To evaluate the outcome of otitis media in dogs after video-otoscopic lavage of the tympanic bulla and long-term antimicrobial drug treatment . DESIGN: Retrospective study . ANIMALS: 44 dogs with otitis media treated in an academic referral practice . PROCEDURE: Medical records were reviewed for signalment, duration of ear canal disease, previous medical treatments, dermatologic diagnosis, results of cytologic examination and microbial culture of ear canal exudate, findings during video-otoscopy, medical treatment, days to resolution, and maintenance treatments prescribed . Four independent variables (age, duration of ear canal disease prior to referral, use of corticosteroids in treatment regimens, and infection with Pseudomonas aeruginosa) were evaluated statistically for potential influence on time to resolution . RESULTS: Mean +/- SD (range) duration of ear canal disease prior to referral was 24.9 +/- 21.6 (3 to 84) months . Otitis media in 36 dogs resolved after lavage of the tympanic bulla and medical management; mean +/- SD (range) time to resolution was 117 +/- 86.7 (30 to 360) days . Time to resolution was not significantly influenced by any variable evaluated . Three dogs were lost to follow-up, and 4 dogs eventually required surgical intervention . Seven of 36 dogs in which otitis had resolved relapsed; 4 required additional lavage procedures . CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that lavage of the tympanic bulla combined with medical management is an effective and viable option for treatment of otitis media in dogs.

J Bacteriol, 2004 Sep, 186(18), 6142 - 9
Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase; Fernandez-Murga ML et al.; To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine . Therefore, NAGK is in T . maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase . The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK) . At 75 degrees C, the inhibition by arginine of T . maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme . The NAGKs of T . maritima and P . aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes . In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity . Potential thermostability determinants of T . maritima NAGK are also discussed.

Protein Sci, 2004 Oct, 13(10), 2706 - 15 Epub 2004 Aug 31.
Probing the influence on folding behavior of structurally conserved core residues in P . aeruginosa apo-azurin; Engman KC et al.; The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied . A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31 . Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus . In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls . All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble . Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state . Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state.

Surg Today, 2004, 34(9), 725 - 31
Recovery of the susceptibility of isolated bacterium achieved by giving long-established antibiotics as prophylaxis against postoperative infections; Kusachi S et al.; PURPOSE: To evaluate the effectiveness of operative antibiotic therapy we changed the antibiotics and examined the susceptibility of the postoperative infection . METHODS: We studied 4 593 patients who underwent gastrointestinal surgery during the last 12.5 years, and examined the changes in intraoperative antibiotics, the return of bacteria isolated from infectious sites, the trends in frequency of Methicillin-resistant Staphylococcus aureus (MRSA) isolation, and changes in the antibiotic susceptibility of Pseudomonas aeruginosa and Bacterioides fragilis . We changed the antibiotics to Cefazolin (CEZ) for upper gastrointestinal tract surgery and cholecystectomy, and to Cefotiam (CTM) for colonic, liver, and pancreatic surgery . We also reduced the period of drug administration . RESULTS: The rate of MRSA infections decreased, the rate of P . aeruginosa infections was always under 20% and the rate of B . fragilis infections increased . After the guidelines of drug susceptibility were prepared, the minimum inhibitory concentrations (MICs) of Cefsulodin (CFS) and Piperacillin (PIPC) for P . aeruginosa and of Latamoxef (LMOX) and PIPC for B . fragilis, decreased . CONCLUSION: The use of antibiotics considered comparatively old for the prophylaxis of postoperative infection prevented the emergence of MRSA-like resistant strains, influenced changes in Gram-negative bacteria drug susceptibility, and led to an overall reduction in multiple drug resistance.

Mol Ther, 2004 Sep, 10(3), 562 - 73
Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell Line; Virella-Lowell I et al.; Mutations in CFTR lead to a complex phenotype that includes increased susceptibility to Pseudomonas infections, a functional deficiency of IL-10, and an exaggerated proinflammatory cytokine response . We examined the effects of CFTR gene correction on the gene expression profile of a CF bronchial epithelial cell line (IB3-1) and determined which CF-related gene expression changes could be reversed by IL-10 expression . We performed microarray experiments to monitor the gene expression profile of three cell lines over a time course of exposure to Pseudomonas . At baseline, we identified 843 genes with statistically different levels of expression in CFTR-corrected (S9) cells compared to the IB3-1 line or the IL-10-expressing line . K-means clustering and functional group analysis revealed a primary up-regulation of ubiquitination enzymes and TNF pathway components and a primary down-regulation of protease inhibitors and protein glycosylation enzymes in CF . Key gene expression changes were confirmed by real-time RT-PCR . Massive reprogramming of gene expression occurred 3 h after Pseudomonas exposure . Changes specific to CF included exaggerated activation of cytokines, blunted activation of anti-proteases, and repression of protein glycosylation enzymes . In conclusion, the CFTR genotype changes the expression of multiple genes at baseline and in response to bacterial challenge, and only a subset of these changes is secondary to IL-10 deficiency.

Clin Ther, 2004 Jul, 26(7), 1046 - 54
Efficacy of ofloxacin otic solution once daily for 7 days in the treatment of otitis externa: a multicenter, open-label, phase III trial; Torum B et al.; BACKGROUND: Otitis externa (OE) is an infection of the external auditory canal that is typically treated with topically applied broad-spectrum antibiotics . Twice-daily topical treatment with ofloxacin otic 0.3% solution for 10 days has been reported to be as effective and well tolerated as the standard of care, neomycin sulfate/polymyxin B sulfate/hydrocortisone solution administered 4 times daily for 10 days . OBJECTIVE: This study evaluated the efficacy and safety profile of 7 days of a once-daily regimen of ofloxacin otic 0.3% solution in the treatment of OE . METHODS: This multicenter, open-label, Phase III study was conducted from June 12, 2002, to October 14, 2002 . Eligible patients were aged > or = 6 months and had OE of <2 weeks' duration with moderate to severe edema and tenderness involving 1 or both ears and sufficient exudate for microbiologic culture . Ofloxacin otic solution was instilled once daily for 7 days (5 drops for children aged 6 months to <13 years, 10 drops for adolescents/adults aged > or = 13 years) . Assessments were conducted at the end-of-treatment visit and 7 to 10 days later (the test-of-cure visit) . Medication was supplied free of charge to study participants who incurred no costs for physician visits . RESULTS: Of 489 patients enrolled at 58 sites in 3 countries, 439 were clinically evaluable (173 children, 266 adolescents/adults; 52 % males, 48% females; 47% Hispanic, 45% white; 5% black, and 3% other) . The cure rate among clinically evaluable patients was 91% (95% of children, 88% of adolescents/adults); 68% of patients were cured within 7 days . Forty-three potentially pathogenic strains were isolated from 253 microbiologically evaluable patients . Pseudomonas aeruginosa was isolated from 158 (62%) microbiologically evaluable patients and Staphylococcus aureus from 32 (13%) . Eradication rates were 96% overall . No serious adverse events were observed . Minor adverse events were experienced by 15 (3%) of 489 patients included in the safety population . The most common adverse events were pruritus (5 patients), increased earache (4 patients), and application-site reactions (3 patients) . Overall mean (SD) adherence to therapy was 98% (11.9) . CONCLUSIONS: Ofloxacin otic 0.3% solution administered once daily for 7 days was well tolerated and effective in achieving clinical and microbiologic cure of OE . The compliance rates in this study suggests that this regimen may be better accepted by patients than longer, more repetitive regimens.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 49 - 55
The interaction of Pseudomonas aeruginosa PAK with human and animal respiratory tract cell lines; Hambrook J et al.; A major virulence factor of a common human pathogen Pseudomonas aeruginosa, was investigated to determine if it dominated attachment interactions in a variety of in vitro cell culture systems . It was found that Type-IV pilus-type mechanisms, which mediated the attachment of P . aeruginosa to three human respiratory tract cell lines (A549, BEAS-2B and RPMI 2650) also mediated attachment to two respiratory tract cell lines from mouse (C57) and rat (L-2) to a similar degree . Significant differences were found in the number of P . aeruginosa associated with the human, rat and mouse cell lines . Additionally, differences were also found between A547, C57 and L-2 cells with respect to the moieties that P . aeruginosa interacted with at the level of the cell surface, suggesting that asialo-GM1 ligands were not the only structure that this bacterium could interact with in order to associate with host cells.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 23 - 8
MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA; Matsuo Y et al.; MexZ is a transcriptional regulator of the mexXY multidrug transporter operon, which confers aminoglycoside resistance on Pseudomonas aeruginosa . Highly purified MexZ showed direct binding with a specific site of the mexZ-mexX intergenic DNA when probed by a gel retardation assay . Both in vitro chemical cross-linking experiments and an in vivo two-hybrid expression system showed that the active form of MexZ, which is capable of binding the intergenic DNA, appeared to be a dimer . These results explain the mechanism by which MexZ represses transcription of the mexXY operon, but do not explain the substrate-induced hyperproduction of MexXY . The presence of inducer antibiotic in the gel-retardation assay mixture failed to detect altered MexZ-probe DNA interaction suggesting the possible involvement of an additional regulator.

Pediatr Pulmonol, 2004 Oct, 38(4), 277 - 84
Longitudinal pulmonary status of cystic fibrosis children with meconium ileus; Li Z et al.; Although meconium ileus (MI) is the earliest manifestation of cystic fibrosis (CF), and is associated with poorer growth, the longitudinal pulmonary progression of CF children with MI is not clear . To test the hypothesis that MI is associated with worse pulmonary outcomes, we prospectively compared from diagnosis to 12 years of age 32 CF children with MI to 50 CF children without MI who were diagnosed during early infancy through neonatal screening . Pulmonary outcome measures included respiratory symptoms, respiratory infections, pathogens, antibiotic usage, hospitalizations, quantitative chest radiology, spirometry, and lung volume determinations . Obstructive lung disease was defined as percent predicted spirometry values below the lower limits of normal . Longitudinal analyses revealed no significant differences in cough, wheezing, respiratory infections, prevalence of and median times to acquisition of Pseudomonas aeruginosa or Staphylococcus aureus, antibiotic usage, and chest radiograph scores between the two groups . However, MI children showed significantly worse forced expiratory volume in 1 sec (FEV(1)), forced vital capacity (FVC), forced expiratory flow between 25-75% of FVC (FEF(25-75)), % predicted FEV(1), % predicted FEF(25-75), and total lung capacity (TLC) . These differences were particularly apparent beginning at age 8-10 years . MI children also had higher rates of and shorter median times to obstructive lung disease . Subgroup analyses showed MI children treated surgically and those treated medically had similar pulmonary outcomes . In conclusion, MI children have worse lung function and more obstructive lung disease than those without MI . Such abnormalities are accompanied by reduced lung volume . MI is a distinct CF phenotype with more severe pulmonary dysfunction.

Am J Otolaryngol, 2004 Sep-Oct, 25(5), 323 - 8
The microbiology and antimicrobial resistance patterns in chronic rhinosinusitis; Kingdom TT et al.; OBJECTIVES: The purpose of this study was to review the microbiology of chronic rhinosinusitis (CRS) in patients undergoing endoscopic sinus surgery (ESS) and comment on antimicrobial resistance trends . METHODS: A retrospective review of 101 patients undergoing ESS during the period of 1997 to 2001 was performed . Patients were divided into groups based on their surgical history . Fifty-five patients without prior ESS history were placed in the primary group; 46 patients who had undergone prior ESS were placed in the revision group . Intraoperative microbiology culture data were reviewed and antimicrobial resistance data analyzed . RESULTS: Data on 101 patients were analyzed . There were 182 total cultures sent, yielding 257 isolates . The most common isolates were coagulase-negative Staphylococcus (SCN) (45% of cultures), gram-negative rods (25% of cultures), and Staphylococcus aureus (24% of cultures) . Pseudomonas aeruginosa was isolated in 9% of cultures . When comparing the 2 patient groups, we did not find consistent trends in the differences in the prevalence of these isolates . Antimicrobial resistance for SCN (P = .01) and S aureus (P < .001) was greater in the revision surgery . Overall, 62% of patients were found to have at least 1 isolate with decreased antibiotic sensitivity . CONCLUSION: The most prevalent microorganisms in patients with CRS are SCN, S aureus, and gram-negative rods . Perhaps more importantly, the antimicrobial sensitivities of these microorganisms appear to be a growing problem . These findings suggest increased antimicrobial resistance in patients undergoing revision ESS when compared with patients undergoing surgery for the first time.

East Mediterr Health J, 2001 Jul-Sep, 7(4-5), 738 - 43
Infection following orthopaedic implants and bone surgery; Mousa HA; Forty-seven patients were investigated for early or late postoperative infections of orthopaedic implants and/or bone . A total of 88 isolates were recovered (64 aerobes and 24 anaerobes) . Pseudomonas aeruginosa and Staphylococcus epidermidis were the most common causative agents . Anaerobic bacteria were isolated from 16 (34%) patients; 50% of patients with late-onset infection and 10.5% with early-onset infection . In 6 (12.8%) patients, infection was with anaerobic organisms alone . All these patients had retained an extramedullary internal fixation device . Anaerobic microorganisms appear to play a significant role in the pathogenesis of late-onset postoperative infection, especially where there is an extramedullary internal fixation device.

Eur Respir J, 2004 Aug, 24(2), 286 - 91
Airway iron and iron-regulatory cytokines in cystic fibrosis; Reid DW et al.; Iron availability is critical to Pseudomonas aeruginosa . The current authors determined sputum iron, ferritin, microalbumin levels and total cell counts (TCC) in 19 adult patients with cystic fibrosis (CF) during an acute exacerbation and repeated analyses following a median of 12 days antibiotic treatment . The current authors also determined sputum interleukin (IL)-1beta and tumour necrosis factor (TNF)-alpha levels because of their putative role in intracellular iron homeostasis . Additional data were obtained from 17 stable CF patients, eight patients with stable chronic obstructive pulmonary disease (COPD) and six normal subjects . Overall, sputum iron, ferritin, microalbumin, IL-1beta and TNF-alpha concentrations and TCCs were significantly elevated in the CF patients compared to those with COPD and normal controls . Sputum ferritin levels were significantly elevated in acute versus stable CF patients and there was a trend for sputum TCC to be higher, but all other inflammatory indices were similar . In the CF patients, sputum iron was positively and strongly related to IL-1beta, TNF-alpha, ferritin and microalbumin levels, but negatively related to forced expiratory volume in one second % predicted . In those acute patients who clinically improved with antibiotics (n=14), there were significant decreases in sputum TCC, iron, ferritin and IL-1beta content, but not TNF-alpha or albumin levels . However, changes in sputum TNF-alpha in acute patients were still closely related to changes in iron, ferritin and albumin content, and changes in IL-1beta were related to changes in sputum ferritin content . Iron and iron-regulatory cytokines may play a role in cystic fibrosis lung disease and the increased iron content may even facilitate Pseudomonas aeruginosa infection.

J Neurooncol, 2004 Jul, 68(3), 245 - 8
Case report: Pseudomonas aeruginosa-related intervertebral discitis in a young boy with medulloblastoma; Mazza E et al.; We report a case of a 15-year-old boy with desmoplastic medulloblastoma of the posterior fossa (T3M3, according to Chang classification) incompletely resected, with leptomeningeal and nodular spread in the posterior fossa and in the cervical and thoracic tracts of the spine, treated with sequential high dose iv chemotherapy and with hyperfractionated cranio-spinal radiotherapy . While on maintenance chemotherapy, the boy developed fever and septic status caused by Pseudomonas aeruginosa, and 1 week later also low back pain . Magnetic resonance imaging (MRI) demonstrated abnormal signal in the fourth ventricle and in the dorso-lumbar tract suggesting medulloblastoma recurrence, so he started with a chemotherapy program . Due to a worsening of back pain, a second MRI of the spine was performed that showed a spondilodiscitis of T11-T12 and L1-L2 discs . The histological and cultural examination of a fine-needle biopsy of the L1-L2 disc revealed the presence of P . aeruginosa . So patient was treated with intensive antibiotic therapy with resolution of the infection . Spondilodiscitis is a rare complication in neoplastic patients, maybe due to either immunodeficient status or invasive procedures such as lumbar puncture . This case demonstrates that MRI is a useful method for differentiating between infection and malignancy in the spine, but sometimes it may be difficult to distinguish metastatic tumor from a lesion due to spondilodiscitis . In this case surgicopathological assessment is crucial and mandatory.

Int J Hyg Environ Health, 2004 Jul, 207(3), 259 - 66
Surveillance of Pseudomonas aeruginosa-isolates in a neonatal intensive care unit over a one year-period; Zabel LT et al.; Outbreaks of gram-negative bacteria such as Pseudomonas aeruginosa in neonatal intensive care units (NICU) can be life-threatening to pre-term infants, which are highly susceptible to serious infections with bacteria . Forty-two ventilated neonates in the NICU of the University Children's Hospital of Tuebingen were found to be colonized (n = 40) or infected (n = 2) with P . aeruginosa within a sampling period of one year . To investigate the colonization patterns and identify potential outbreak sources, epidemiological investigations, environmental surveillance and typing by serotyping and pulsed-field gel electrophoresis of the recovered isolates were performed . The investigation demonstrated a genetically related cluster of P . aeruginosa isolates during the surveillance period in 39 neonates and a second cluster at the end of the period in two neonates . A third strain representing a genetically distinct group was found in only one patient . Environmental investigations demonstrated the presence of P . aeruginosa in the ventilation equipment of 22 patients: binasal prongs (n = 22), water reservoir (n = 9), and heater (n = 1) . In one case, P . aeruginosa was found in breast milk . Other environmental investigations revealed no P . aeruginosa . Although no evidence for a unique source was found, a series of intervention steps were initiated by the NICU personnel, medical microbiologists and infection control experts . The intervention steps included reinforced training of health care staff and a change from chemical to thermal disinfection of binasal prongs . Implementation of these measurements successfully stopped the recurrent occurrence of P . aeruginosa colonization.

J Chemother, 2004 Jun, 16(3), 282 - 7
Microbiological activity and clinical efficacy of a colistin and rifampin combination in multidrug-resistant Pseudomonas aeruginosa infections; Tascini C et al.; The aim of the study was to assess the microbiological activity and clinical efficacy of colistin and rifampin combination against multidrug-resistant (MDR) Pseudomonas aeruginosa infections . The antimicrobial activity of the colistin/rifampin combination was evaluated using the checkerboard and time-kill curve methods against different MDR P . aeruginosa strains . The combination of rifampin and colistin resulted fully (1 strain) or partially (5 strains) synergistic for 6/7 strains and minimum inhibitory concentrations (MICs) in combination were reduced to easily obtainable therapeutic levels . The time-kill curves showed that the combination was bactericidal against the strains tested . The clinical efficacy of the combination was tested in four patients with difficult-to treat infections (sepsis or pneumonia) caused by MDR P . aeruginosa . All infections were successfully treated . Our microbiological and clinical observations suggest that the addition of rifampin to colistin may result in a synergistic bactericidal combination that may be useful in patients with infections caused by MDR P . aeruginosa which are difficult to cure.

J Chemother, 2004 Jun, 16(3), 264 - 8
Susceptibility patterns in Pseudomonas aeruginosa causing nosocomial infections; Astal Z; Nosocomial infection caused by Pseudomonas aeruginosa has not been reported previously in the Gaza Strip . This study aims to determine the distribution of antimicrobial drug resistance in P . aeruginosa causing nosocomial infections . One hundred thirty-one P . aeruginosa isolates were collected from various nosocomial infection clinical samples . The study was conducted between April and October 2003 . The results of this study reveal that the most common resistance was to ampicillin, followed by cephalexin . The most effective antimicrobial agents were meropenem and amikacin, respectively . The highest resistance to ciprofloxacin was found among ICU and surgery sections . The data analysis shows that no remarkable difference was reported with respect to previous admission and prior antimicrobial treatment for most antibiotics . The results of this study emphasize the need for constant monitoring of antimicrobial effectiveness to correctly guide empiric therapy and local intervention programs in an attempt to reduce antimicrobial resistance.

Acta Microbiol Pol, 2004, 53(1), 45 - 52
Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme; Saeed HM et al.; Urate oxidase (uricase) was isolated and purified from Pseudomonas aeruginosa to apparent homogeneity using ammonium sulphate precipitation followed by ion exchange and gel filtration chromatography . The specific activity of the purified uricase enzyme was found to be 636.36 with the use of uric acid as a substrate . The purified uricase enzyme is a monomeric protein with molecular weight of 64 kilodaltons . The optimal pH and temperature of the purified enzyme is 9.0 and 30 degrees C, respectively . The effect of some metal ions was studied . Sulphate forms of Fe+2, Zn+2 and Co+2 inhibit the uricolytic activity whereas; NaCl and CaCl2 enhance the enzyme activity . Moreover, the purified enzyme is inhibited by EDTA and KCN.

Ann Acad Med Singapore, 2004 Jul, 33(4), 484 - 8
Contact lens microbial keratitis and prior topical steroid use: a disaster in the making?
Wang JC, Su D, Lim L.
INTRODUCTION: To review the best-corrected visual acuity, ulcer size, microbiological profile and morbidity of contact lens-related microbial keratitis with and without prior topical steroid use . MATERIALS AND METHODS: Retrospective case review of admitted cases of contact lens-related microbial keratitis in a tertiary hospital . Data pertaining to demographics, pre-admission treatment with or without topical steroids, ulcer size, duration of admission, Gram stain and culture results as well as the final best-corrected visual acuity were recorded . Patients are classified into 3 groups: Group 1 received no treatment prior to presentation, Group 2 received topical antibiotics only from their general practitioners and Group 3 prescribed both topical antibiotics and steroids . RESULTS: Forty-six cases were enrolled in the study, 41.3% had prior topical steroids (all dexamethasone) in combination with antibiotics . None of them had topical steroids alone . Large ulcers were associated with steroid use, odds ratio = 7.74 {95% confidence interval (CI), 1.18-50.56} and positivity of Gram stains odds ratio = 7.74 {95% CI, 1.18-50.56} whereas loss of more than 2 Snellen lines was associated with Pseudomonas aeruginosa infection, odds ratios of 21.70 {95% CI,2.09-225.03} and presence of central ulcer, 13.51 {95% CI, 2.33-78.3} . Prior topical steroid use was associated with longer duration of symptoms prior to admission but not duration of stay or surgical intervention . CONCLUSION: Patients with prior topical combined antibiotics-steroids present slightly later and with larger ulcers . However, the duration of stay, final visual acuity, treatment failure and complication rates were not statistically different from the non-treated group . This might be due to 1) early presentation and therefore early treatment of contact lens-related microbial keratitis and 2) the short duration of use of combined antibiotic-steroid eye drops.

Nature, 2004 Aug 26, 430(7003), 1024 - 7
Cooperation and competition in pathogenic bacteria; Griffin AS et al.; Explaining altruistic cooperation is one of the greatest challenges for evolutionary biology . One solution to this problem is if costly cooperative behaviours are directed towards relatives . This idea of kin selection has been hugely influential and applied widely from microorganisms to vertebrates . However, a problem arises if there is local competition for resources, because this leads to competition between relatives, reducing selection for cooperation . Here we use an experimental evolution approach to test the effect of the scale of competition, and how it interacts with relatedness . The cooperative trait that we examine is the production of siderophores, iron-scavenging agents, in the pathogenic bacterium Pseudomonas aeruginosa . As expected, our results show that higher levels of cooperative siderophore production evolve in the higher relatedness treatments . However, our results also show that more local competition selects for lower levels of siderophore production and that there is a significant interaction between relatedness and the scale of competition, with relatedness having less effect when the scale of competition is more local . More generally, the scale of competition is likely to be of particular importance for the evolution of cooperation in microorganisms, and also the virulence of pathogenic microorganisms, because cooperative traits such as siderophore production have an important role in determining virulence.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002 Aug, 37(7), 1379 - 90
Antibacterial properties of chitosan in waterborne pathogen; Chen YM et al.; The antimicrobial properties of chitosan, a derivative of chitin, were investigated in the solid and liquid culture against bacteria associated with waterborne disease in order to assess the potential for using chitosan as a natural disinfectant . Six strains which included three gram-negative and three gram-positive bacteria were studied . The effects of the deacetylation degree, concentration, and molecular weight of chitosan on antibacterial activities were assessed . Chitosan exhibited the highest antibacterial activity against the Pseudomonas aeruginosa on the solid agar . Similar tendency was found when the bacteria were cultivated in liquid broth . The higher deacetylation degree and higher concentration of chitosan cause the higher antibacterial activities . The effect of molecular weight of chitosan on the inhibition efficacy of bacteria is dependent on the species of bacteria . Escherichia coli is sensitive to chitosan during its death phase and logarithmic phase . The antibacterial mechanism of chitosan was illustrated by the surface charge and persistence length . Results indicated that chitosan is potential as a natural disinfectant.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3576 - 8
Isolation of an integron-borne blaVIM-4 type metallo-beta-lactamase gene from a carbapenem-resistant Pseudomonas aeruginosa clinical isolate in Hungary; Libisch B et al.; The first integron-borne metallo-beta-lactamase gene was isolated in Hungary . The bla(VIM-4) gene is located on a class 1 integron that also carries a novel bla(OXA)-like gene . The integron is harbored by a serotype O12 Pseudomonas aeruginosa strain and shows high structural similarity to integrons isolated in Greece and Poland.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3573 - 5
Lack of association between hypermutation and antibiotic resistance development in Pseudomonas aeruginosa isolates from intensive care unit patients; Gutierrez O et al.; Hypermutation is a common feature of Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis patients that is linked with antibiotic resistance development . In this work, using a large collection of sequential P . aeruginosa isolates from ICU patients, we found that despite the fact that mutational antibiotic resistance development is a frequent outcome, the prevalence of hypermutable strains is low (found in isolates from only 1 of 103 patients) and there is no evidence of coselection of the hypermutable and antibiotic resistance phenotypes.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3457 - 61
Azithromycin inhibits MUC5AC production induced by the Pseudomonas aeruginosa autoinducer N-(3-Oxododecanoyl) homoserine lactone in NCI-H292 Cells; Imamura Y et al.; The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus . Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease . The quorum-sensing signal molecule {N-(3-oxododecanoyl) homoserine lactone (3O-C(12)-HSL)} not only regulates bacterial virulence but also is associated with the immune response . In this study, we investigated whether 3O-C(12)-HSL could stimulate the production of a major mucin core protein, MUC5AC . The effect of a macrolide on MUC5AC production was also studied . 3O-C(12)-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners . A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C(12)-HSL . 3O-C(12)-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-kappa B phosphorylation in cells, and this induction was suppressed by azithromycin . 3O-C(12)-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059 . Our findings suggest that the P . aeruginosa autoinducer 3O-C(12)-HSL contributes to excessive mucin production in chronic bacterial infection . Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3402 - 6
Sequence-selective recognition of extended-spectrum beta-lactamase GES-2 by a competitive, peptide nucleic acid-based multiplex PCR assay; Weldhagen GF; Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa, such as GES-2, which compromises the efficacy of imipenem, tend to be geographically restricted . The CC-to-AA base pair substitution at positions 493 and 494 of the bla(GES-2)-coding region distinguishes this ESBL from bla(GES-1) and the bla(IBC)-type genes, making it an ideal target for the development of a novel sequence-specific, peptide nucleic acid (PNA)-based multiplex PCR detection method . By using two primer pairs in conjunction with a PNA probe, this method provided an accurate means of identification of bla(GES-2) compared to standard PCR and gene sequencing techniques when it was used to test 100 P . aeruginosa clinical isolates as well as previously published, well-described control strains encompassing all presently known genes in the bla(GES-IBC) ESBL family . This novel method has the potential to be used in large-scale, cost-effective screening programs for specific or geographically restricted ESBLs.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3396 - 401
A mucoadhesive polymer extracted from tamarind seed improves the intraocular penetration and efficacy of rufloxacin in topical treatment of experimental bacterial keratitis; Ghelardi E et al.; Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities . Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth . However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization . In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental Pseudomonas aeruginosa and Staphylococcus aureus keratitis in rabbits . The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes . Rufloxacin delivered by the polysaccharide reduced P . aeruginosa and S . aureus in the cornea at a higher rate than that obtained by rufloxacin alone . In particular, use of the polysaccharide allowed a substantial reduction of S . aureus in the cornea to be achieved even when the time interval between drug administrations was extended . These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs . The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3284 - 90
Functional and structural characterization of the genetic environment of an extended-spectrum beta-lactamase blaVEB gene from a Pseudomonas aeruginosa isolate obtained in India; Aubert D et al.; A Pseudomonas aeruginosa clinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam . A bla(VEB-1)-like gene named bla(VEB-1a), which codes for the extended-spectrum beta-lactamase VEB-1a, was identified . The genetic environment of bla(VEB-1a) was peculiar: (i) no 5' conserved sequence (5'-CS) region was present upstream of the beta-lactamase gene, whereas bla(VEB-1)-like genes are usually associated with class 1 integrons; (ii) bla(VEB-1a) was inserted between two truncated 3'-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the bla(VEB-1a) gene . Expression of the bla(VEB-1a) gene was driven by a strong promoter located in one of these repeated sequences . In addition, cloning of the beta-lactamase content of this P . aeruginosa isolate followed by expression in Escherichia coli identified the naturally occurring AmpC beta-lactamase and a gene encoding an OXA-2-like beta-lactamase located in a class 1 integron, In78, in which an insertion sequence, ISpa7, was inserted within its 5'-CS region.

Invest Ophthalmol Vis Sci, 2004 Sep, 45(9), 3177 - 84
Caspase-1 inhibitor reduces severity of pseudomonas aeruginosa keratitis in mice; Thakur A et al.; PURPOSE: To test an inhibitor of IL-1beta converting enzyme (ICE), with or without ciprofloxacin, in a C57BL/6 mouse model of keratitis induced by Pseudomonas aeruginosa in which corneal perforation is expected . METHODS: Clinical score, histopathology, myeloperoxidase (MPO) activity, bacterial counts, and ELISA analysis were used to assess the efficacy of treatment initiated at 18 hours postinfection (p.i.) with ICE inhibitor versus placebo; and with ICE inhibitor plus ciprofloxacin versus placebo plus ciprofloxacin . Efficacy of the ICE inhibitor was also tested and evaluated for clinical score in experimental corneal infection induced by a clinical isolate and a ciprofloxacin-resistant bacterial strain . RESULTS: Clinical scores were reduced at 3, 5, and 7 days p.i . in ICE inhibitor versus placebo-treated mice; reduced scores also were observed with a combined treatment (ICE inhibitor and ciprofloxacin) . Further testing (MPO assay) revealed reduced PMN number, particularly striking in ICE inhibitor and ciprofloxacin versus placebo and ciprofloxacin-treated mice . Corneal protein levels for IL-1beta and MIP-2 also were reduced in mice treated with the ICE inhibitor versus placebo and in ICE inhibitor and ciprofloxacin versus ciprofloxacin and placebo-treated mice . Treatment with ICE inhibitor also reduced clinical scores after corneal infection with a clinical isolate, KEI-1025, and with a ciprofloxacin-resistant P . aeruginosa strain . CONCLUSIONS: Downregulation of IL-1beta by ICE together with ciprofloxacin to kill bacteria may provide alternate therapy to current treatment . Copyright Association for Research in Vision and Ophthalmology

Int J Antimicrob Agents, 2004 Sep, 24(3), 219 - 25
Effect of adjunctive treatment with gamma interferon against Pseudomonas aeruginosa pneumonia in neutropenic and non-neutropenic hosts; Babalola CP et al.; To evaluate the adjunctive effect of interferon-gamma (IFN-gamma) in treatment of Pseudomonas aeruginosa pneumonia, neutropenic and non-neutropenic mice received LD(100) of the organism intratracheally, followed by subcutaneous administration of IFN-gamma, ceftazidime (TAZ) or a combination of both agents 2 h post-inoculation . Treatment with IFN-gamma alone showed no significant increase in survival when compared with control . Addition of IFN-gamma to TAZ resulted in no significant change in survival compared with TAZ alone . Survival in TAZ and TAZ + IFN-gamma groups, was significantly higher than in control and IFN-gamma groups . This suggests that adjunctive treatment with IFN-gamma in combination with ceftazidime may not be more beneficial than the antibiotic alone when managing acute P . aeruginosa pneumonia in both immunocompromised and immunocompetent hosts.

Biochem Biophys Res Commun, 2004 Sep 17, 322(2), 483 - 9
Role of the membrane fusion protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa; Mokhonov VV et al.; The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner membrane transporter (e.g., MexB, MexY), the periplasmic membrane-fusion-protein (e.g., MexA, MexX), and the outer membrane channel protein (e.g., OprM) . These subunits were assumed to assemble into a transporter unit during export of the substrates . However, subunit interaction and their specificity in native form remained to be elucidated . To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits . We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures . Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM . None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction . These results implied that the membrane fusion protein is central to the tripartite xenobiotic transporter assembly.

J Immunol, 2004 Sep 1, 173(5), 2909 - 12
Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression; Wang TT et al.; The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14 . 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements . In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses . The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression . 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils . Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa . 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.

Infect Immun, 2004 Sep, 72(9), 5433 - 8
Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells; Frisk A et al.; The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology . Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

Infect Immun, 2004 Sep, 72(9), 5012 - 8
Microarray analysis reveals induction of lipoprotein genes in mucoid Pseudomonas aeruginosa: implications for inflammation in cystic fibrosis; Firoved AM et al.; The main cause of the high morbidity and mortality of cystic fibrosis (CF) is the progressive lung inflammation associated with Pseudomonas aeruginosa colonization . During the course of chronic CF infections, P . aeruginosa undergoes a conversion to a mucoid phenotype . The emergence of mucoid P . aeruginosa in CF is associated with increased inflammation, respiratory decline, and a poor prognosis . Here we show, by the use of microarray analysis, that upon P . aeruginosa conversion to mucoidy, there is a massive and preferential induction of genes encoding bacterial lipoproteins . Bacterial lipoproteins are potent agonists of Toll-like receptor 2 (TLR2) signaling . The expression of TLR2 in human respiratory epithelial cells was ascertained by Western blot analysis . Human respiratory epithelial cells responded in a TLR2-dependent manner to bacterial lipopeptides derived from Pseudomonas lipoproteins induced in mucoid strains . The TLR2 proinflammatory response was further augmented in CF cells . Thus, the excessive inflammation in CF is the result of a global induction in mucoid P . aeruginosa of lipoproteins that act as proinflammatory toxins (here termed lipotoxins) superimposed on the hyperexcitability of CF cells . Blocking the signaling cascade responding to bacterial lipotoxins may provide therapeutic benefits for CF patients.

J Coll Physicians Surg Pak, 2004 Aug, 14(8), 459 - 61
Microbiology and drug sensitivity patterns of chronic suppurative otitis media; Aslam MA et al.; OBJECTIVE: To identify the commonest microorganisms associated with chronic discharging ears and their antimicrobial sensitivities . DESIGN: Descriptive study . PLACE AND DURATION OF STUDY: This study was carried out from August 2003 to February 2004 at the Department of Otorhinolaryngology and Head and Neck Surgery, Fauji Foundation Hospital, Rawalpindi . MATERIALS AND METHODS: A total of 124 patients with unilateral or bilateral active chronic suppurative otitis media attending the outpatient clinic were included in the study . All patients were evaluated through detailed history and clinical examination . Pus samples were collected from the discharging ear(s) and sent to the hospital laboratory where culture and sensitivity studies were done for aerobes, anaerobes and fungi and antibiotic sensitivity patterns . RESULTS: Overall microbiology of 142 samples was studied . Among them, 108 (76%) were pure cultures and 34 (23.9%) were mixed . There were 186 isolates including 182 (97.8%) aerobes, nil anaerobes and only 4 (2.1%) fungi . Pseudomonas aeruginosa 94(50.5%) was the most common isolate, followed by Staphylococcus aureus 44 (23.6%) . Drug sensitivities pattern of Pseudomonas aeruginosa showed that ciprofloxacin was active against majority 95.8% of isolates followed by amikacin 83.3%, gentamicin and tobarmycin 60% and cefotaxime 41.6% . Staphylococcus aureus isolates were resistant to penicillin, ampicillin and amoxicillin in 77.2% whereas majority was sensitive to coamoxiclav 81.8% and cephradine 86.3% . CONCLUSION: Commonest organisms isolated from chronic discharging ears were Pseudomonas aeruginosa and Staphylococcus aureus . Majority of isolates of Pseudomonas aeruginosa were sensitive to ciprofloxacin . Majority of strains of Staphylococcus aureus were resistant to penicillin . Cephradine and coamoxiclav were effective against most of the isolates of Staphylococcus aureus.

Clin Exp Immunol, 2004 Sep, 137(3), 478 - 85
Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection; Jensen PO et al.; Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis . We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P . aeruginosa . These parameters were correlated with the quantitative bacteriology and histopathology of the lungs . After challenge, the content of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein-2 (MIP-2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood . However, 2 days after challenge the concentration of G-CSF and MIP-2 was higher in the lungs and sera of BALB/c mice . CD11b expression was higher on the PMNs of the C3H/HeN mice . The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only . These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice . In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up-regulation of CD11b . The severe lung inflammation in BALB/c mice may be caused by the early higher content of G-CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP-2 in the lungs.

J Laryngol Otol, 2004 Jul, 118(7), 576 - 9
Use of magnetic resonance imaging as the primary imaging modality in the diagnosis and follow-up of malignant external otitis; Ismail H et al.; Malignant external otitis (MEO) is a severe infection of the external auditory meatus caused by Pseudomonas aeruginosa . Classical features include unrelenting deep otalgia, otorrhoea and granulations in the floor of the ear canal . Treatment is generally protracted antibiotic therapy and monitoring of inflammatory markers; the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) . Traditionally computed tomography (CT) has been the imaging modality of choice . The authors present a case where magnetic resonance imaging (MRI) has been crucial in the diagnosis and follow up of a patient with MEO.

J Biol Chem, 2004 Nov 19, 279(47), 48671 - 9 Epub 2004 Aug 17.
Structure-activity relationships in defensin dimers: a novel link between beta-defensin tertiary structure and antimicrobial activity; Campopiano DJ et al.; Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system . We recently described a beta-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines . Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C) . Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C . Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mm NaCl, whereas Defr1 Y5C was inactive . By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers . Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds . In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture . This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond . Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have beta-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities . Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity . We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond . This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

J Bacteriol, 2004 Sep, 186(17), 5672 - 84
Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis; Lizewski SE et al.; The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression . In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P . aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P . aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid . Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes . Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2) . An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1 . Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P . aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain . AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557 . Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant . This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557.

J Bacteriol, 2004 Sep, 186(17), 5596 - 602
Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1; Vandenende CS et al.; The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and l-2,4-diaminobutyrate . Both pvdH and asd (encoding aspartate beta-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of l-2,4-diaminobutyrate in the culture media . The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P . aeruginosa PAO1 . PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate beta-semialdehyde and l-2,4-diaminobutyrate . Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for alpha-ketoglutarate . The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower . The enzyme has negligible activity toward other keto acids tested . Homologues of PvdH were present in the genomes of other Pseudomonas spp . These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes . This suggests that there is a general mechanism of l-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.

J Antimicrob Chemother, 2004 Oct, 54(4), 767 - 71 Epub 2004 Aug 18.
Comparison of in vivo intrinsic activity of cefepime and imipenem in a Pseudomonas aeruginosa rabbit endocarditis model: effect of combination with tobramycin simulating human serum pharmacokinetics; Navas D et al.; OBJECTIVES: The purpose of this experimental study was first to compare the in vivo intrinsic activity of imipenem and cefepime administered as a continuous infusion and to determine their lowest effective serum steady-state concentration (LESSC) . Secondly, we studied the effect of combining therapy with tobramycin . METHODS: In a Pseudomonas aeruginosa (ATCC 27853) rabbit endocarditis model, beta-lactam antibiotics were administered by continuous infusion over a 24 h treatment period at different doses until the LESSC was reached, i.e . able to achieve a 2-log drop of cfu/g of vegetations versus untreated animals . The effect of adding tobramycin (3 mg/kg once daily) was then studied . RESULTS: The LESSC was between 3 x and 4 x MIC of cefepime for P . aeruginosa and about 0.2 5x MIC of imipenem . Combination of tobramycin with each of the two beta-lactams did not result in any further significant killing . CONCLUSION: The optimal Css/MIC ratio might differ from one molecule to another . The LESSC of imipenem is lower than that of cefepime, giving a better intrinsic activity in vivo, despite a higher MIC in vitro.

Am J Respir Crit Care Med, 2004 Dec 1, 170(11), 1164 - 71 Epub 2004 Dec 1.
Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection; Drannik AG et al.; The object of this study was to investigate the impact of cigarette smoke on bacterial clearance and immune inflammatory parameters after infection with Pseudomonas aeruginosa in mice . We observed a delayed rate of bacterial clearance in smoke-exposed compared with sham-exposed mice . This was associated with increased inflammation characterized by greater numbers of neutrophils and mononuclear cells in the bronchoalveolar lavage . After infection, we observed increased levels of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) and chemokines (monocyte chemoattractant protein-1 {MCP-1} and macrophage inflammatory protein-2 {MIP-2}) as well as myeloperoxidase and proteolytic activity in the lungs of smoke-exposed compared with sham-exposed animals . Delayed clearance was associated with increased morbidity and greater weight loss of smoke-exposed mice . After delivery of inactivated bacteria, we observed a similar inflammatory response, clinical score, and tumor necrosis factor-alpha expression in smoke- and sham-exposed animals, suggesting that increased inflammation and altered clinical presentation are due to the delayed rate of bacterial clearance . Our findings suggest that cigarette smoke affects respiratory immune-inflammatory responses elicited by bacteria . We postulate that altered respiratory host defense may be implicated in smoking-related diseases such as chronic obstructive pulmonary disease.

Hautarzt, 2004 Nov, 55(11), 1067 - 73
{Pseudomonas aeruginosa infection presenting as gonorrhea}; Schugt I et al.; A 68 year old man presented with urethritis and a purulent discharge, carrying the tentative diagnosis of gonorrhea . He had already been treated with multiple antibiotics . Microbiological investigation revealed Pseudomonas aeruginosa, a relatively frequent Gram-negative bacteria in hospitals, which can cause several nosocomial diseases such as pneumonia, wound infections and urogenital infections . Therapy can be difficult because of frequent antibiotic resistance . Guided by sensitivity studies, the patient was successfully treated with gyrase inhibitors . Pseudomonas aeruginosa-induced urogenital infections in ambulatory patients are extremely rare and usually not associated with a gonorrhea-like discharge.

Dermatology, 2004, 209(2), 111 - 6
Early cutaneous alterations in experimental sepsis by Pseudomonas aeruginosa; Petropoulou H et al.; BACKGROUND: To evaluate whether histopathologic findings of skin in sepsis by Pseudomonas aeruginosa correlate with the clinical course . METHODS: Histological alterations after bacterial challenge by one susceptible (A) and two multidrug-resistant isolates (B and C) of P . aeruginosa were studied in 18 rabbits . Sepsis was induced by the intravenous infusion of 1 x 10(8) CFU by a catheter in the right jugular vein; blood was sampled for the estimation of tumor necrosis factor alpha (TNF-alpha) and malondialdehyde (MDA) . Skin biopsies were collected along with a subcutaneous fat specimen for culture . RESULTS: The mean survival was 0.85, 1.75 and 11.00 days after challenge by isolates A, B and C, respectively . The main histologic findings of skin were: inflammation and swelling of the dermis; thickening of the endothelium and infiltration of vessel wall and lumen by polymorphonuclear leukocytes; extravasation of red blood cells, and necrobiotic changes of the hair follicles . Serum TNF-alpha was elevated in animals challenged by isolate A compared to challenge by isolates B and C . Concentrations of MDA were similar for all isolates . Mean log(10) of viable cells isolated from subcutaneous fat were 5.74, 2.74 and 1.40 after challenge by isolates A, B and C, respectively . CONCLUSIONS: Prolongation of survival was accompanied by lower serum TNF-alpha, decreased viable cells from subcutaneous fat and intensified inflammatory response in the dermis and subcutaneous tissue . These findings might be of importance for immunomodulatory intervention.

J Biol Chem, 2004 Oct 29, 279(44), 45919 - 25 Epub 2004 Aug 16.
Crystal structure of ADP-ribosylated ribosomal translocase from Saccharomyces cerevisiae; Jorgensen R et al.; The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 A resolution . The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor . We have obtained an electron density map of ADP-ribosylated translation factor 2 revealing both the ADP-ribosylation and the diphthamide . This is the first structure showing the conformation of an ADP-ribosylated residue and confirms the inversion of configuration at the glycosidic linkage . Binding experiments show that the ADP-ribosylation has limited effect on nucleotide binding affinity, on ribosome binding, and on association with exotoxin A . These results provide insight to the inhibitory mechanism and suggest that inhibition may be caused by erroneous interaction of the translation factor with the codon-anticodon area in the P-site of the ribosome.

J Med Microbiol, 2004 Sep, 53(Pt 9), 915 - 20
Effect of carbapenems on the transcriptional expression of the oprD, oprM and oprN genes in Pseudomonas aeruginosa; Kolayli F et al.; The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes oprD, oprM and oprN in Pseudomonas aeruginosa were studied by quantitative real-time PCR . Four strains were examined: the type strain PT5 (PAO1), its derivatives M7 and PT149, and a clinical isolate, PaKT3 . The derivative M7 is a nalB mutant, overexpressing the MexAB-OprM pump, and the derivative PT149 is a nfxC-type mutant, overexpressing the MexEF-OprN pump while it is down-regulated for the OprD protein . After 18 h incubation in broth, the cultures were divided into three portions . Two were supplemented with antibiotics and the other was left antibiotic-free as the control . After a further 45 min incubation, total RNA was isolated from the strains by guanidine denaturation and acid-phenol/chloroform extraction . DNA-free total RNAs were immediately reverse-transcribed by MMuLV reverse transcriptase . Concentrations of mRNAs obtained by quantitative PCR were expressed relative to uninduced portions of the strains . The results showed that oprD was relatively stable against carbapenem antibiotics . oprM was induced significantly by imipenem in only one strain and oprN was induced by imipenem in most of the strains . The responses at the mRNA level found here were unexpected and suggested a chaotic, unpredictable regulatory mechanism.

J Mol Biol, 2004 Jul 30, 341(1), 171 - 84
Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer--dimer equilibrium; Plevin MJ et al.; In mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is implicated in the regulation of the cellular levels of asymmetric methylarginines, small molecule metabolites that themselves represent a family of endogenous inhibitors of nitric oxide synthase (NOS) . The involvement of DDAH function in the regulation of NOS makes this enzyme a potentially attractive therapeutic target . DDAH from the bacterium Pseudomonas aeruginosa (PaDDAH) is so far the only structurally tractable homologue of mammalian DDAH isoforms . To complement the recent crystal structure of this protein, we show by hydrodynamic measurements that PaDDAH exists in dynamic equilibrium between monomer (ca 29 kDa) and symmetric homodimer (ca 58 kDa) states with a dimer dissociation constant, K(d) approximately 500nM . For the purposes of NMR-based approaches to the study of this enzyme's interactions with substrate and inhibitor ligands, it would be useful to obtain the protein in monomeric form . Through detailed analysis of the homodimer PaDDAH crystal structure we identified key residues involved in the protomer-protomer interface and targeted these for mutation . The hydrodynamic and self-associative properties of a series of PaDDAH interface mutants were analyzed by concentration-dependent analytical size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation . The individual substitution of several of the interface residues shifts the equilibrium position towards the monomer, which allowed the design of a double mutant variant (Arg40-->Glu, Arg98-->His) that behaves exclusively as a stable monomer, yet retains greater than 95% catalytic activity compared to wild-type . Comparative two-dimensional (1)H, (15)N heteronuclear NMR spectra indicate that the double mutant remains a monomer even at approximately 1 mM concentration . Accordingly, the double mutant PaDDAH is an attractive template for further NMR-based investigations of the enzyme mechanism and characterization of ligand-binding and inhibitor-binding profiles . These results indicate that dimerization of PaDDAH is not critical for the maintenance of the biological function of the protein . These results are discussed in the context of known modes of self-association between structurally related, but functionally distinct, members of the beta/alpha-propeller fold class.

Chemosphere, 2004 Oct, 57(3), 165 - 9
Mechanism of Navitan Fast Blue S5R degradation by Pseudomonas aeruginosa; Valli Nachiyar C et al.; The mechanism by which Pseudomonas aeruginosa degraded Navitan Fast Blue S5R was studied using TLC, FTIR, HPLC and GC-MS analysis . Degradation started with the reduction of azo bonds producing metanilic acid and peri acid whose presence was confirmed by HPLC . Aniline, the desulfonated product from metanilic acid and salicylic acid could also be detected by HPLC . GC-MS analysis of the degradation products confirmed the presence of aniline and revealed the presence of beta-ketoadipic acid . Based on these products a probable pathway has been proposed for the degradation of Navitan Fast Blue S5R by Pseudomonas aeruginosa.

Nat Struct Mol Biol, 2004 Sep, 11(9), 868 - 76 Epub 2004 Aug 15.
How bacterial ADP-ribosylating toxins recognize substrates; Sun J et al.; ExoS and ExoT are bifunctional type III cytotoxins of Pseudomonas aeruginosa that contain an N-terminal RhoGAP domain and a C-terminal ADP-ribosylation domain . Although they share 76% amino acid identity, ExoS and ExoT ADP-ribosylate different substrates . Using protein modeling and site-directed mutagenesis, the regions of ExoS and ExoT that define substrate specificity were determined . Regions B (active site loop), C (ARTT motif) and E (PN loop) on ExoS are necessary and sufficient to recognize ExoS targets, whereas regions B, C and E on ExoT are necessary but not sufficient to recognize ExoT targets, such as the Crk proteins . A specific Crk recognition motif on ExoT was defined as region A (helix alpha1) . The electrostatic properties of regions A, B, C and E define the substrate specificity of ExoS and ExoT and these interactions can explain how other bacterial ADP-ribosylating toxins recognize their unique substrates.

J Biol Chem, 2004 Oct 29, 279(44), 45791 - 802 Epub 2004 Aug 15.
The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa; Wegele R et al.; For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron . After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin . The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta . The gene for a second putative heme oxygenase in P . aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome . Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P . aeruginosa . HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha . The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases . Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product . In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism . Because P . aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.

Microbes Infect, 2004 Aug, 6(10), 875 - 81
Role of alphavbeta5 integrins and vitronectin in Pseudomonas aeruginosa PAK interaction with A549 respiratory cells; Leroy-Dudal J et al.; Bacterial adherence to mammalian cells and their internalization are thought to participate in Pseudomonas aeruginosa pathogenicity . In this study, we explored the role of alpha5beta1 and alphavbeta5 integrins and their natural ligands, fibronectin (Fn) and vitronectin (Vn), in P . aeruginosa interaction with epithelial cells by using the PAK reference bacterial strain, A549 respiratory, and SKOV-3 human ovarian cell lines . The host cell cytoskeleton and cellular tyrosine kinases seem to be solicited during the PAK-respiratory cell interaction: cytochalasin D and genistein decreased the bacterial adherence and internalization . Blocking antibodies to alphavbeta5 integrins were the only antibodies tested to have inhibitory activity against PAK adherence to A549 cells . PAK internalization by A549 and SKOV-3 cells was markedly decreased in the presence of blocking antibodies to Vn and alphavbeta5 integrins . Addition of Vn in excess restored PAK invasion of both A549 and SKOV-3 cells in the presence of anti-Vn antibodies . Immunofluorescence experiments revealed that, in the presence of bacteria, the Vn fibrillar network disappeared, and alphavbeta5 staining was concentrated in sites where adherent bacteria were present . Taken together, these findings suggest that alphavbeta5 integrins, and their natural ligand Vn, are involved in PAK entry into human epithelial cells.

Mol Microbiol, 2004 Aug, 53(4), 1135 - 46
Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI; Gould TA et al.; The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa . Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine . The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP . This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI . Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine . Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150 . This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents.

Mol Microbiol, 2004 Aug, 53(4), 1089 - 98
A novel extracellular phospholipase C of Pseudomonas aeruginosa is required for phospholipid chemotaxis; Barker AP et al.; Pseudomonas aeruginosa and other bacterial pathogens express one or more homologous extracellular phospholipases C (PLC) that are secreted through the inner membrane via the twin arginine translocase (TAT) pathway . Analysis of TAT mutants of P . aeruginosa uncovered a previously unidentified extracellular PLC that is secreted via the Sec pathway (PlcB) . Whereas all presently known PLCs of P . aeruginosa (PlcH, PlcN and PlcB) hydrolyse phosphatidylcholine (PC), only PlcB is active on phosphatidylethanolamine (PE) . plcB candidates were identified based on deductions made from bioinformatics data and extant DNA microarray data . Among these candidates, a gene (PA0026) required for the expression of an extracellular PE-PLC was identified . The protein encoded by PA0026 has limited, but significant similarity, over a short region (approximately 60aa of 328), to a class of zinc-dependent prokaryotic PLCs . A conserved His residue of PlcB (His216) that is required for coordinate binding of zinc in this class of PLCs was mutated . Analysis of this mutant established that the protein encoded by PA0026 is PlcB . Three in-dependent recently published reports indicate that homoserine lactone-mediated quorum sensing regulates the expression of PA0026 (i.e . plcB) . PlcB, but not PlcH or PlcN, is required for directed twitching motility up a gradient of certain kinds of phospholipids . This response shows specificity for the fatty acid moiety of the phospholipid.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 243 - 51
Siderophore production by a marine Pseudomonas aeruginosa and its antagonistic action against phytopathogenic fungi; Manwar AV et al.; A marine isolate of fluorescent Pseudomonas sp . having the ability to produce the pyoverdine type of siderophores under low iron stress (up to 10 microM iron in the succinate medium) was identified as Pseudomonas aeruginosa by using BIOLOG Breathprint and siderotyping . Pyoverdine production was optimum at 0.2% (w/v) succinate, pH 6.0, in an iron-deficient medium . Studies carried out in vitro revealed that purified siderophores and Pseudomonas culture have good antifungal activity against the plant deleterious fungi, namely, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium oxysporum, and Sclerotium rolfsii . Siderophore-based maximum inhibition was observed against A . niger . These in vitro antagonistic actions of marine Pseudomonas against phytopathogens suggest the potential of the organism to serve as a biocontrol agent.

J Biol Chem, 2004 Oct 15, 279(42), 43595 - 603 Epub 2004 Aug 09.
Amino acid residues involved in autophosphorylation and phosphotransfer activities are distinct in nucleoside diphosphate kinase from Mycobacterium tuberculosis; Tiwari S et al.; Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools . We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase . The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of approximately 16.5 kDa . The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate . Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5'-adenosine 3'-phosphate, and adenosine 3'-phosphate 5'-phosphosulfate, had no effect on the activity . Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation . However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution . Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa . Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M . tuberculosis protein in maintaining phosphotransfer ability.

Chest, 2004 Aug, 126(2), 412 - 9
Lung function decline in cystic fibrosis patients and timing for lung transplantation referral; Rosenbluth DB et al.; STUDY OBJECTIVES: To determine risk factors associated with an accelerated decline in lung function in cystic fibrosis (CF), and whether longitudinal changes in FEV(1) would be a better predictor of the need for referral for lung transplantation than any single value for FEV(1.) DESIGN: The rate of decline in pulmonary function was determined by standard linear regression from each patient's calendar year's best percentage of predicted FEV(1) (%FEV(1)) over at least 4 years, and patients were classified into three cohorts based on their rate of decline . Differences between groups in age, weight-for-age z score, gender, genotype, pancreatic status, diabetes, and the presence of various lung microbial isolates were analyzed . A subset of 30 patients referred for lung transplantation were further analyzed, and a prediction model for lung transplantation referral was created using the patient's rate of decline in lung function, the mean waiting time for donor organs, and the average level of lung function of patients prior to lung transplantation . PATIENTS: One hundred fifty-three patients with CF followed up at the Washington University Adult Cystic Fibrosis Center . RESULTS: Younger age, malnutrition, and concurrent infection with both Pseudomonas aeruginosa and Staphylococcus aureus were significant (p < 0.05) risk factors for rapidly declining lung function . Among patients with rapidly declining lung function, referral for lung transplantation would have occurred 8.4 months earlier than actual referral age (p < 0.05) if the prediction model had been used, possibly resulting in additional patient salvage in several cases . CONCLUSIONS: Rate of decline in lung function should be routinely evaluated in patients with CF, and a prediction model utilizing the rate of decline in %FEV(1), and the median regional waiting period for donor lungs for patients with CF may assist in the timing of referral for lung transplantation and more rapidly declining lung function.

Clin Microbiol Infect, 2004 Aug, 10(8), 705 - 8
Effect of chronic Pseudomonas aeruginosa infection on the development of atherosclerosis in a rat model; Turkay C et al.; In order to investigate the possible relationship between atherosclerosis and chronic Pseudomonas aeruginosa infection, 66 Wistar rats were given five separate intratracheal inoculations of either P . aeruginosa or sterile saline at 4-week intervals . The rats were divided into four groups: group 1 was infected with P . aeruginosa and fed a diet containing cholesterol 1% w/v; group 2 was infected with P . aeruginosa and fed a normal diet; group 3 was not infected and was fed a diet containing cholesterol 1% w/v; and group 4 (the control group) was not infected and was fed a normal diet . One month after the final inoculation, the rats were killed humanely; computerised image analysis was used to evaluate sections of the aorta and heart, and the maximal wall thickness of the aorta and coronary artery . The aortic wall thickness was significantly greater for group 1 (329.53 +/- 58.06 microm) compared to groups 2 (190.59 +/- 27.81 microm; p < 0.0001), 3 (262.90 +/- 61.12 microm; p < 0.0004) and 4 (158.00 +/- 30.30 microm; p < 0.0001) . Similarly, the coronary artery wall thickness was significantly greater for group 1 (72.96 +/- 10.67 microm) compared to groups 2 (35.07 +/- 8.53 microm; p < 0.0001), 3 (41.45 +/- 10.22 microm; p < 0.0001) and 4 (32.30 +/- 5.27 microm; p < 0.0001) . These findings strengthen the hypothesis that chronic infection plays a role in the pathogenesis of atherosclerosis.

Biochemistry, 2004 Aug 17, 43(32), 10400 - 13
Real-time probing of membrane transport in living microbial cells using single nanoparticle optics and living cell imaging; Xu XH et al.; Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication . Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR . Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood . In this study, we directly measure real-time change of membrane permeability and pore sizes of P . aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes . The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells . In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity . Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells . Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes . Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.

South Med J, 2004 Jul, 97(7), 705 - 6
Pseudomonas sternoclavicular pyarthrosis; Kaw D et al.; Most cases of Pseudomonas pyarthrosis affecting the sternoclavicular joint have been reported in immunosuppressed intravenous drug users . We report a case of Pseudomonas pyarthrosis in a man who was otherwise immunocompetent, except for his age . A 66-year-old white man presented to the clinic with a 1-month history of right-sided shoulder and arm pain associated with swelling of the upper part of the chest in the region of the right sternoclavicular joint . The chest radiograph revealed opacity in the right superior mediastinum . Computed tomography scan of the chest confirmed a mass in the right sternoclavicular region with associated osteolysis of the clavicular head . A needle biopsy of the mass was negative for malignancy . An open biopsy specimen showed evidence of chronic inflammation without evidence of malignancy, and culture of the tissue grew Pseudomonas aeruginosa . The patient's symptoms improved after extensive incision and drainage of the affected area followed by treatment with antibiotics for 6 weeks.

Niger Postgrad Med J, 2004 Jun, 11(2), 116 - 20
Profile of aerobic bacteria isolated in chronic maxillary sinusitis patients; Aneke EC et al.; OBJECTIVES: To determine the pattern of aerobic bacteria isolated in patients with chronic maxillary sinusitis at the Ear, Nose and Throat Clinic of the University of Nigeria Teaching Hospital, Enugu, and the antibiotics sensitivity pattern of these organisms . METHODS: A prospective hospital-based clinical study . RESULTS: Fifty - four patients with clinical diagnosis of chronic maxillary sinusitis were evaluated . Out of 54 maxillary sinus aspirate specimens studied, 31 yielded bacterial growths and 32 no bacterial growth . The common aerobic bacteria isolated were Staphylococcus aureus (32.3% ), Pseudomonas aeruginosa and Escherichia coli were 16.1% each . Staphylococcus aureus was sensitive to Erythromycin, and the gram-negative organisms to Gentamicin . CONCLUSION: Bacteria isolated in chronic maxillary sinusitis and their sensitivity patterns varied . Bacteriologic study of the antral washings / aspirates should be done in every patient with chronic maxillary sinusitis . Combination chemotherapy that included Erythromycin and Gentamicin was recommended.

Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 711 - 7
Structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa at 2.2 A resolution; Tsai LC; The structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa has been determined by difference Fourier synthesis using phases from the wild-type azurin model . The final crystallographic R value is 0.170 for 17 394 reflections to a resolution of 2.2 A . The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1), a = 57.4, b = 80.4, c = 110.3 A . The four molecules in the asymmetric unit are packed as a dimer of dimers . The nickel metal site of this mutant structure is similar to the zinc metal site in the azurin Asp47 mutant . The site-specific mutation was performed at residue Trp48, which is located in the center of the protein in a highly hydrophobic environment, to investigate its suggested role in the long-range electron-transfer pathway between the disulfide bond on one side of the protein to the Cu centre . The structure around the mutation site Met48 showed no significant change compared with the wild-type structure.

Acta Crystallogr D Biol Crystallogr, 1996 Sep, 52(Pt 5), 950 - 8
Mutant Met121Ala of Pseudomonas aeruginosa Azurin and Its Azide Derivative: Crystal Structures and Spectral Properties; Tsai LC; The crystal structures of the azurin mutant Met121Ala and its azide derivative Met121Ala-azide from Pseudomonas aeruginosa have been determined . The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively . In the Met121Ala mutant, the distance between the copper ion and His117 increases by 0.34 A compared with the wild-type structure . The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74 A . In the Met121Ala-azide structure, the azide anion occupies the cavity created by replacing the Met121 side chain with the smaller methyl group of Ala . The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A . In addition, the copper ion has moved out of the trigonal plane by about 0.26 A towards the azide anion . Thus, the copper site in this structure has a distorted tetrahedral arrangement . The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different . The Met121Ala mutant still maintains the properties of an ordinary type 1 copper site while the Met121Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

Acta Crystallogr D Biol Crystallogr, 1993 Sep, 49(Pt 5), 449 - 57
Structure of Pseudomonas aeruginosai zinc azurin mutant Asn47Asp at 2.4 A resolution; Sjolin L; The Pseudomonas aeruginosa azurin mutant Asn47Asp has been isolated, its spectroscopic and kinetic properties characterized, and the X-ray crystal structure of its zinc derivative determined . While the optical and electron paramagnetic resonance spectra as well as the electron-transfer activity of the mutant are very similar to the wild-type values, the Asn47Asp reduction potential is slightly increased by 20 mV . The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 57.8, b = 81.5 and c = 112.6 A . There are four molecules in the asymmetric unit, packed as a tetramer which consists of two independent dimers . The zinc site of this mutant structure is similar to the wild-type zinc azurin and, in particular, the metal-binding site is almost identical to the site found in the wild-type zinc-azurin structure {Nar, Huber, Messerschmidt, Filippou, Barth, Jaquinod, Kamp & Canters (1992) . Eur . J . Biochem . 205, 1123-1129} . The Asp47 side chain at that mutation site takes on a very similar orientation to Asn47 in the wild-type structure preserving the two hydrogen bonds with the neighbouring Thr113 NH and O(gamma)H . Therefore, the increased reduction potential of the mutant is probably a result of an altered charge distribution close to the metal site.

Acta Crystallogr D Biol Crystallogr, 1994 Jan, 50(Pt 1), 37 - 9
Crystallization and preliminary crystallographic data for the azurin mutant End-121 from Pseudomonas aeruginosa; Strange RW; Pseudomonas aeruginosa azurin has been crystallized from a mutant where residues from Met 121 to Lys128 have been deleted from the protein . The crystals form pale-blue well formed prisms in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 60.79 (5), b = 123.47 (5), c = 187.77 (5) A . The crystals diffract to 3.0 A and there are eight molecules in the asymmetric unit.

Curr Microbiol, 2004 Aug, 49(2), 108 - 14
Isolation, identification, and characterization of a novel, oil-degrading bacterium, Pseudomonas aeruginosa T1; Hasanuzzaman M et al.; A novel, oil-degrading bacterium (strain T1) was isolated from a hot spring in Hokkaido, Japan . It efficiently degrades different types of fats and oils, including edible oil waste . When grown in a mineral salt medium containing 1% triacylglycerol (as salad oil), hydrolysis products were 1,3- and 1,2-diacylglycerols, monoacylglycerol, and free fatty acid . However, these products were almost completely consumed during cultivation at 30 degrees C for 5 days, indicating that extracellular lipase acts randomly at different sn-positions of acylglycerols and that strain T1 has a high capacity to utilize free fatty acids . Secreted lipase activity was induced by salad oil and oleic acid . This strain was a Gram-negative straight rod shaped, aerobic, with a polar flagellum, capable of growing in temperature ranges between 15 degrees C and 55 degrees C . The 16S rRNA gene sequence analysis and DNA-DNA hybridization revealed it as a new strain of Pseudomonas aeruginosa . The type strain was T1.

J Clin Microbiol, 2004 Aug, 42(8), 3857 - 60
V-antigen genotype and phenotype analyses of clinical isolates of Pseudomonas aeruginosa; Allmond LR et al.; The pcrV genotype was analyzed in clinical isolates of Pseudomonas aeruginosa which showed a negative phenotype for secretion of V-antigen PcrV . The suppression of PcrV secretion in these isolates was due not to a lack of the pcrV gene but rather to suppression of PcrV expression.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1233 - 6
Study of the attachment of Pseudomonas aeruginosa on gold and modified gold surfaces using surface plasmon resonance; Jenkins AT et al.; This paper describes how the technique of surface plasmon resonance (SPR) can be utilized to follow (in real time) the attachment of Pseudomonas aeruginosa bacteria on bare gold and gold modified with a self-assembled monolayer (SAM) of mercaptounadecanoic acid . We show that SPR is able to discriminate between the adsorption of live versus dead (thermally shocked) bacteria . Moreover, the SPR distinguishes between the adsorption of wild-type versus mutant bacteria (single gene knockouts), the concentration of the bacterial suspension, and between bacteria adsorbing on SAM-modified and bare gold . SPR is able to measure bacterial adsorption within seconds of the bacterial suspension being introduced . Finally, a qualitative correlation between results from SPR with a crystal violet staining assay for different mutant bacteria was observed.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 207 - 10
Comparative biodegradation examination of Pseudomonas aeruginosa (ATCC 27853) and other oil degraders on hydrocarbon contaminated soil; Szoboszlay S et al.; The soil that we investigated in our experiment was extremly high contaminated with petroleum hydrocarbons . The sample was originated from a former truck factory, next to an oil tank . We tried on this sample three different biodegradation treatments in a soil respirometer . The aim of the experiment was investigate if the clinical strain Pseudomonas aeruginosa ATCC 27853 is able to utilize petroleum hydrocarbons as carbon source . In spite of the relevant literature Pseudomonas aeruginosa ATCC 27853 (clinical isolate, from blood) was proven to be a good oil degrader.

J Antimicrob Chemother, 2004 Sep, 54(3), 684 - 7 Epub 2004 Aug 04.
Phenotypic detection of extended-spectrum and AmpC beta-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test; Shahid M et al.; OBJECTIVES: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories . METHODS: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures . Whole bacterial cells and freeze-thaw preparations, as beta-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E . coli ATCC 25922 . Technical variations of the test included placing the beta-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC beta-lactamases, respectively . RESULTS: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results . However, the best results were given by the spot-inoculation method . In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods . CONCLUSIONS: The method described here is simple and cost-effective . Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.

Protein Expr Purif, 2004 Sep, 37(1), 72 - 82
High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa; Ivanov NV et al.; An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC . The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C . acidovorans . Co-expression of the xdhC gene is required for maximal level of functional expression . Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation . The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression . Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme . This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis . A method is also described to evaluate the suitability of P . aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.

Eur Respir J, 2004 Jul, 24(1), 101 - 6
Clonal strains of Pseudomonas aeruginosa in paediatric and adult cystic fibrosis units; O'Carroll MR et al.; Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious . Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia . All P . aeruginosa isolates were typed using pulsed-field gel electrophoresis . Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded . The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P . aeruginosa . A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains . Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1) . Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function . Treatment requirements were similar in these two groups, as were the rates of multiresistance . In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance . In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance . The clinical significance of clonal strains remains uncertain and requires longitudinal study.

J Biol Chem, 2004 Oct 8, 279(41), 42936 - 44 Epub 2004 Aug 02.
ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor; Sun J et al.; ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains . Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear . The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP . A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases . Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol . A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression . HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP . This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation . A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.

Microbiology, 2004 Aug, 150(Pt 8), 2727 - 37
AlgR functions in algC expression and virulence in Pseudomonas syringae pv . syringae; Penaloza-Vazquez A et al.; Pseudomonas syringae pv . syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees . P . syringae and the human pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid . In P . aeruginosa, the response regulator AlgR (AlgR1) is required for transcription of algC and algD, which encode key enzymes in the alginate biosynthetic pathway . In P . syringae FF5, however, algR is not required for the activation of algD . Interestingly, algR mutants of P . syringae remain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis . In the current study, the algC promoter region was cloned from P . syringae pv . syringae strain FF5, and sequence analysis of the algC promoter indicated the presence of potential binding sites for AlgR and sigma(54), the alternative sigma factor encoded by rpoN . The algC promoter from P . syringae FF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and the PsalgC-uidA transcriptional fusion was used to monitor algC expression in strains FF5.32 (algR mutant of P . syringae FF5) and PG4180.K2 (rpoN mutant of P . syringae pv . glycinea PG4180) . Expression of the PsalgC-uidA fusion was fourfold lower in both the algR and rpoN mutants as compared to respective wild-type strains, indicating that both AlgR and sigma(54) are required for full activation of algC transcription in P . syringae pv . syringae . AlgR from P . syringae was successfully overproduced in Escherichia coli as a C-terminal translational fusion to the maltose-binding protein (MBP) . Gel shift experiments indicated that MBP-AlgR binds strongly to the algC promoter region . Biological assays demonstrated that the algR mutant was significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type strain . These results, along with the gene expression studies, indicate that AlgR has a positive role in the activation of algC in P . syringae and contributes to both virulence and epiphytic fitness . Furthermore, the symptoms observed with wild-type P . syringae FF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy of algR is required for systemic movement.

Microbiology, 2004 Aug, 150(Pt 8), 2653 - 61
Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa; Shan Z et al.; Mu transposition complexes were used for transposon mutagenesis of Pseudomonas aeruginosa strain PA68 . Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon in vitro, and introduced into Pseudomonas aeruginosa by electroporation . Eight mutants deficient in twitching motility were isolated . Southern blotting confirmed that the insertions had occurred as single events . DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, PAO171, PA1822, PAO413, PA4959, PA4551 and PA5040 . Four of these have previously been proven to be needed for twitching motility, whereas the PA1822 and PA0171 genes have not previously been shown to be required for twitching motility . The twitching-motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the defect in the PA0171 mutant was fully complemented when PA0171 was provided . A PA0171 mutant and a PA1822 mutant were constructed by gene replacement in the P . aeruginosa PAO1 strain . These mutants were deficient in twitching motility, showing that both the PA1822 and the PA0171 gene are involved in twitching motility.

Am J Ophthalmol, 2004 Aug, 138(2), 226 - 30
A laboratory evaluation of antibiotic therapy for ciprofloxacin-resistant Pseudomonas aeruginosa; Rhee MK et al.; PURPOSE: The emergence of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) has created a new therapeutic challenge in ophthalmology . We evaluated ophthalmic antibiotics in vitro and in a rabbit keratitis model to determine effective therapy . DESIGN: Experimental laboratory investigation . METHODS: The susceptibilities of 12 CRPA isolates were determined in vitro for amikacin, ceftazidime, tobramycin, polymyxin B, gentamicin, ticarcillin, and the fluoroquinolones (that is, ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin) using E-tests and National Committee of Clinical Laboratory Standards . A rabbit keratitis model was used to determine the reduction in colony counts of CRPA and ciprofloxacin-susceptible P . aeruginosa (CSPA) isolates following topical treatment with polymyxin B/trimethoprim, tobramycin (14 mg/ml), ceftazidime (50 mg/ml), and ciprofloxacin (3 mg/ml) . RESULTS: For 12 CRPA isolates, the susceptibilities and median minimum inhibitory concentrations ({MIC}microg/ml) were as follows: amikacin (92%, 14.0), ceftazidime (75%, 4.0), tobramycin (67%, 1.75), polymyxin B (42%, 7.0), gentamicin (17%, 7.0), ticarcillin (0%, >32.0), and all fluoroquinolones (0%, >32.0) . While no antibiotic regimen reduced colony counts in the time frame of the animal model for CRPA, ciprofloxacin alone demonstrated a significant decrease in colony counts for CSPA . Comparing CRPA with CSPA, both tobramycin and ciprofloxacin demonstrated a significant decrease in colony counts for CSPA . CONCLUSION: Our laboratory studies suggest that current antibiotics may be suboptimal in treating CRPA keratitis . Until new antibiotics are available, combination therapy such as fortified tobramycin and ticarcillin, and others may prove effective in aggressive topical long-term therapy.

Lancet Infect Dis, 2004 Aug, 4(8), 519 - 27
Does combination antimicrobial therapy reduce mortality in Gram-negative bacteraemia? A meta-analysis; Safdar N et al.; The use of combination antimicrobial therapy for bacteraemia caused by Gram-negative bacilli is controversial . We did a meta-analysis of published studies to determine whether a combination of two or more antimicrobials reduces mortality in patients with Gram-negative bacteraemia . Criteria for inclusion were: analytic studies of patients with documented Gram-negative bacteraemia that included patients receiving a single antibiotic (monotherapy) and patients receiving two or more antibiotics (combination therapy) . Data on mortality (outcome) had to be provided . A pooled odds ratio was calculated with the random effects model of DerSimonian and Laird . Assessment of heterogeneity was done with the Breslow-Day test and reasons for heterogeneity were explored . 17 studies met the inclusion criteria, five prospective cohort studies, two prospective randomised trials, and ten retrospective cohort studies . Most studies used beta-lactams or aminoglycosides alone and in combination . The summary odds ratio was 0.96 (95% CI 0.70-1.32), indicating no mortality benefit with combination therapy . Subgroup analyses adjusting for year of publication, study design, and severity of illness did not change the results . Considerable heterogeneity was present in the main analyses . Analysis of only Pseudomonas aeruginosa bacteraemias showed a significant mortality benefit (OR 0.50, 95% CI 0.30-0.79) . Our analysis does not support the routine use of combination antimicrobial therapy for Gram-negative bacteraemia, beyond settings where infection by P aeruginosa is strongly suspected or more than one drug would be desirable to assure in-vitro efficacy.

J Mol Biol, 2004 Aug 13, 341(3), 839 - 52
The crystal structure of ZapA and its modulation of FtsZ polymerisation; Low HH et al.; FtsZ is part of a mid-cell cytokinetic structure termed the Z-ring that recruits a hierarchy of fission related proteins early in the bacterial cell cycle . The widely conserved ZapA has been shown to interact with FtsZ, to drive its polymerisation and to promote FtsZ filament bundling thereby contributing to the spatio-temporal tuning of the Z-ring . Here, we show the crystal structure of ZapA (11.6 kDa) from Pseudomonas aeruginosa at 2.8 A resolution . The electron density reveals two dimers associating via an extensive C-terminal coiled-coil protrusion to form an elongated anti-parallel tetramer . In solution, ZapA exists in a dimer-tetramer equilibrium that is strongly correlated with concentration . An increase in concentration promotes formation of the higher oligomeric state . The dimer is postulated to be the predominant physiological species although the tetramer could become significant if, as FtsZ is integrated into the Z-ring and is cross-linked, the local concentration of the dimer becomes sufficiently high . We also show that ZapA binds FtsZ with an approximate 1:1 molar stoichiometry and that this interaction provokes dramatic FtsZ polymerisation and inter-filament association as well as yielding filaments, single or bundled, more stable and resistant to collapse . Whilst in vitro dynamics of FtsZ are well characterised, its in vivo arrangement within the ultra-structural architecture of the Z-ring is yet to be determined despite being fundamental to cell division . The ZapA dimer has single 2-fold symmetry whilst the bipolar tetramer displays triple 2-fold symmetry . Given the symmetry of these ZapA oligomers and the polar nature of FtsZ filaments, the structure of ZapA carries novel implications for the inherent architecture of the Z-ring in vivo.

Eur J Intern Med, 2004 Jul, 15(4), 238 - 241
Phenotype and genotype of French cystic fibrosis patients with long survival and follow-up; Badet F et al.; Background: The aim of this study was to assess the clinical features and the genotype characteristics of French patients diagnosed with cystic fibrosis (CF) before their fifth year and who were still alive after 30 years . It is the first descriptive study of 114 CF patients with long survival and follow-up . We compared this subgroup of French CF patients with the overall French CF population and with French adult (> 18 years) CF patients regardless of their age at diagnosis . Methods: Data were obtained from the French CF registry . Results: The 67 men and 47 women studied were 30-59 years old . Some 56% of the patients had DeltaF508 homozygous genotype, 90% had a pancreatic insufficiency, and 81% were colonized with Pseudomonas aeruginosa . The mean body mass index (BMI) was 19.5 for both female and male patients . Mean forced expiratory volume in 1 s was 46% (S.D . 29.2) of the predicted value for men and 53% (S.D . 20.6) for women . Eleven patients underwent a lung transplantation . Conclusions: The data on the patients with long survival and long follow-up were very similar to the data of the overall CF adult population in terms of clinical status . Therefore, criteria such as DeltaF508 homozygous genotype, pancreatic insufficiency, and P . aeruginosa colonization are not sufficient to serve as prognostic criteria for life expectancy in CF patients.

Int J Antimicrob Agents, 2004 Aug, 24(2), 173 - 7
Comparative pharmacodynamics of the new fluoroquinolone ABT492 and ciprofloxacin with Escherichia coli and Pseudomonas aeruginosa in an in vitro dynamic model; Zinner SH et al.; The killing kinetics of Escherichia coli and Pseudomonas aeruginosa were compared when exposed to ABT492 and ciprofloxacin . E . coli ATCC 25922 and a clinical isolate of P . aeruginosa 4226 were exposed to ABT492 (single dose) and ciprofloxacin (two 12 h doses) at the ratios of area under the curve (AUC) to MIC varying from 60 to 480 h and at clinically achievable AUC/MIC ratios of ABT492 (1,740 and 140 h, respectively) and ciprofloxacin (2,200 and 120 h, respectively) that correspond to a 400 mg dose of ABT492 and two 500 mg doses of ciprofloxacin . In addition, a double dose of ABT492 (800 mg; AUC/MIC 280 h) and two 12 h doses of ABT492 (2 x 400 mg) were used with P . aeruginosa . Maximal reductions in the starting inoculum of E . coli and P . aeruginosa were greater with ABT492 than with ciprofloxacin at a given AUC/MIC ratio (60-480 h), whereas the times to regrowth were shorter with ABT492 . A specific AUC/MIC relationship of the antimicrobial effect was inherent in each quinolone-pathogen pair . With both E . coli and P . aeruginosa, AUC/MIC plots of the area between the control growth and the time-kill curves (I(E)) were steeper for ciprofloxacin than ABT492 and they were species-independent . The effect of ABT492 on E . coli at the clinically achievable AUC/MIC ratio (1740h) was more pronounced than the respective AUC/MIC of ciprofloxacin (2,200 h) . With P . aeruginosa, a 140 h AUC/MIC of ABT492 (400 mg as a single dose) provided 1.8-fold less effect than a 120 h AUC/MIC of ciprofloxacin (2 x 500 mg) . However, two 12 h doses of ABT492 (AUC/MIC 2 x 140 h) but not a double single dose (800 mg) were more efficient than ciprofloxacin . These findings predict comparable efficacies of clinically achievable AUC/MICs of ABT492 and ciprofloxacin against E . coli (q.d . versus b.i.d . quinolone dosing) and P . aeruginosa at b.i.d . but not at q.d . ABT492.

Lett Appl Microbiol, 2004, 39(3), 274 - 7
Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa; Barnini S et al.; AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa . METHODS AND RESULTS: Ps . aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions . The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining . CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps . aeruginosa strains . SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.

Scand J Infect Dis, 2004, 36(5), 342 - 9
PCR identification and automated ribotyping of Pseudomonas aeruginosa clinical isolates from intensive care patients; Sarwari A et al.; Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant . P . aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period . Antimicrobial susceptibility of the 56 P . aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p < 0.01) . In addition, 55% of the P . aeruginosa clinical isolates were resistant to 4 or more antibiotics . Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p < 0.05) . PCR (using P . aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P . aeruginosa isolates tested . Automated ribotyping was used to investigate the clonal diversity of the 56 P . aeruginosa isolates . Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one . The P . aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time . The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P . aeruginosa infections.

Phytother Res, 2004 Jun, 18(6), 468 - 70
Biological screening of Nigella damascena for antimicrobial and molluscicidal activities; Fico G et al.; The essential oil, various extracts at different polarity, fractions, and pure compounds obtained from Nigella damascena plants and seeds were screened for biological activity . Antimicrobial tests showed the essential oil to be active only against Gram positive bacteria; among the extracts, the BuOH was active against Pseudomonas aeruginosa and Staphylococcus aureus . Molluscicidal activity was absent.

Rev Hosp Clin Fac Med Sao Paulo, 2004 May-Jun, 59(3), 99 - 103 Epub 2004 Jul 28.
Effect of clarithromycin on the cell profile of bronchoalveolar lavage fluid in mice with neutrophil-predominant lung disease; Pinto LA et al.; OBJECTIVE: Macrolide antibiotics have anti-inflammatory properties in lung diseases . The aim of this study was to investigate the effect of clarithromycin in pulmonary cellular inflammatory response in mice . METHOD: Eight adult Swiss mice were studied . All animals received an intranasal challenge (80 micro L) with dead Pseudomonas aeruginosa (1.0 x 10(12) CFU/mL) . Bronchoalveolar lavage was performed 2 days later, with total cell count and differential cell analysis . The study group (n = 4) received clarithromycin treatment (50 mg/kg/day, intraperitoneal) for 5 days . Treatment was initiated 2 days before intranasal challenge . RESULTS: There was no significant difference in total cell count between the groups (mean: 2.0 x 10(6) and 1.3 x 10(6), respectively) . In both groups, there was a predominance of neutrophils . However, the study group had a higher percentage of lymphocytes in the bronchoalveolar lavage than the control group (median of 19% vs 2.5%, P =.029) . CONCLUSION: Clarithromycin alters the cytological pattern of bronchoalveolar lavage of Swiss mice with neutrophil pulmonary inflammation, significantly increasing the percentage of lymphocytes.

Am J Physiol Lung Cell Mol Physiol, 2004 Dec, 287(6), L1199 - 206 Epub 2004 Jul 30.
Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells; Arita Y et al.; Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality . Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process . A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence . Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections . IL-8 expression was also increased in cells exposed to both hyperoxia and PA . Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA . These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species . Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.

Immunol Cell Biol, 2004 Aug, 82(4), 383 - 92
Pseudomonas aeruginosa-induced production of free radicals by IFNgamma plus TNFalpha-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?
De Assis MC, Saliba AM, Vidipo LA, De Salles JB, Plotkowski MC.
We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNgamma plus TNFalpha to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed . In this study we investigated the fate of IFNgamma plus TNFalpha-activated HUVEC that have harboured IC bacteria for up to 24 h . At 10 h, the endothelial cell killing of P . aeruginosa isolates exceeded 90% . IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC . P . aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH) . Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability . Treatment of cells with the antioxidant alpha-lipoic acid significantly increased the survival of infected cells . Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells . Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.

Avian Dis, 2004 Apr-Jun, 48(2), 238 - 43
Effect of BioSentry 904 and ethylenediaminetetraacetic acid-tris disinfecting during incubation of chicken eggs on microbial levels and productivity of poultry; Walker SE et al.; Proper sanitation practices and the use of efficacious disinfectants in a hatchery have an effect on chick quality . Aerosol bacterial counts, egg moisture loss, hatchability, chick quality, and broiler productivity were evaluated when egg surfaces were contaminated by immersion of each egg into a broth medium containing a field isolate of Pseudomonas aeruginosa and incubated with exposure to one of three disinfectant treatments administered by fine spray: distilled water, BioSentry 904 (904), and a 1:1 ratio of 904 and ethylenediaminetetraacetic acid (EDTA)-Tris . The aerosol bacteria levels were statistically greater on day 21 of incubation in the group treated with distilled water than in those receiving disinfectants . Overall hatch of fertile eggs and egg moisture loss were comparable among all three treatments . At 1 day of age, the chicks incubated with 904 had a statistically lower yolk sac contamination rate than those incubated with 904+EDTA-Tris or distilled water . The 2-wk mortality rates, body weights, feed conversion ratios, yolk sac weights, and yolk sac contamination rates were all similar among the three treatments.

Ned Tijdschr Geneeskd, 2004 Jul 3, 148(27), 1351 - 4
{Three patients with complications following piercing of the auricular cartilage}; Janssen K et al.; In three patients, a 21-year-old man and females aged 16 and 18 years, piercing through the auricular cartilage was followed by an infection . Treatment left behind a residual deformity, for which a reconstruction was carried out with a satisfactory result . The risk of infection following piercing through the avascular cartilage of the helix and tragus of the pinna is greater than following piercing of non-cartilaginous tissue such as the ear lobe . (Peri)chondritis leads to chondral necrosis and subsequent deformities . It is important to recognise the early features of perichondritis, which include local heat, erythema and pain, before swelling appears . Treatment should focus on eradicating Pseudomonas aeruginosa and Staphylococcus aureus . Surgical intervention is required at the earliest sign of an abscess . Reconstruction for a post-piercing deformity can be considered at a later stage.

Arch Microbiol, 2004 Sep, 182(1), 7 - 17 Epub 2004 Jul 28.
Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs; Brichta DM et al.; Pseudomonas aeruginosa PAO1 was shown to contain three pyrC sequences . Two of these genes, designated pyrC (PA3527) and pyrC2 (PA5541), encode polypeptides with dihydroorotase (DHOase) activity, while the third, pyrC' (PA0401), encodes a DHOase-like polypeptide that lacks DHOase activity, but is necessary for the structure and function of ATCase . Both pyrC and pyrC2 were cloned and complemented an Escherichia coli pyrC mutant . In addition, pyrC and pyrC2 were individually inactivated in P . aeruginosa by homologous exchange with a mutated allele of each . The resulting mutant strains were prototrophic . A pyrC, pyrC2 double mutant was also constructed, and this strain had an absolute requirement for pyrimidines . The transcriptional activity of pyrC and pyrC2 was measured using lacZ promoter fusions . While pyrC was found to be constitutively expressed, pyrC2 was expressed only in the pyrC mutant background . An in vitro transcriptional/translational system was used to estimate the size of the pyrC2 gene product . The expressed polypeptide was approximately 47 kDa, which is in keeping with the theoretical molecular mass of 48 kDa, making it the largest prokaryotic DHOase polypeptide identified to date . To our knowledge, this is the first report of a true DHOase mutant in P . aeruginosa and also the first confirmation that pyrC2 encodes a polypeptide with DHOase activity.

Invest Ophthalmol Vis Sci, 2004 Aug, 45(8), 2569 - 76
Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells; Zhang J et al.; PURPOSE: To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs) . METHODS: Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001 . The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody . Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis . Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes . Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP . RESULTS: P . aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation . Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P . aeruginosa-infected HUCL cells or primary culture of HCECs . Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis . CONCLUSIONS: Bacterial infection of HCECs induces EGFR transactivation through HB-EGF ectodomain shedding . EGFR and its downstream ERK and PI3K signaling pathways play a role in preventing epithelial apoptosis in the early stage of bacterial infection.

Br J Sports Med . 2004 Aug;38(4):E11.
An unusual presentation of immersion foot; Macgregor DM; We report a case of "green foot" in a child with a plaster cast applied for a fractured metatarsal who subsequently re-presented with circulatory compromise . The foot was green and smelly and profuse Pseudomonas aeruginosa was cultured . The infection cleared with simple exposure to air . Perhaps this diagnosis should be considered in patients presenting with circulatory compromise in a cast as severe infection can result in amputation.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 3086 - 92
Doripenem versus Pseudomonas aeruginosa in vitro: activity against characterized isolates, mutants, and transconjugants and resistance selection potential; Mushtaq S et al.; Doripenem is a broad-spectrum parenteral carbapenem under clinical development in Japan and North America . Its activities against (i) Pseudomonas aeruginosa isolates with graded levels of intrinsic efflux-type resistance, (ii) mutants with various combinations of AmpC and OprD expression, (iii) PU21 transconjugants with class A and D beta-lactamases, and (iv) P . aeruginosa isolates with metallo-beta-lactamases were tested by the agar dilution method of the National Committee for Clinical Laboratory Standards . Selection of resistant P . aeruginosa mutants was investigated in single- and multistep procedures . Doripenem MICs for isolates without acquired resistance mostly were 0.12 to 0.5 microg/ml, whereas meropenem MICs were 0.25 to 0.5 microg/ml and imipenem MICs were 1 to 2 microg/ml . The MICs of doripenem, meropenem, ertapenem, and noncarbapenems for isolates with increased efflux-type resistance were elevated, whereas the MICs of imipenem were less affected . The MICs of doripenem were increased by the loss of OprD but not by derepression of AmpC; nevertheless, and as with other carbapenems, the impermeability-determined resistance caused by the loss of OprD corequired AmpC activity and was lost in OprD- mutants also lacking AmpC . The TEM, PSE, PER, and OXA enzymes did not significantly protect P . aeruginosa PU21 against the activity of doripenem, whereas MICs of > or =16 microg/ml were seen for clinical isolates with VIM and IMP metallo-beta-lactamases . Resistant mutants seemed to be harder to select with doripenem than with other carbapenems (or noncarbapenems), and the fold increases in the MICs were smaller for the resistant mutants . Single-step doripenem mutants were mostly resistant only to carbapenems and had lost OprD; multistep mutants had broader resistance, implying the presence of additional mechanisms, putatively including up-regulated efflux . Most mutants selected with aminoglycosides and quinolones had little or no cross-resistance to carbapenems, including doripenem.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2918 - 23
Therapeutic efficacy of "nubiotics" against burn wound infection by Pseudomonas aeruginosa; Dale RM et al.; "Nubiotics" are a novel class of proprietary protonated nucleic acid-based drugs shown to have potent in vitro antibacterial activities against a number of gram-positive and gram-negative bacteria . These nubiotics are evaluated here for their in vivo therapeutic efficacy for the treatment of burn wound infection caused by Pseudomonas aeruginosa . To achieve this, a burn wound infection model was established in mice by using a highly pathogenic burn wound clinical isolate of P . aeruginosa . Lethal doses of the bacteria were determined for two routes of infection (subcutaneous and topical), representing systemic and local forms of infection, respectively . Using this infection model, treatment with nubiotics by various routes of drug administration was evaluated and optimized . A total of 12 nubiotics and their analogues were tested and of these, Nu-2, -3, -4, and -5 were found to be extremely efficacious in the postexposure treatment of burn wound infection (60 to 100% survival rates versus 0% for untreated control {P < 0.05}) . These nubiotics were effective when given either systemically by intravenous and/or subcutaneous administration or given locally to the affected site in the skin by topical application . Treatment by these two routes resulted in almost 100% survival rates and complete eradication of the bacteria from infection sites in the livers, spleens, and blood . These nubiotics were found to be as effective as intravenously administered ciprofloxacin, a potent and broad-spectrum fluoroquinolone . These results suggest that nubiotics may be a promising and effective approach for the treatment of burn wound infection caused by P . aeruginosa.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1467 - 9 Epub 2004 Jul 21.
Crystallization and preliminary X-ray analysis of the outer membrane pyoverdine receptor FpvA from Pseudomonas aeruginosa; Cobessi D et al.; FpvA, the pyoverdine outer-membrane receptor from Pseudomonas aeruginosa, is involved in iron uptake when bacteria grow under iron limitation . Crystals of the in vivo pyoverdine-loaded FpvA were obtained under several crystallization conditions using different detergents . A native data set was collected at 3.6 A resolution and a three-wavelength MAD data set was collected at 3.6 A resolution using crystals of selenomethionine-substituted protein . The crystals grew under similar conditions and both belong to space group C2, but have different unit-cell parameters.

Infect Immun, 2004 Aug, 72(8), 4741 - 50
The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes; Goure J et al.; Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS) . Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P . aeruginosa strains with macrophages and TTSS-dependent hemolysis . Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin . Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation . Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts . Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs . Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV . Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.

Infect Immun, 2004 Aug, 72(8), 4561 - 9
Toll-like receptor 2 represses nonpilus adhesin-induced signaling in acute infections with the Pseudomonas aeruginosa pilA mutant; Lorenz E et al.; Expression of pili and associated proteins is an important means of host invasion by bacterial pathogens . Recent evidence has suggested that the binding of Pseudomonas aeruginosa through nonpilus adhesins may also be important in respiratory diseases, since adhesins bind mucins . Using wild-type C57BL/6 and TLR2KO mice, we compared the induction levels of the host response to P . aeruginosa that either expressed pili or lacked pilus expression due to a mutation in the structural gene pilA . In C57BL/6 mice, deletion of pili led to a decreased immune response, evidenced by a lower secretion of cytokines and a lack of neutrophil chemotaxis . By contrast, the P . aeruginosa pilA mutant induced a hyperresponsive phenotype in TLR2KO mice . TLR2KO mice showed an increased number of neutrophils in lavage fluid compared to the levels seen when either mouse strain was exposed to wild-type P . aeruginosa . Further analysis indicated that the increased neutrophil influx was associated with an increased expression of calgranulins, possibly through an induction of Toll-like receptor 4 (TLR4) expression . The hyperresponsive phenotype of TLR2KO mice exposed to the P . aeruginosa pilA mutant was associated with TLR4 induction and indicated that nonpilus adhesin-induced signaling was repressed by TLR2 function and, if not blocked by the host, could induce airway hyperresponsiveness.

Chem Biol, 2004 Jul, 11(7), 971 - 80
Characterization and genetic manipulation of peptide synthetases in Pseudomonas aeruginosa PAO1 in order to generate novel pyoverdines; Ackerley DF et al.; PvdD, a nonribosomal peptide synthetase (NRPS) of Pseudomonas aeruginosa PAO1, incorporates two L-threonines into the siderophore pyoverdine . A pvdD mutant did not synthesize pyoverdine and lacked a high Mr iron-regulated cytoplasmic protein (IRCP) . Analysis of other IRCPs and the P . aeruginosa genome enabled the remaining pyoverdine NRPSs to be identified . The pvdD mutation could be complemented in trans, enabling design of plasmid-based systems for the generation of novel pyoverdines . Introduction of a truncated pvdD gene resulted in attenuated forms of pyoverdine, and introduction of L-threonine-incorporating NRPSs from other organisms restored pyoverdine production to mutant cells . This is the first successful rational in vivo modification of NRPS modules outside of Bacillus subtilis . The systems employed did not allow incorporation of other residues into pyoverdine, indicating that there are multiple elements contributing toward substrate specificity in NRPSs.

Clin Otolaryngol, 2004 Aug, 29(4), 321 - 3
Emergence of ciprofloxacin-resistant pseudomonas in chronic suppurative otitis media; Jang CH et al.; The most frequently isolated organism in chronic suppurative otitis media (CSOM) is Pseudomonas aeruginosa . Ototopical ciprofloxacin has proven effectiveness against P . aeruginosa . The purpose of this study is to evaluate the patients with recurrent otorrhoea caused by CSOM that was unresponsive to topical ciprofloxacin . Eighty-eight patients (18-77 years of age) with otorrhoea due to CSOM were reviewed retrospectively . All of them were initially treated with ciprofloxacin eardrops but the otorrhoea failed to resolve . Bacteriological specimens were processed and identified with standard cultures . In vitro antimicrobial susceptibility of these bacterial isolates was assessed by an agar disc diffusion method . Isolates were tested against 16 antibiotics . Ciprofloxacin-resistant P . aeruginosa were isolated in all cases . Imipenem was the most sensitive antibiotic agent with an overall susceptibility rate of 96.5%, followed by amikacin (55.6%), piperacillin/tazobactam (37.5%) and ceftazidime (31.8%) . In our series, ciprofloxacin-resistant P . aeruginosa is increasing recently . Continuous surveillance is necessary to monitor antimicrobial resistance and to guide antibacterial therapy.

Thromb Haemost, 2004 Aug, 92(2), 281 - 7
Heparin binding protein is increased in chronic leg ulcer fluid and released from granulocytes by secreted products of Pseudomonas aeruginosa; Lundqvist K et al.; Recently it was demonstrated by Gautam, et al . that release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, induced endothelial hyperpermeability and neutrophil efflux . Here, we show that chronic leg ulcer fluid, in contrast to wound fluid from acute wounds, contains highly increased levels of HBP . Immunohistochemistry demonstrated the presence of HBP in chronic ulcer tissues . Furthermore, secreted products of Pseudomonas aeruginosa were found to induce release of HBP from human neutrophils . Our data suggest a possible link between bacterial presence and HBP-release in chronic ulcers . Thus, targeting HBP offers an interesting option for reduction of endothelial barrier dysfunction in chronic ulcers.

J Small Anim Pract, 2004 Jul, 45(7), 357 - 61
Successful intravenous human immunoglobulin treatment of drug-induced Stevens-Johnson syndrome in a dog; Nuttall TJ et al.; A two-year-old, male English springer spaniel developed severe mucocutaneous ulceration following treatment with trimethoprim-potentiated sulphadiazine . The clinical signs were consistent with Stevens-Johnson syndrome (SJS): there were no target or arciform lesions typical of erythema multiforme minor and major; more than one mucosal surface was affected; epidermal detachment affected less than 10 per cent of the body surface area; and there was a clear history of drug exposure . Systemic signs included a severe hepatopathy, dyspnoea, pyrexia and cachexia . Glucocorticoid therapy was associated with secondary infection by Pseudomonas aeruginosa . The clinical signs rapidly resolved following a single intravenous infusion of 0.51 g/kg human immunoglobulin (ivHIG) as a 5 per cent solution . By blocking FAS/FAS ligand (CD95/CD95L) interactions, ivHIG is thought to prevent keratinocyte apoptosis . It also binds to immunoglobulin G Fc receptors, inhibiting cell activation and cytokine synthesis, neutralises autoantibodies and immune complexes, blocks complement activity, is antimicrobial and increases colloid osmotic pressure . To the authors' knowledge, this is the first report of successful treatment of canine SJS using ivHIG, although it has been used to treat erythema multiforme in a cat and toxic epidermal necrolysis in a dog.

Rev Biol Trop, 2000 Dec, 48 Suppl 1, 199 - 206
{Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)}; Morales T et al.; Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) . The minimal inhibitory concentration (MIC) and toxic activity of extract were determined . Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E . coli, while CHCl3 fraction inhibited E . coli and S . aureus growth . The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria . Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E . coli and S . aureus . In Sf2 EtOAc/MeOH, MIC was moderate for S . aureus (MIC > 256 g/ml) . F4 CHCl3/MeOH produced a high inhibition in S . aureus (MIC = 0.125 g/ml) and for E . coli (MIC > 16 g/ml) . F10 CHCl3/MeOH showed a moderate activity against S . aureus (MIC > 128 g/ml) and low activity against E . coli (MIC = 512 g/ml) . F10 CHCL3/MeOH did no present toxic activity against Artemia salina . The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml . LC50 in both cases was 548.4 and 243.4 microg/ml respectively . Secondary metabolites of medium polarity obtained from A . fistularis have a wide spectrum of anti bacterial activity . Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.

J Immunol, 2004 Aug 1, 173(3), 2031 - 40
Different domains of Pseudomonas aeruginosa exoenzyme S activate distinct TLRs; Epelman S et al.; Some bacterial products possess multiple immunomodulatory effects and thereby complex mechanisms of action . Exogenous administration of an important Pseudomonas aeruginosa virulence factor, exoenzyme S (ExoS) induces potent monocyte activation leading to the production of numerous proinflammatory cytokines and chemokines . However, ExoS is also injected directly into target cells, inducing cell death through its multiple effects on signaling pathways . This study addresses the mechanisms used by ExoS to induce monocyte activation . Exogenous administration resulted in specific internalization of ExoS via an actin-dependent mechanism . However, ExoS-mediated cellular activation was not inhibited if internalization was blocked, suggesting an alternate mechanism of activation . ExoS bound a saturable and specific receptor on the surface of monocytic cells . ExoS, LPS, and peptidoglycan were all able to induce tolerance and cross-tolerance to each other suggesting the involvement of a TLR in ExoS-recognition . ExoS activated monocytic cells via a myeloid differentiation Ag-88 pathway, using both TLR2 and the TLR4/MD-2/CD14 complex for cellular activation . Interestingly, the TLR2 activity was localized to the C-terminal domain of ExoS while the TLR4 activity was localized to the N-terminal domain . This study provides the first example of how different domains of the same molecule activate two TLRs, and also highlights the possible overlapping pathophysiological processes possessed by microbial toxins.

J Struct Funct Genomics, 2004, 5(3), 179 - 94
Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells; Perez A et al.; To test the hypothesis that the excess inflammatory response in cystic fibrosis airway epithelial cells is the result of differential activation of genes in response to a laboratory strain of Pseudomonas aeruginosa (PAO1), a 48-h time course of genes expressed following PAO1 stimulation (10(9) CFU for 1 h) was studied in two pairs of airway epithelial cells: 9/HTEo- and 16HBE14o-, each with a matched normal and CF phenotype pair . cRNA was hybridized to Affymetrix HG-U95Av2 chips and pairwise comparisons against zero time (no PAO1) were calculated for each time point . PAO1 elicited profound changes in both cell pairs: for 9/HTEo-, 144 genes changed significantly in the normal pair, and 116 for the CF pair; for the 16HBE14o- pair, 57 genes changed significantly for the normal pair and 53 for the CF pair . Changes were much greater in the 9/HTEo- than in the 16HBE14o- pair, but basal levels of expression of inflammatory genes are higher in the 16HBE14o- pair, so 16HBE14o- was used mainly to corroborate the results of 9/HTEo- . Clustering analysis indicated that the pattern of gene expression is similar in the CF cells and their normal counterparts . However, there were substantial quantitative differences in gene expression . Thus, the difference between CF and normal resides in the magnitude, not the pattern, of the changes.

J Bacteriol, 2004 Aug, 186(15), 4903 - 9
Ribosomal protein S1 specifically binds to the 5' untranslated region of the Pseudomonas aeruginosa stationary-phase sigma factor rpoS mRNA in the logarithmic phase of growth; Sevo M et al.; The rpoS gene encodes the stationary-phase sigma factor (RpoS or sigma(s)), which was identified in several gram-negative bacteria as a central regulator controlling the expression of genes involved in cell survival in response to cessation of growth (stationary phase) and providing cross-protection against various stresses . In Pseudomonas aeruginosa, the levels of sigma(s) increase dramatically at the onset of the stationary phase and are regulated at the transcriptional and posttranscriptional levels . The P . aeruginosa rpoS gene is transcribed as a monocistronic rpoS mRNA transcript comprised of an unusually long 373-bp 5' untranslated region (5' UTR) . In this study, the 5' UTR and total protein extracts from P . aeruginosa logarithmic and stationary phases of growth were used in order to investigate the protein-RNA interactions that may modulate the translational process . It was observed that a 69-kDa protein, which corresponded to ribosomal protein S1, preferentially binds the 5' UTR of the rpoS mRNA in the logarithmic phase and not in the stationary phase . This is the first report of a protein-rpoS mRNA 5' UTR interaction in P . aeruginosa, and the possible involvement of protein S1 in translation regulation of rpoS is discussed.

J Spinal Disord Tech, 2004 Apr, 17(2), 112 - 4
Histopathologic effects of discitis on neural tissues: an experimental study; Yucesoy K et al.; To determine the cause of neurologic symptoms and signs seen in discitis, the neural histopathologic effects of discitis were investigated in an experimental study carried out on rats . Groups of seven rats each had their intervertebral discs inoculated with either Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, or a control solution . Histopathologic examinations of the spinal cord and nerve roots were performed after 3 weeks . On histopathologic examination, vacuolar myelopathy in the spinal cord and vacuolar neuropathy within the nerve roots near the junction with the spinal cord were found . The severity and form of vacuolar myelopathy varied according to the bacteria used for inoculation . The myelopathy and neuropathy seen in this rat model of bacterial discitis might be the result of an immunologic mechanism and could be responsible for the neurologic signs and symptoms of discitis in patients.

Biometals, 2004 Aug, 17(4), 409 - 14
The stereoisomers of pyochelin, a siderophore of Pseudomonas aeruginosa; Schlegel K et al.; The Pseudomonas aeruginosa siderophore pyochelin is obtained from the bacterial culture medium as a mixture of two epimers . Chromatically isolated pure stereoisomers equilibrate readily in most solvents . Experiments will be reported which allow to isolate one of the isomers in pure form and which shed some additional light on the epimerization reaction.

Intensive Care Med, 2004 Oct, 30(10), 1964 - 8 Epub 2004 Jul 15.
Faucets as a reservoir of endemic Pseudomonas aeruginosa colonization/infections in intensive care units; Blanc DS et al.; OBJECTIVE: To evaluate the role of faucets as a reservoir for Pseudomonas aeruginosa colonization/infection of patients hospitalized in intensive care units (ICUs) . DESIGN: Prospective epidemiological investigation performed during a nonepidemic period of 1 year . The inner part of the ICU faucets were swabbed for P . aeruginosa . Data were recorded on all patients with at least one culture of a clinical specimens positive for P . aeruginosa . Pulsed-field gel electrophoresis was used to characterize the strains . SETTING: Five ICUs of a university hospital which are supplied by two separate water distribution networks . PATIENTS: During a 1-year period 132 cases were investigated . RESULTS: In 42% of cases (56/132) there were isolates identical to those found in the faucets, with a total of nine different genotypes . Among the nine genotypes isolated from both patients and faucets one of them, the most prevalent, was isolated in the two networks and in 30 cases . The other eight genotypes were recovered almost exclusively from either one (three genotypes in 12 cases) or the other (five genotypes in 12 cases) network and from the patients in the corresponding ICUs . CONCLUSIONS: These results suggest that the water system of the ICUs was the primary reservoir of patient's colonization/infection with P . aeruginosa in a substantial proportion of patients, although the exact mode of acquisition could not be determined.

Shock, 2004 Aug, 22(2), 131 - 6
Beneficial effect of an inhibitor of leukocyte elastase (EPI-hNE-4) in presence of repeated lung injuries; Honore S et al.; Persistence of alveolar neutrophil influx and activation may enhance the fibrotic process after acute lung injury . On the other hand, elastase has an antimicrobial activity and could participate in neutrophil migration, both events being critically important in host defense, explaining the controversial issue of therapeutic elastase inhibition in the setting of acute lung injury . We assessed the effect of a neutrophil elastase inhibitor, EPI-hNE-4, in single (bleomycin, 1.2 mg/rat intratracheally) and repeated (bleomycin, 1.2 mg/rat plus endotoxin and 1 mg/kg intratracheally 24 h later) lung injuries to assess the role of neutrophil in fibrosis . Subsequently, the effect of EPI-hNE-4 on bacterial clearance was evaluated during Pseudomonas aeruginosa-induced pneumonia . In the single injury model, despite a dramatic reduction of alveolar neutrophil influx with EPI-hNE-4 treatment, no significant inhibition of the decrease in respiratory system compliance, an index of lung fibrosis, was demonstrated at day 14 . In the repeated injury model, EPI-hNE-4 treatment afforded a significant protective effect on compliance and alveolar inflammation at day 14 . During bacterial pneumonia, EPI-hNE4 did not modify alveolar neutrophil recruitment or bacterial clearance from bronchoalveolar lavage fluid and lung homogenate . In conclusion, EPI-hNE-4, a specific inhibitor of leukocyte elastase, afforded a partial protective effect on the respiratory system compliance during repeated lung injuries, and had no detrimental effect during a gram-negative bacterial pneumonia.

Microbiology, 2004 Jul, 150(Pt 7), 2161 - 9
DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential; Renelli M et al.; Natural membrane vesicles (n-MVs) produced by Pseudomonas aeruginosa PAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were purified and analysed for DNA content . The MVs were isolated by a procedure designed to ensure no cellular contamination from the parent MV-producing cells . Fluorometry analysis revealed that p-MVs were associated with 7.80 ng DNA (20 microg MV protein)(-1) . PCR analysis using specific primers for pAK1900 sequences and a chromosomal target, oprL, indicated that only plasmid DNA was contained within the lumen of p-MVs after exogenous DNA was digested by DNase . MVs have previously been shown to be capable of fusing into the outer membrane (OM) of PAO1 and Escherichia coli DH5 alpha . Accordingly, p-MVs should deliver the plasmid into the periplasm, where it would only have to by-pass the plasma membrane (PM) for effective transformation . It was speculated that p-MVs should increase transformation efficiency but the data suggested otherwise . p-MVs did not transform PAO1 nor DH5 alpha under a variety of transforming conditions . To characterize p-MVs and to ensure that membrane-encapsulated pAK1900 was not derived from a small proportion of lysed cells within the culture and bound by PM instead of OM, which typically forms n-MVs, the physical and ultrastructural differences between n- and p-MVs were determined . Cryo-transmission electron microscopy (cryo-TEM) revealed that n-MVs and p-MVs closely resembled isolated OM . Buoyant density measurements using isopycnic sucrose gradients on isolated PM, OM, n- and p-MVs demonstrated that isolated OM and n-MVs both fractionated into two bands (rho=1.240 and 1.260 g ml(-1)) . p-MVs also produced two bands but at two different densities (rho=1.250 and 1.265 g ml(-1)) which may be attributed to the presence of DNA . SDS-PAGE showed that p-MVs possessed most major OM proteins and also contained 43.70 nmol 3-deoxy-d-manno-octulosonic acid (KDO) (mg protein)(-1) as an LPS marker . The amount of NADH oxidase activity, a PM enzyme, in the p-MVs was barely detectable . These data strongly suggest that p-MVs are OM-based, with little if any PM material associated with them . The possibility of whether exogenous plasmid DNA could enter n-MVs once the vesicles had departed from cells was also tested; surprisingly, a small amount of DNA could . Accordingly, the data suggest that DNA can be taken up by MVs using two separate routes: (1) via a periplasmic route and (2) via an extracellular, exogenous route.

J Antimicrob Chemother, 2004 Aug, 54(2), 386 - 92 Epub 2004 Jul 14.
Tolerance of Pseudomonas aeruginosa to Melaleuca alternifolia (tea tree) oil is associated with the outer membrane and energy-dependent cellular processes; Longbottom CJ et al.; OBJECTIVES: The essential oil of Melaleuca alternifolia (tea tree oil) and its components have antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria, fungi and viruses . The mechanism(s) by which Pseudomonas aeruginosa NCTC 10662 maintains a decreased susceptibility to tea tree oil and components was investigated . RESULTS: Ethylene diamine tetraacetic acid enhanced the antimicrobial activity of tea tree oil and terpinen-4-ol against stationary phase P . aeruginosa while polymyxin B nonapeptide enhanced the activity of tea tree oil and gamma-terpinene . Pre-treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone increased the susceptibility of exponential phase cells to sub-inhibitory concentrations of tea tree oil, terpinen-4-ol and gamma-terpinene, indicating that intrinsic tolerance to tea tree oil and components is substantially energy dependent . CONCLUSIONS: Increased tolerance to tea tree oil in P . aeruginosa is directly related to the barrier and energy functions of the outer membrane, and may involve efflux systems.

Kaohsiung J Med Sci, 2004 Jun, 20(6), 261 - 7
In vitro activities of antibiotic combinations against clincal isolates of Pseudomonas aeruginosa; Chen YH et al.; Combination therapy has been recommended to treat Pseudomonas aeruginosa infections worldwide . The purpose of the present study was to determine the in vitro activities of piperacillin, cefepime, aztreonam, amikacin, and ciprofloxacin alone and in combination against 100 clinical isolates of P . aeruginosa from one medical center in southern Taiwan . The combination susceptibility assay was performed using the checkerboard technique . The percentage of resistance of P . aeruginosa to single agents in our study was relatively high for the Asia-Pacific area, except to aztreonam . Piperacillin plus amikacin exhibited the highest potential for synergy (59/100) in this study . Moreover, a high percentage of synergism was also noted with amikacin combined with cefepime (7/100) or aztreonam (16/100) . The combination of two beta-lactams, such as cefepime with piperacillin, and aztreonam with cefepime or piperacillin, showed synergistic effects against some P . aeruginosa isolates . Although ciprofloxacin is a good anti-pseudomonal agent, a very low potential for synergy with other antibiotics was demonstrated in this study . No antagonism was exhibited by any combination in our study . Among piperacillin-resistant strains, there was synergy with a beta-lactam plus amikacin, including the combination of piperacillin and amikacin . However, the combination of two beta-lactams, such as piperacillin and cefepime or aztreonam, did not have any synergistic activity against these strains . In summary, the combinations of amikacin with the tested beta-lactams (piperacillin, aztreonam, cefepime) had a greater synergistic effect against P . aeruginosa, even piperacillin-resistant strains, than other combinations . Understanding the synergistic effect on clinical strains may help clinicians choose better empirical therapy in an area with high prevalence of multidrug-resistant P . aeruginosa.

J Proteome Res, 2004 May-Jun, 3(3), 434 - 44
Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry; Blonder J et al.; Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer . Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P . aeruginosa membrane subproteome . Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins . The application of these approaches resulted in the identification of 786 proteins . A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32) . Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex . This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P . aeruginosa and for prokaryotes in general.

J Proteome Res, 2004 May-Jun, 3(3), 393 - 402
Using proteomics to mine genome sequences; Arthur JW et al.; We present a method for mining unannotated or annotated genome sequences with proteomic data to identify open reading frames . The region of a genome coding for a protein sequence is identified by using information from the analysis of proteins and peptides with MALDI-TOF mass spectrometry . The raw genome sequence or any unassembled contigs of an organism are theoretically cleaved into a number of equal sized but overlapping fragments, and these are then translated in all six frames into a series of virtual proteins . Each virtual protein is then subjected to a theoretical enzymatic digestion . Standard proteomic sample preparation methods are used to separate, array, and digest the proteins of interest to peptides . The masses of the resulting peptides are measured using mass spectrometry and compared to the theoretical peptide masses of the virtual proteins . The region of the genome responsible for coding for a particular protein can then be identified when there are a large number of hits between peptides from the protein and peptides from the virtual protein . The method makes no assumptions about the location of a protein in a particular gene sequence or the positions or types of start and stop codons . To illustrate this approach, all 773 proteins of Pseudomonas aeruginosa contained in SWISS-PROT were used to theoretically test the method and optimize parameters . Increasing the size of the virtual proteins results in an overall improvement in the ability to detect the coding region, at the cost of decreasing the sensitivity of the method for smaller proteins . Increasing the minimum number of matching peptides, lowering the mass error tolerance, or increasing the signal-to-noise ratio of the simulated mass spectrum, improves the ability to detect coding regions . The method is further demonstrated on experimental data from Mycobacterium tuberculosis and is also shown to work with eukaryotic organisms (e.g., Homo sapiens).

J Biol Chem, 2004 Sep 10, 279(37), 38402 - 8 Epub 2004 Jul 12.
Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS; Maresso AW et al.; Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin . The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity . The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation . Infection of HeLa cells with P . aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes . Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation . Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins . ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras . Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS . Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells . The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.

Transplant Proc, 2004 Jun, 36(5), 1569 - 73
Hickman catheter site infections after allogeneic stem cell transplantation: a single-center experience; Kim DH et al.; Hickman catheter site infections are known to increase transplant-related mortality . A retrospective analysis of 103 patients who received allogeneic stems cell transplants was performed to define the incidence and outcomes of Hickman infections . Seventy-six patients received peripheral blood stem cells (PBSC) (73.8%) and 29 patients (28.2%) nonmyeloablative conditioning . During the median follow-up of 9 months, Hickman infections were observed in 10 patients (9.7%) at a median onset of 32 days posttransplant (range 2 to 102 days) . The causative organisms identified in five cases included Staphylococcus species (n = 4) and Pseudomonas aeruginosa (n = 1) . Six events successfully resolved with antibiotic treatment, while the other four required the removal of the Hickman catheter with subsequent death in two cases . The survival duration for infected patients was shorter than that for the noninfected group (83 days vs 366 days, P < .001) . Myeloid engraftment was delayed in the infected group (18.0 days vs 15.0 days, P = .038) and this complication was more frequently observed among the BMT compared with PBSC group (22.2% vs 5.3%, P = .019) . Hickman infections were associated with transplant-related mortality especially during the first 3 months posttransplant . As such, the current results emphasize both the importance of Hickman catheter care and the need for surveillance cultures after stem cell transplantation .

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 197 - 204
Mutations in the chloramphenicol acetyltransferase (S61G, Y105C) increase accumulated amounts and resistance in Pseudomonas aeruginosa; Wang J et al.; A chloramphenicol acetyltransferase (catB7) gene containing two point mutations, 181A/G and 314A/G, has been recently reported to be a determinant for high-level chloramphenicol resistance phenotype in a Pseudomonas aeruginosa strain PAhcr1 . The mutant CATB7 was further characterized in vitro and in vivo to elucidate the molecular basis of high-level resistance . CAT assay demonstrated that the mutant and wild-type recombinant CATB7 had similar specific activities . Dot blotting revealed that the accumulated amounts of CATB7 in P . aeruginosa strains PAO1 and PAhcr1 were proportionate to the respective anti-chloramphenicol level . Site-directed mutagenesis showed that G61S and Y105C contributed synergistically to the PAhcr1 resistance phenotype . It could be proposed that the mutant CATB7 was more structurally stable than catalytically efficient as a chloramphenicol resistance determinant in PAhcr1.

Clin Infect Dis, 2004 Jul 15, 39 Suppl 1, S65 - 7
Antimicrobial prophylaxis in febrile neutropenia; Yoshida M et al.; Antibiotics generally considered for antibacterial prophylaxis for immunosuppressed patients are trimethoprim-sulfamethoxazole and the quinolones . Trimethoprim-sulfamethoxazole can significantly reduce infections and is highly effective in preventing pneumonia due to Pneumocystis carinii . However, it can cause sulfonamide-related reactions, myelosuppression, oral candidiasis, and development of bacterial resistance, and it lacks activity against Pseudomonas aeruginosa . Quinolones can reduce the occurrence of fever and infections in patients with neutropenia but do not provide adequate coverage against gram-positive bacteria, and inappropriate use can induce resistance among gram-negative organisms . Routine antibacterial prophylaxis is not recommended for patients likely to develop neutropenia . Antifungal prophylaxis is appropriate in settings in which fungal infections are frequent . Fluconazole is recommended for patients who are to undergo hematopoietic stem cell transplantation; it can be considered for elderly patients with acute leukemia who are to receive intensive chemotherapy . Itraconazole can also be used . Prophylaxis with antiviral agents is generally not indicated; however, it should be given to hematopoietic stem cell transplant recipients.

Clin Infect Dis, 2004 Jul 15, 39 Suppl 1, S25 - 31
Changes in the etiology of bacteremia in febrile neutropenic patients and the susceptibilities of the currently isolated pathogens; Ramphal R; The etiology of bacteremia in febrile neutropenic patients in the past few decades has shifted from gram-negative to gram-positive organisms . Potential reasons include the use of indwelling catheters, local environmental conditions, and the administration of specific antibiotic agents, especially as prophylaxis . Other factors may emerge from new studies, such as the categorization of febrile neutropenic patients into groups at low risk and at high risk of developing serious complications, continuing changes in resistance in the community, the use of antibiotic-coated catheters, and future changes in cytotoxic chemotherapy or antineoplastic therapy . In addition, there has been a drift in susceptibility patterns, with resistance issues seen in the general population of hospitalized patients now emerging in febrile neutropenic patients, as well as some issues specific to these patients . These changes affect empirical therapy as it was practiced a decade ago . Among the most commonly used agents, cefepime and carbapenems continue to show the highest rates of in vitro susceptibility, providing coverage against most gram-positive and gram-negative organisms and reducing the need for glycopeptides . Older agents continue to show degradation of their effectiveness . Among Pseudomonas aeruginosa strains, susceptibility to all agents continues to decline.

Clin Infect Dis, 2004 Jul 15, 39 Suppl 1, S7 - S10
Microbiological data for patients with febrile neutropenia; Kanamaru A et al.; The pattern of bacterial infections and antimicrobial susceptibility has changed significantly during the past 20-30 years . The causative organisms for bacteremia or fungemia identified at Kinki University Hospital in 1985-1996 were compared with the isolates identified during 1997-2002 . The prevalence of gram-negative organisms decreased, whereas the prevalence of gram-positive organisms increased . Staphylococcal species predominated in the second period, accounting for 22% of isolates, and methicillin-resistant Staphylococcus aureus (MRSA) increased from 5% to 14% of isolates . Pseudomonas aeruginosa ranked second, although the prevalence decreased in the second period compared with the first . Candida species were also relatively frequent (11%) . Enterococcal species had an 8% prevalence . A comparison of all culture isolates showed that gram-negative isolates still predominated among the general patient population, whereas almost equal prevalence was observed in patients with hematological diseases . MRSA was the organism most frequently isolated in the general patient population, followed by P . aeruginosa . Among staphylococcal species, MRSA accounted for as much as 90% of isolates.

Protein Expr Purif, 2004 Aug, 36(2), 217 - 25
Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems; Murthy TV et al.; Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system . Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions . Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions . The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h . A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.

Am J Physiol Lung Cell Mol Physiol, 2004 Nov, 287(5), L944 - 52 Epub 2004 Jul 09.
Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice; van Heeckeren AM et al.; Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients . The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear . Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads . Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages . The inflammatory responses to P . aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-alpha, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X . Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice . Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P . aeruginosa.

Bioorg Med Chem, 2004 Aug 1, 12(15), 4221 - 31
New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa . Part 3: 7beta-{2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido} cephalosporins bearing 4-{3-(aminoalkyl)-ureido}-1-pyridinium at C-3'; Yoshizawa H et al.; Among the prepared C-3' substituted-pyridinium cephalosporins, a series of 7beta-{2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido} cephalosporins bearing 4-{3-(aminoalkyl)-ureido}-1-pyridinium at C-3' showed highly potent antibacterial activity against MRSA and Pseudomonas aeruginosa.

Bioorg Med Chem, 2004 Aug 1, 12(15), 4211 - 9
New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa . Part 2: Synthesis and structure-activity relationships in the S-3578 series; Yoshizawa H et al.; Among the prepared novel cephalosporin derivatives related to S-3578, a series of 7beta-{2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetamido}-3-{1-(aminoalkyl)-1H-pyrazolo{4,3-b}pyridinium-4-yl}methyl-3-cephem-4-carboxylate showed potent activity against both MRSA and Pseudomonas aeruginosa, and displayed good water solubility.

Anal Biochem, 2004 Aug 1, 331(1), 115 - 21
The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis; Karmali K et al.; A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy . This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((2)H(2)O) . The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis . The extinction coefficients epsilon of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1)cm(-1) at 1625, 1605, and 1365 cm(-1), respectively . The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P . aeruginosa . The kinetic constants (V(max), K(m), and K(cat)) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4mM, and 806.36 s(-1), respectively . These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media . Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.

Intensive Care Med, 2004 Sep, 30(9), 1768 - 75 Epub 2004 Jul 09.
Patterns of colonization by Pseudomonas aeruginosa in intubated patients: a 3-year prospective study of 1,607 isolates using pulsed-field gel electrophoresis with implications for prevention of ventilator-associated pneumonia; Valles J et al.; OBJECTIVE: To identify routes and patterns of colonization with Pseudomonas aeruginosa in intubated patients to design strategies of prevention for respiratory infection . DESIGN AND SETTING: Prospective and observational study in the 16-bed intensive care unit of a teaching hospital . PATIENTS AND PARTICIPANTS: Ninety-eight intubated patients were investigated over a 3-year period . Those ventilated less than 72 h were excluded . MEASUREMENTS AND RESULTS: Samples from the tap water from each patient's room, stomach, oropharynx, subglottic secretions, trachea, and rectum were collected when the patient was intubated, and then three times per week . Pulsed-field gel electrophoresis was performed to type the strains . We identified 1,607 isolates pertaining to 35 different pulsotypes . Overall 54.2% of patients presented colonization, and tracheal colonization was present in 30.5% . Ten patients had colonization at intubation, and four of these developed ventilator-associated pneumonia (VAP) after a mean of 4+/-2 days . ICU-acquired colonization occurred in 31 patients, and 4 of these developed VAP after a median of 10+/-5 days . P . aeruginosa was isolated from the room's tap water in 62.4% of samples . More than 90% of tap water samples had pulsotypes 1 and 2, which were frequently isolated in the stomach (59%) but were only rarely associated with VAP . CONCLUSIONS: Although colonization/infection with P . aeruginosa in intubated patients tends to be endogenous, exogenous sources should not be ruled out . A combination of early identification (and eradication) of airways colonization by P . aeruginosa plus infection control measures targeted to reduce cross-contamination should be the basis to prevent pulmonary infection.

J Clin Microbiol, 2004 Jul, 42(7), 3225 - 31
Antigenic evidence of prevalence and diversity of Mycobacterium tuberculosis arabinomannan; Glatman-Freedman A et al.; Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule . The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development . In the present study we examined the prevalence and diversity of AM among Mycobacterium tuberculosis strains using four AM-binding monoclonal antibodies (MAbs) . One of these MAbs, MAb 9d8, is known to bind to AM specifically . By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8 . However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11 M . tuberculosis strains tested, as demonstrated by capture ELISA . Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains . Mice immunized with an experimental AM-recombinant Pseudomonas aeruginosa exoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge . Our results indicate that AM was detected in all M . tuberculosis strains tested, with differences in epitope distributions of certain strains . In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.

J Antimicrob Chemother, 2004 Aug, 54(2), 546 - 8 Epub 2004 Jul 08.
Anomaly and correlation of killing in the therapeutic properties of silver (I) chelation with glutamic and tartaric acids; Batarseh KI; OBJECTIVES: To investigate whether silver chelates or silver ions are more effective as therapeutic agents, and to examine their mode of action so that safer and stable compounds that have a broad spectrum of therapeutic activities can be developed . METHODS: Efficacy was investigated against Pseudomonas aeruginosa (ATCC 15442) by determining MIC via a broth macrodilution procedure using NCCLS methods for antibiotic susceptibility testing . RESULTS: It was found that the responsible agent for silver therapeutic properties is the silver chelates rather than silver ions, contradicting previous findings, and the efficacy profiles mimic that of free silver ions present in solution . CONCLUSIONS: Silver therapeutic activities seem to be more effective as complexes-an intracellular package-rather than free silver ions, demonstrating that the effect of silver is linked to cells' DNA unwinding, and not respiratory or membrane functionality as was traditionally recognized.

Blood Purif, 2004, 22(4), 329 - 37
Reprocessed (high-flux) Polyflux dialyzers resist trans-membrane endotoxin passage and attenuate inflammatory markers; Teehan GS et al.; BACKGROUND: Bacterial contamination of dialysis water can contribute to the chronic microinflammatory state observed in dialysis patients . This study characterized the selective permeability of new and peroxyacetic acid/acetic acid/hydrogen peroxide (Renalin) reprocessed high-flux, polyarylethersulfone-polyvinylpyrrolidone (Polyflux-17R) dialyzers after exposure to endotoxin-contaminated dialysate during in vitro dialysis . Clinical correlation with pre-dialysis levels of systemic markers of inflammation, and clearance of middle molecules was also assessed in vivo . METHODS: Six hemodialysis (HD) patients were enrolled in the study . After reuses 0, 1, 5, 10, and 15, the dialyzers were reclaimed and submitted to an in vitro dialysis circuit using standard dialysate and blood from healthy volunteers . New and reprocessed dialyzers were sequentially exposed to escalating doses of Pseudomonas aeruginosa endotoxin in the dialysate compartment, and whole blood tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production was used as an index of reverse passage of endotoxin . In vivo, IL-6, C-reactive protein (CRP) and serum amyloid A (SAA) levels were measured to assess the impact of reprocessing on the systemic inflammatory response . Finally, pre- and post-dialysis samples were collected to measure urea and beta(2)-microglobulin (beta(2)-M) clearances . RESULTS: During in vitro dialysis, blood-side endotoxin levels were undetectable following dialysate contamination . TNF-alpha production remained unchanged (p = NS), and IL-6 production fell significantly on reuses 0, 1, 10, and 15 (p = 0.03) suggesting membrane adsorption, as a result of reuse-dependent surface binding . In vivo, whereas IL-6 and SAA levels did not significantly differ (p = 0.90 and 0.59, respectively), CRP levels fell near significantly, over the course of 15 reuses (p = 0.06) . In vivo, beta(2)-M clearance was not affected by the reuse technique (p = 0.28) . CONCLUSIONS: This study provides in vitro and in vivo evidence arguing that high-flux Polyflux dialyzers provide more than adequate dialysis, while preventing the in vitro back-diffusion of bacterial endotoxin despite 15 reuses with Renalin . Clinically, this may translate into an attenuation of the microinflammatory milieu.

Appl Environ Microbiol, 2004 Jul, 70(7), 4356 - 62
Use of fluorescent lectin probes for analysis of footprints from Pseudomonas aeruginosa MDC on hydrophilic and hydrophobic glass substrata; Bejarano EM et al.; Microbial footprints of Pseudomonas aeruginosa MDC attached for 1 h to clean or silanized glass were analyzed with fluorescently labeled lectin probes . Footprint composition varied, depending on cell physiology and substratum surface chemistry . This suggests that substratum physicochemistry affected the structure of cell surfaces of adsorbed organisms.

Appl Environ Microbiol, 2004 Jul, 70(7), 4004 - 11
Effect of pyocyanin on a crude-oil-degrading microbial community; Norman RS et al.; Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments . In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P . aeruginosa and is a redox-active compound capable of inhibiting microbial growth . To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 microM pyocyanin and microbial community structure and oil degradation were monitored for 50 days . Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures . Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively . Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture . Pyocyanin also affected the overall degradation of the crude oil . At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C(29) and C(30) hopanes . This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.

Protein Sci, 2004 Aug, 13(8), 2130 - 8 Epub 2004 Jul 06.
Evolutionary trace analysis of the alpha-D-phosphohexomutase superfamily; Shackelford GS et al.; The alpha-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates . The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans . Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition . Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the alpha-D-phosphohexomutase superfamily . These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P . aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family . Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family . In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.

Liver Transpl, 2004 Jul, 10(7), 844 - 9
Bacteremias in liver transplant recipients: shift toward gram-negative bacteria as predominant pathogens; Singh N et al.; During the 1990s, gram-positive bacteria emerged as major pathogens after liver transplantation . We sought to determine whether the pathogens associated with bacteremias in liver transplant recipients have changed . Patients included 233 liver transplant recipients transplanted between 1989 and 2003 . The proportion of all infections due to bacteremias increased significantly over time (P <.0001) . Of other major infections, a trend toward a decrease in fungal infections (P =.089) and a significant decrease in cytomegalovirus (CMV) disease (P =.0004) were documented . Whereas the proportion of bacteremias due to gram-negatives increased from 25% in the period of 1989-1993 to 51.8% in 1998-03, that of gram-positive bacteria decreased from 75% in the period of 1989-93 to 48.2% in the period of 1998-2003 . Methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, and Pseudomonas aeruginosa were the most frequent pathogens in bacteremic patients . The incidence of bacteremias due to MRSA and Pseudomonas aeruginosa has remained unchanged (P <.20); however, that due to enteric gram-negative bacteria, particularly Klebsiella pneumoniae has increased (P =.02) . Klebsiella pneumoniae isolates in the current quartile were not clonally related . In conclusion, bacteremias as a proportion of all infections in liver transplant recipients have increased significantly over time, due in part to a decline in infections due to other major pathogens, e.g., fungi, primarily Candida species, and CMV . Gram-negative bacteria have emerged as predominant pathogens in bacteremic liver transplant recipients.

J Hosp Infect, 2004 Jul, 57(3), 217 - 22
The influence of laboratory adaptation on test strains, such as Pseudomonas aeruginosa, in the evaluation of the antimicrobial efficacy of ortho-phthalaldehyde; Herruzo-Cabrera R et al.; The microbiocidal efficacy of 0.55% ortho-pthalaldehyde (OPA) was evaluated in a rough carrier test, using more than 200 strains of bacteria and yeasts from patients and reference ATCC strains . This test was then compared with the European carrier test (prEN14561) using Pseudomonas aeruginosa . We also sought to determine whether recently isolated P . aeruginosa had the same susceptibility to OPA, after laboratory adaptation . It was shown that P . aeruginosa was less susceptible to OPA (being reduced by a factor of 10(3.8)) than the other strains (reduced by a factor of 10(4)) . The surface test used, produced a lesser reduction of P . aeruginosa than the European test . For recently isolated strains (N = 66), the rough model demonstrated that the number of survivors increased both quantitatively and qualitatively from day one to day seven . It was concluded that disinfectant efficacy should be confirmed with recently isolated organisms.

Biochemistry, 2004 Jul 13, 43(27), 8662 - 9
Detailed kinetic studies of an aggregating inhibitor; inhibition of phosphomannomutase/phosphoglucomutase by disperse blue 56; Liu HY et al.; Phosphomannomutase/phosphoglucomutase occupies a central position in the pathways by which several virulence factors are synthesized in Pseudomonas aeruginosa . Virtual screening was used to identify potential inhibitors of phosphomannomutase/ phosphoglucomutase, and one compound, the anthraquinone-based dye Disperse Blue 56, showed potent inhibition in vitro . The kinetics of inhibition was complex; the time courses for reactions in the presence of the inhibitor were biphasic, suggestive of slow-binding inhibition . Quantitative analysis of the progress curves and preincubation experiments demonstrated that slow-binding inhibition was not occurring, however . Initial velocity kinetic studies indicated that Disperse Blue 56 was a parabolic, noncompetitve inhibitor . Progress curves for reactions in the presence of Disperse Blue 56 could be fitted very well by a model in which 2 equiv of the inhibitor bound to free enzyme or the enzyme-substrate complex . The inhibition was largely relieved by the inclusion of 0.01% Triton X-100 in the assay solutions, which has been suggested to be the hallmark for inhibition by compounds that exert their effect through aggregates {McGovern, S . L., Caselli, E., Grigorieff, N., and Shiochet, B . K . (2002) J . Med . Chem . 45, 1712-1722} . Our kinetic data appear to be consistent with either inhibition by a dimer of Disperse Blue 56 or inhibition by a Disperse Blue 56 aggregate, but the latter appears much more likely . We present a detailed analysis of the system to provide further information that may help in the recognition of inhibition through aggregation.

Biol Trace Elem Res, 2004 Summer, 99(1-3), 269 - 77
Bioaccumulation properties of nickel-, cadmium-, and chromium-resistant mutants of Pseudomonas aeruginosa NBRI 4014 at alkaline pH; Gupta A et al.; Nickel-, cadmium-, and chromium-resistant mutants of Pseudomonas aeruginosa sp . NBRI 4014 are potent growth promontory strains . In the presence of metal(s), they have shown increased cell morphology and could accumulate nickel (2.0874 mM/g), cadmium (2.295 mM/g), and chromium (2.997 mM/g) at pH 9 . However, chromium accumulation was maximum, but the toxicity levels of nickel, cadmium, and chromium was ranged from 90 to 504 microM of used metal(s) . Bioaccumulation increases up to the fifth day at the highest used metal concentration and then became constant . This suggests their exploitation for detoxification and subsequently loaded biomass treatment for the recovery of accumulated metal(s).

J Microbiol Methods, 2004 Aug, 58(2), 203 - 12
Development of tools for the genetic manipulation of Pseudomonas aeruginosa; Suh SJ et al.; To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed . These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P . aeruginosa via conjugation, (b) a minimal sequence for P . aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P . aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis . Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P . aeruginosa.

J Theor Biol, 2004 Aug 7, 229(3), 339 - 47
Application of formal methods to biological regulatory networks: extending Thomas' asynchronous logical approach with temporal logic; Bernot G et al.; Based on the discrete definition of biological regulatory networks developed by Rene Thomas, we provide a computer science formal approach to treat temporal properties of biological regulatory networks, expressed in computational tree logic . It is then possible to build all the models satisfying a set of given temporal properties . Our approach is illustrated with the mucus production in Pseudomonas aeruginosa . This application of formal methods from computer science to biological regulatory networks should open the way to many other fruitful applications.

Klin Mikrobiol Infekc Lek, 2004 Jun, 10(3), 124 - 129
{Multiresistant nosocomial bacterial strains and their "in vitro" susceptibility to chloramphenicol and colistin}; Niks M et al.; Objective: A multicenter study was conducted to obtain "in vitro" chloramphenicol and colistin susceptibility data on multiresistant hospital bacterial pathogens in Slovak Republic . Material and methods: During the period of April-June 2001, 628 clinical bacterial multiresistant isolates from patients with serious infections were selected in 10 hospitals and tested to a large scale of antibiotics by means of a microdilution method . The strains expressed either a significant resistance phenotype (ESBL, MRSA, CoNMRS, MLSB/c, efflux in Ps . aeruginosa), or were resistant to one or more preparations in at least half of reliable unrelated antibiotic groups (beta-lactams, aminoglycosides, quinolons, macrolides) . Results: Both chloramphenicol and colistin retained significant "in vitro" activity against many multiresistant hospital bacterial pathogens . The highest activity of chloramphenicol was documented for isolates of Stenotrophomonas maltophilia (76,5 % susceptible, MIC50 = 4 mg/L, MIC90 = 16 mg/L) and of Staphylococcus aureus (76,2 % susceptible, MIC50 = 8 mg/L, MIC90 = 16 mg/L) . In tested Pseudomonas aeruginosa (82,5 % susceptible, MIC50 = 2 mg/L, MIC90 = 16 mg/L) and Stenotrophomonas maltophilia (88,2 % susceptible, MIC50 = 1 mg/L, MIC90 = 8 mg/L) isolates colistin represented the most "in vitro" effective antibiotic . Colistin was the only "in vitro" effective antimicrobial in four of 120 multiresistant Pseudomonas aeruginosa isolates tested in our study . Conclusions: The study confirmed a good "in vitro" susceptibility of many multiresistant hospital bacterial pathogens to chloramphenicol and colistin in Slovak Republic . The clinical application of chloramphenicol and colistin might be reconsidered in infections caused by extremely resistant bacteria with prooved susceptibility to these antibiotics . It is important to consider, that the infection danger has to exceed the risk of antibiotic toxicity.

Proc Natl Acad Sci U S A, 2004 Jul 6, 101(27), 9994 - 9 Epub 2004 Jun 28.
Structure of the periplasmic component of a bacterial drug efflux pump; Higgins MK et al.; Multidrug resistance among Gram-negative bacteria is conferred by three-component membrane pumps that expel diverse antibiotics from the cell . These efflux pumps consist of an inner membrane transporter such as the AcrB proton antiporter, an outer membrane exit duct of the TolC family, and a periplasmic protein known as the adaptor . We present the x-ray structure of the MexA adaptor from the human pathogen Pseudomonas aeruginosa . The elongated molecule contains three linearly arranged subdomains; a 47-A-long alpha-helical hairpin, a lipoyl domain, and a six-stranded beta-barrel . In the crystal, hairpins of neighboring MexA monomers pack side-by-side to form twisted arcs . We discuss the implications of the packing of molecules within the crystal . On the basis of the structure and packing, we suggest a model for the key periplasmic interaction between the outer membrane channel and the adaptor protein in the assembled drug efflux pump.

J Biol Chem, 2004 Sep 3, 279(36), 37551 - 8 Epub 2004 Jun 28.
Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1; Miller WL et al.; WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5) . The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography . Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD(+) as a coenzyme . The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy . Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid . WbpA requires either NH(4)(+) or K(+) for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series . Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K(0.5) of 94 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.8 . In addition, WbpA has a K(0.5) for NAD(+) of 220 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.1 . The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution . However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy . The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.

Int J Antimicrob Agents, 2004 Jul, 24(1), 35 - 8
Molecular detection of GES-2 extended spectrum Beta-lactamase producing Pseudomonas aeruginosa in Pretoria, South Africa; Weldhagen GF et al.; Screening for and detection of the novel extended spectrum Beta-lactamase (ESBL), GES-2 produced by Pseudomonas aeruginosa remains a problem in the clinical microbiology laboratory . This study aimed to compare the normally used ESBL screening agent ceftazidime, with molecular detection, to demonstrate the presence of GES-2 ESBL production in clinical isolates of P . aeruginosa . Ceftazidime was found unreliable as an ESBL screening agent, with a specificity of 34.4%, when National Committee for Clinical Laboratory Standards resistance criteria for P . aeruginosa were employed . An improved PCR detection method was devised, that amplified a 371 bp segment of bla(GES-2) . This should lead to more cost-effective DNA sequencing and sequence interpretation in the laboratory.

Mol Microbiol, 2004 Jul, 53(1), 297 - 308
A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system; Dasgupta N et al.; Expression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells . Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed . Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion . ExsA is an activator of TTSS gene transcription, and ExsD is an anti-activator of ExsA . In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription . Here, we describe the characterization of ExsC as an anti-anti-activator of TTSS expression . Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression . The dependence on exsC for transcription is relieved in the absence of exsD, suggesting that ExsC and ExsD function together to regulate transcription . Consistent with this idea, ExsC interacts with ExsD in bacterial two-hybrid and co-purification assays . We propose a model in which the anti-anti-activator (ExsC) binds to and sequesters the anti-activator (ExsD) under low Ca(2+) conditions, freeing ExsA and allowing for transcription of the TTSS . The P . aeruginosa system represents the first example of an anti-activator/anti-anti-activator pair controlling transcription of a TTSS.

Ann Otol Rhinol Laryngol, 2004 Jun, 113(6), 462 - 4
Ecthyma gangrenosum: a rare cutaneous manifestation of a potentially fatal disease; Solowski NL et al.; Ecthyma gangrenosum is a cutaneous lesion frequently associated with Pseudomonas aeruginosa bacteremia, although it may develop in the absence of bacteremia and may originate from other bacterial and fungal organisms . Ecthyma gangrenosum most often occurs in patients with neutropenia and other immunocompromised hosts . It typically occurs on the extremities and gluteal and perineal regions . We report a rare case of ecthyma gangrenosum presenting as an aggressive necrotic skin lesion on the nasal ala of a patient with myelofibrosis . Tissue and blood cultures were positive for P aeruginosa . This clinical entity should be considered when otolaryngologists are asked to evaluate necrotic cutaneous lesions of the head and neck.

Environ Sci Technol, 2004 Jun 1, 38(11), 3148 - 52
Cadmium removal from contaminated soil by tunable biopolymers; Prabhukumar G et al.; An elastin-like polypeptide (ELP) composed of a polyhistidine tail (ELPH12) was exploited as a tunable, metal-binding biopolymer with high affinity toward cadmium . By taking advantage of the property of ELPH12 to undergo a reversible thermal precipitation, easy recovery of the sequestered cadmium from contaminated water was demonstrated as the result of a simple temperature change . In this study, batch soil washing experiments were performed to evaluate the feasibility of using ELPH12 as an environmentally benign strategy for removing cadmium from contaminated soil . The stability constant (log KL) for the cadmium-ELPH12 complex was determined to be 6.8, a value similar to that reported for the biosurfactant rhamnolipid . Two washings with 1.25 mg/mL of ELPH12 were able to remove more than 55% of the bound cadmium as compared to only 8% removal with ELP containing no histidine tail or 21% removal using the same concentration of EDTA . Unlike rhamnolipid from Pseudomonas aeruginosa ATCC 9027, which adsorbs extensively to soil, less than 10% of ELPH12 was adsorbed under all soil washing conditions . As a result, a significantly lower concentration of ELPH12 (0.036 mM as compared to 5-10 mM of biosurfactants) was required to achieve similar extraction efficiencies . However, cadmium recovery by simple precipitation was incomplete due to the displacement of bound cadmium by zinc ions present in soil . Owing to its benign nature, ease of production, and selective tailoring of the metal binding domain toward any target metals of interest, ELP biopolymers may find utility as an effective extractant for heavy metal removal from contaminated soil or ore processing.

Blood Cells Mol Dis, 2004 Jul-Aug, 33(1), 57 - 63
Low doses of dexamethasone constantly delivered by autologous erythrocytes slow the progression of lung disease in cystic fibrosis patients; Rossi L et al.; OBJECTIVE: To evaluate the safety and efficacy of the administration of low doses of glucocorticoids in patients with cystic fibrosis (CF) by using autologous erythrocytes loaded with dexamethasone 21-phosphate . STUDY DESIGN: Nine consecutive CF patients (patients nos . 1-9) received autologous erythrocytes loaded with increasing amounts of dexamethasone 21-phosphate to obtain a slow delivery of dexamethasone in circulation . The appearance of possible adverse effects, the reproducibility of the procedure, and the dexamethasone pharmacokinetics were evaluated . Subsequently, patient no . 9 and eight additional patients (patient nos . 10-17) received dexamethasone 21-phosphate-loaded erythrocytes at 1-month intervals to evaluate the efficacy of continuous release in circulation of low doses of dexamethasone . RESULTS: Erythrocytes from CF patients can be processed to be loaded with increasing dexamethasone 21-P concentrations . Once reinfused in respective donors, a slow and prolonged delivery of dexamethasone in the blood stream was measured up to 28 days . Repeated administrations of drug-loaded erythrocytes at 4-week intervals for 15 months showed that very low doses of glucocorticoids provide significant improvement in FEV1 values and significant reduction of infective relapses due to Pseudomonas aeruginosa without adverse effects . CONCLUSIONS: The administration of very low doses of glucocorticoids using autologous erythrocytes is possible, with benefits for patients and without side effects . This method is likely to be extended to other chronic diseases.

Proteomics, 2004 Jul, 4(7), 1996 - 2004
Proteomic analysis of agar gel-entrapped Pseudomonas aeruginosa; Vilain S et al.; We have compared the protein maps of agar-entrapped Pseudomonas aeruginosa cells to those of free counterparts grown in the presence or absence of the immobilized-cell gel support . Principal component analyses (PCAs) were used to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis . PCA of the data matrix (923 rows x 6 columns) in which spot density values were standardized horizontally extracted three principal components (PCs) with eigenvalues higher than 1, accounting together for 71.6% of the variability in the data . Principal component 1 (PC1) opposed free (F) and agar-entrapped (AE) cultures, with a low contribution of agar-released, free (ARF) cultures to PC1 . Inversely, the contribution of ARF cultures to PC2 was high, opposing those of AE and F cultures . Component 3 was related to the duration of incubation . Only 10% of total proteins were upregulated in AE cells during the first 18 h of incubation, the number of underexpressed peptides balancing that of overexpressed ones . Downregulation clearly became the dominant tendency when the incubation time was extended to 48 h . These results demonstrate that AE and ARF bacteria are physiologically different from F organisms.

Am J Physiol Lung Cell Mol Physiol, 2004 Oct, 287(4), L809 - 15 Epub 2004 Jun 25.
Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase; Lillehoj EP et al.; We reported previously that Muc1 on the surface of epithelial cells was a receptor for Pseudomonas aeruginosa (Lillehoj EP, Kim BT, and Kim KC . Am J Physiol Lung Cell Mol Physiol 282: L751-L756, 2002) . Other studies showed that the Muc1 cytoplasmic tail (CT) contains multiple phosphorylation sites, some of which are phosphorylated constitutively and associated with signaling proteins . However, the relationship between extracellular P . aeruginosa binding and intracellular signaling is unknown . To investigate the signaling mechanism of Muc1, this study examined phosphorylation of its CT and activation of the extracellular signal-regulated kinase (ERK) in response to stimulation by P . aeruginosa or purified flagellin . Our results showed 1) the Muc1 CT was phosphorylated constitutively on serine and tyrosine, 2) serine phosphorylation was stimulated by bacterial cells or flagellin, and 3) binding of P . aeruginosa or flagellin to Muc1 induced phosphorylation of ERK . These results are the first to demonstrate Muc1 CT phosphorylation and ERK activation in response to a clinically important airway pathogen.

Nefrologia, 2004, 24 Suppl 3, 26 - 9
{Multiple complications after renal transplantation}; Manrique J et al.; This is the case of a 32-year-old male patient, diagnosed with end stage renal disease secondary to a focal and segmental glomerulonephritis . After four years of haemodialysis, he received a renal graft from a cadaveric donor . During the following sixteen years, he developped many different complications . In the early post-transplant period, he developed a severe acute tubular necrosis and two episodes of acute rejection took place, both of them with later recovery . Among the outstanding infectious complications were a virus herpes zoster dorsal infection and a Pseudomonas aeruginosa nosocomial pneumonia . Twelve months later, a series of severe digestive complications took place: cholecystitis that required cholecystectomy, pancreatic pseudocyst which required laparotomy because of an abdominal complication, two separate episodes of upper digestive bleeding that finally required gastric surgery, and an hemorrhagic subphrenic abscess that required a second laparotomy . Currently he has developed a calcified chronic pancreatitis . Moreover, metabolic complications must be mentioned carbohydrate intolerance, cataracts and an avascular bone necrosis, all of them closely related to the immunosuppressive therapy . In spite of these multiple complications, he mantains a good renal function and his quality of life is acceptable.

Jpn J Infect Dis, 2004 Jun, 57(3), 119 - 20
Contribution of exotoxin A of Pseudomonas aeruginosa in acute and chronic experimental renal infection; Sharma S et al.; The role of Pseudomonas aeruginosa exotoxin A was studied in acute and chronic pyelonephritis models employing an exotoxin A-producing strain PAO, and its toxin deficient mutant PAOT1 . Interestingly, the mutant strain was found to be at an advantage in its ability to induce acute pyelonephritis and it induced severer renal pathology . No significant differences were observed in the ability of the parent strain and its mutant to induce chronic renal inflammation.

Clin Transplant, 2004, 18 Suppl 12, 61 - 6
Conversion to sirolimus-based maintenance immunosuppression using daclizumab bridge therapy in renal transplant recipients; Sundberg AK et al.; INTRODUCTION: Conversion from calcineurin inhibitor (CI)-based maintenance immunosuppression to sirolimus (SRL)-based immunosuppression may be beneficial in selected renal transplant recipients . The purpose of this study was to evaluate the safety and efficacy of a daclizumab (DAC) bridge protocol in patients converted from CI- to SRL-based maintenance immunosuppression . METHODS: We conducted a retrospective chart review of renal transplant recipients who were converted to SRL at least 2 months post-transplant . The protocol consisted of an abrupt discontinuation of either cyclosporin (CsA) or tacrolimus (TAC), initiation of SRL within 48 h of CI discontinuation, and DAC 2 mg/kg at the time of CI discontinuation and again at 14 d (depending on the SRL serum concentration) . The SRL starting dose was based on risk stratification in each patient . RESULTS: Twenty-one renal transplant patients were converted to SRL (11 from TAC, 10 from CsA) between October 2001 and July 2003 . Conversion occurred at a mean of 23 months post-transplant . Indications for SRL conversion included 12 for chronic allograft nephropathy (CAN), four for CI-associated neurotoxicity, two for thrombotic microangiopathy (TMA), two for post-transplant diabetes mellitus (PTDM), and one for polyomavirus interstitial nephritis (PVN) . Mean follow-up was 16 months from time of conversion . Therapeutic SRL levels were reached at a mean of 14 d . Total serum cholesterol levels increased from a mean of 205 (+/- 47) to 234 (+/- 55) mg/dL (P = 0.014), and serum triglyceride levels increased from a mean of 186 (+/- 66) to 257 (+/- 88) mg/dL (P = 0.002) . In addition, mean haemoglobin level decreased from 12.0 (+/- 2.3) to 10.5 (+/- 2.1) g/dL (P = 0.002); total white blood cell count decreased from 8300 (+/- 4300) to 4700 (+/- 1400)/mm(3) (P < 0.001); and platelet count decreased from 238 000 (+/- 72 800) to 186 000 (+/- 51 900)/mm(3) (P = 0.002) from before to after conversion . Patients experienced the following side-effects while taking SRL: diarrhoea (n = 6), peripheral oedema (n = 5), arthralgias (n = 4), anaemia (n = 4), oral ulcers (n = 1), deep vein thrombosis (n = 1), shortness of breath (n = 1), and mild increase in serum transaminases (n = 1) . Two patients (9.5%) discontinued SRL due to side-effects, both secondary to severe arthralgias . There were two serious infections noted after conversion: one Pseudomonas aeruginosa urosepsis, and one PVN (that was ongoing prior to conversion) . Patient survival was 100%, and kidney graft survival was 76% . Five patients (24%) lost their allograft after conversion due to progression of CAN (n = 2), persistent TMA in the kidney (n = 1), patient self-discontinuation of sirolimus (n = 1), and preexisting PVN unresponsive to cidofovir therapy (n = 1) . Of the five patients who lost their allograft, the mean serum creatinine at the time of conversion was 3.5 (+/-1.1) mg/dL compared with 2.2 (+/- 0.8) mg/dL in patients who did not lose their allograft (P = 0.034) . No acute rejection episodes occurred after conversion to sirolimus . CONCLUSIONS: DAC bridge therapy provides safe and effective immunosuppressive coverage while converting renal transplant recipients from CI- to SRL-based maintenance immunosuppressive therapy . A pharmacoeconomic analysis, however, is necessary to determine the cost-effectiveness of this conversion protocol.

J Infect Dis, 2004 Jul 15, 190(2), 356 - 64 Epub 2004 Jun 21.
Potential therapeutic role of histatin derivative P-113d in experimental rat models of Pseudomonas aeruginosa sepsis; Cirioni O et al.; BACKGROUND: Morbidity and mortality from Pseudomonas aeruginosa sepsis remain high despite the availability of antibiotics to which the microorganism is sensitive . METHODS: The in vitro activity of histatin derivative P-113d was investigated against Pseudomonas aeruginosa . In addition, its in vivo efficacy was studied in 3 rat models of infection: intraperitoneal injection of 1 mg of P . aeruginosa 10 lipopolysachharide, intraperitoneal injection of 2 x 10(10) cfu of P . aeruginosa ATCC 27853, and intra-abdominal sepsis induced by cecal ligation and puncture . Rats received isotonic sodium chloride solution parenterally (control groups), 1 mg of P-113d/kg of body weight, 1 mg of polymyxin B/kg of body weight, or 20 mg of imipenem/kg of body weight . Main outcomes measured were abdominal exudate and plasma bacterial growth, plasma concentrations of endotoxin and tumor necrosis factor (TNF)- alpha, and lethality . RESULTS: The in vivo studies showed that all compounds reduced lethality, when compared with results for the control group . Overall, P-113d exhibited a slightly lower antimicrobial activity than did imipenem, even though P-113d achieved a substantial decrease in plasma concentrations of endotoxin and TNF- alpha, compared with the imipenem . No statistically significant differences for antimicrobial and antiendotoxin activities were noted between P-113d and polymyxin B . DISCUSSION: These results provide evidence for double antiendotoxin and antimicrobial activity for P-113d and point to its potential use for the treatment of severe infections.

Microbiol Immunol, 2004, 48(5), 435 - 9
A quorum-sensing autoinducer enhances the mexAB-oprM efflux-pump expression without the MexR-mediated regulation in Pseudomonas aeruginosa; Sawada I et al.; We have previously described that the quorum-sensing autoinducer, N-butyryl-L-homoserine lactone (C4-HSL) enhances mexAB-oprM expression, and this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon . In this study, we investigated the interaction between C4-HSL and mexR by using a knockout mutant . It was indicated that the C4-HSL-mediated enhancement of mexAB-oprM expression occurred without MexR-mediated regulation . Furthermore, it was observed that the C4-HSL-mediated enhancement of mexAB-oprM expression without the MexR-mediated regulation was repressed by MexT.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2665 - 72
Detection and susceptibility testing of hypermutable Pseudomonas aeruginosa strains with the Etest and disk diffusion; Macia MD et al.; Resistance development in Pseudomonas aeruginosa from chronically colonized cystic fibrosis (CF) patients has been linked to the presence of a high proportion of mismatch repair-deficient hypermutable strains . The detection of hypermutable strains by microbiology laboratories may be useful for establishing adequate antimicrobial therapies . In this work, we find that the Etest and disk diffusion can be used as simple methods for the detection and susceptibility testing of hypermutable P . aeruginosa isolates . Strain PAO1 and its hypermutable derivative strain PAODeltamutS were used to standardize the procedure, which was tested with 35 P . aeruginosa isolates from 21 CF patients . Mutation frequencies were estimated by standard methods, and 29% of the isolates were found to be hypermutable . MICs and inhibition zone diameters were determined for ceftazidime, imipenem, meropenem, ciprofloxacin, and tobramycin by using Etest strips and conventional disks, respectively . The presence (or absence) of resistant mutant subpopulations, as well as their relative numbers and the highest MICs for them (or smallest inhibition zone diameters), was recorded . The presence of resistant mutant subpopulations within the inhibition zones of three or more antibiotics clearly identified the strains as hypermutable (they were present in 10 of 10 hypermutable strains and 0 of 25 nonhypermutable strains) with both methods . Additionally, these methods allowed us to differentiate between dual effects of hypermutation in antibiotic resistance, namely, that (i) hypermutable isolates were substantially more resistant than nonhypermutable isolates and that (ii) the resistance of hypermutable isolates was dramatically increased by the presence of resistant mutant subpopulations . This differentiation may be relevant for the design of adequate treatments, since the second effect, in contrast to the first, may be overcome by antibiotic combinations.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2431 - 6
Factors associated with relative rates of antibiotic resistance in Pseudomonas aeruginosa isolates tested in clinical laboratories in the United States from 1999 to 2002; Flamm RK et al.; For the period from 1999 to 2002 in the United States, the in vitro susceptibilities of 52,637 Pseudomonas aeruginosa isolates to 10 antimicrobial agents were evaluated . The isolates were from 29 laboratories, 11 of which participated in The Surveillance Network for four consecutive years . Isolates were collected from adult patients (> or =18 years of age) in intensive care units (ICU), non-ICU inpatients, nursing home patients, and outpatients; data were analyzed to evaluate factors, such as year of isolation, patient age group, isolate specimen source, and patient type (hospitalized patients {ICU, non-ICU, or nursing home} or outpatients) . Rates of resistance for the 4-year period were highest for isolates from patients in ICU and 18- to 39-year-old patients and for isolates from the lower respiratory tract . Resistance decreased with age . Resistance was lowest in isolates from outpatients, in isolates from > or =70-year-old patients, and from specimens from the upper respiratory tract . Multidrug resistance (MDR) (resistance to three or more antimicrobial agents) accounted for 24.9% of all isolates . The MDR rate was highest in isolates from patients in nursing homes (29.9%) and ICU (29.5%).

Can J Microbiol, 2004 May, 50(5), 375 - 81
Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines; Abdi-Ali A et al.; Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P . aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin . Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P . aeruginosa 42A . Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates . Pyocin activity of the fractions was detected using the Govan spot testing method . The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected . Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P . aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay . The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF . Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.

Can J Microbiol, 2004 May, 50(5), 303 - 12
Inhibition of Pseudomonas aeruginosa adhesion to fibronectin by PA-IL and monosaccharides: involvement of a lectin-like process; Rebiere-Huet J et al.; Pseudomonas aeruginosa adherence to fibronectin has been shown to be important to bacterial colonization and infection . To better understand the mechanisms involved in this interaction, the role of the carbohydrate moiety of the fibronectin molecule in P . aeruginosa adhesion was studied . Strain NK 125 502 adhered to immobilized fibronectin with an adherence index of 4.8 x 10(5) CFU/ micro g . Periodic oxidation of fibronectin markedly reduced the adhesion of P . aeruginosa, while a neuraminidase treatment increased bacteria adhesion . N-Acetylgalactosamine, N-acetylglucosamine, sialic acid, and also lectin PA-IL worked as efficient inhibitors in adhesion assays: 59%, 70.7%, 100%, and 60% of inhibition, respectively . We have demonstrated here the involvement of a lectin-like process in the interaction of P . aeruginosa NK 125 502 with immobilized fibronectin.

Infect Immun, 2004 Jul, 72(7), 4275 - 8
Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice; Lau GW et al.; Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection . PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells . Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.

Infect Immun, 2004 Jul, 72(7), 4224 - 32
The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung; Priebe GP et al.; Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains . In cystic fibrosis patients, however, LPS-rough strains of P . aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection . It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS) . We previously reported that interruption of the galU gene in P . aeruginosa results in production of a rough LPS and truncated LPS core . Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU . We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103 . The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro . In a corneal infection model, the galU mutants were significantly attenuated . In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread . These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung . Host defenses in the lung appear to be insufficient to control infection with LPS-rough P . aeruginosa when local bacterial levels are high.

FEMS Microbiol Lett, 2004 Jul 1, 236(1), 137 - 43
Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance; Wolter DJ et al.; Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem . These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of oprD transcription . The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains . Expression of oprD could only be detected in two of the strains, but expression was very low as indicated by the high number of RT-PCR cycles required to amplify the product . PCR was performed to amplify the oprD gene using primers upstream of the promoter and downstream of the structural gene . Amplified products were sequenced, and sequences were compared to wild-type P . aeruginosa strain PAO1 . Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon . The other five isolates provided PCR products that were 1.3-1.6 kb larger than expected, suggesting the presence of large inserts . Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within oprD . In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD . Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates . This observation does not explain the observed decrease in transcriptional expression . This is the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by IS elements.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9792 - 7 Epub 2004 Jun 21.
Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis; Wilderman PJ et al.; In many bacteria, iron homeostasis is controlled primarily by the ferric uptake regulator (Fur), a transcriptional repressor . However, some genes, including those involved in iron storage, are positively regulated by Fur . A Fur-repressed regulatory small RNA (sRNA), RyhB, has been identified in Escherichia coli, and it has been demonstrated that negative regulation of genes by this sRNA is responsible for the positive regulation of some genes by Fur . No RyhB sequence homologs were found in Pseudomonas aeruginosa, despite the identification of genes positively regulated by its Fur homolog . A bioinformatics approach identified two tandem sRNAs in P . aeruginosa that were candidates for functional homologs of RyhB . These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and a functional Fur box precedes each . Their expression is induced under iron limitation . Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism . As in E . coli, induction of the PrrF sRNAs leads to the rapid loss of mRNAs for sodB (superoxide dismutase), sdh (succinate dehydrogenase), and a gene encoding a bacterioferritin . Thus, the PrrF sRNAs are the functional homologs of RyhB sRNA . At least one gene, bfrB, is positively regulated by Fur and Fe(2+), even in the absence of the PrrF sRNAs . This work suggests that the role of sRNAs in bacterial iron homeostasis may be broad, and approaches similar to those described here may identify these sRNAs in other organisms.

J Biol Chem, 2004 Aug 27, 279(35), 37201 - 7 Epub 2004 Jun 17.
Pseudomonas aeruginosa exotoxin A induces human mast cell apoptosis by a caspase-8 and -3-dependent mechanism; Jenkins CE et al.; Mast cells play an important role in both allergy and innate immunity . Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines . Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P . aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium . ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process . Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis . ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate . Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not . Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis . Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors . Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.

J Bacteriol, 2004 Jul, 186(13), 4387 - 9
tonB3 is required for normal twitching motility and extracellular assembly of type IV pili; Huang B et al.; Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili . These defects could be complemented with wild-type tonB3.

J Bacteriol, 2004 Jul, 186(13), 4228 - 37
Structure of Pseudomonas aeruginosa populations analyzed by single nucleotide polymorphism and pulsed-field gel electrophoresis genotyping; Morales G et al.; Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings . To analyze the population structure and distribution of P . aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC) . The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P . aeruginosa strains . Thirteen of the 91 identified SNP genotypes were found in two or more strains . In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles . The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats . The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes . The P . aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains . The fact that some SNP genotypes were overrepresented in the P . aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.

J Enzyme Inhib Med Chem, 2004 Feb, 19(1), 51 - 6
Binding of transition metal ions {cobalt, copper, nickel and zinc} with furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-derived cephalexins as potent antibacterial agents; Chohan ZH et al.; A method is described for the preparation of novel cephalexin-derived furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-containing compounds showing potent antibacterial activity . The binding of these newly synthesized antibacterial agents with metal ions such as cobalt(II), copper(II), nickel(II) and zinc(II) has been studied and their inhibitory properties against various bacterial species such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae are also reported . These results suggest that metal ions to possess an important role in the designing of metal-based antibacterials and that such complexes are more effective against infectious diseases compared to the uncomplexed drugs.

J Antimicrob Chemother, 2004 Aug, 54(2), 557 - 62 Epub 2004 Jun 16.
CS-023 (R-115685), a novel carbapenem with enhanced in vitro activity against oxacillin-resistant staphylococci and Pseudomonas aeruginosa; Thomson KS et al.; OBJECTIVE: To compare the in vitro activities of the carbapenem, CS-023, four representative beta-lactam antibiotics and levofloxacin, against 970 Gram-positive or Gram-negative US clinical isolates . METHODS: Susceptibilities of bacteria chosen for their varying levels of resistance to the comparator agents were determined by NCCLS microdilution methodology . RESULTS: CS-023 exhibited activity comparable to that of imipenem against most Gram-positive isolates, but was approximately 8-fold more potent against oxacillin-resistant staphylococci . It was comparable to meropenem against most Gram-negative isolates, but was 4- to 8-fold more potent against five isolates of meropenem-resistant Pseudomonas aeruginosa . CONCLUSIONS: If tissue and body fluid concentrations >8 mg/L can safely be achieved, further studies of CS-023 are warranted to determine its clinical efficacy.

J Endotoxin Res, 2004, 10(3), 163 - 74
C-reactive protein: a predominant LPS-binding acute phase protein responsive to Pseudomonas infection; Ng PM et al.; As a structural component of the outer membrane of Gram-negative bacteria, endotoxin, also known as lipopolysaccharide (LPS) exhibits strong immunostimulatory properties, rendering it a pivotal role in the pathogenesis of Gram-negative septicaemia . Our attempt to identify LPS-binding proteins from the hemolymph of the horseshoe crab led to the isolation and identification of Creactive protein (CRP) as the predominant LPS-recognition protein during Pseudomonas infection . CRP is an evolutionarily ancient member of a superfamily of 'pentraxins' . It is a major protein in acute phase of infection in humans . Our investigation of CRP response to Pseudomonas aeruginosa unveiled a robust innate immune system in the horseshoe crab, which displays rapid suppression of a dosage of 10(6) CFU of bacteria in the first hour of infection and effected complete clearance of the pathogen by 3 days . Such a high dose would have been lethal to mice . Full-length CRP cDNA was cloned . Analysis of the untranslated regions suggests their crucial role in post-transcriptional regulation of CRP transcript levels . Northern blot analysis demonstrated an acute up-regulation of CRP by about 60-fold in 6-48 h of Pseudomonas infection . Taken together, our results provide new insights into the importance of CRP as a conserved molecule for pathogen recognition.

Cancer Biol Ther, 2004 Aug, 3(8), 708 - 14 Epub 2004 Aug 10.
Microbial-based therapy of cancer: a new twist to age old practice; Punj V et al.; The use of bacteria in the regression of tumors has long been known . Various approaches for using bacteria in cancer therapy include the use of bacteria as sensitizing agents for chemotherapy, as delivery agents for cancer drugs and as agents for gene therapy . The tumor regression stimulated by infecting microorganisms has been attributed to activation of the immune system of the host . However, recent studies indicate that when tumor-harboring mice with defective immune systems are infected with certain microorganisms, the regression of the tumor is still observed, suggesting that there are other host factors contributing to the microbial associated regression of tumors . Since the use of live or attenuated bacteria for tumor regression has associated toxic effects, studies are in progress to identify a pure microbial metabolite or any component of the microbial cell that might have anti-cancer activity . It has now been demonstrated that a redox protein from Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and trigger the apoptotic cell death . In vivo, this cupredoxin can lead to the regression of tumor growth in immunodeficient mice harboring xenografted melanomas and breast cancer tumors without inducing significant toxic effects, suggesting that it has potential anti-cancer activity . This bacterial protein interacts with p53 and modulates mammalian cellular activity . Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancer treatments . Here, we focus on diverse approaches to cancer biotherapy, including bacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.

Biochemistry, 2004 Jun 22, 43(24), 7954 - 65
The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status; Clement E et al.; In iron-deficient conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (Pvd), which after chelating iron(III) is transported back into the cell via its outer membrane receptor FpvA . FpvA is a TonB-dependent transport protein and has the ability to bind Pvd in its apo- or iron-loaded form . The fluorescence properties of Pvd were used to determine the binding kinetics of metal-free and metal-loaded Pvd to FpvA and showed two major features . First, the kinetics of formation of the FpvA-Pvd complex, in vivo and in vitro, are markedly slower compared to those observed for FpvA-Pvd-metal . Second, apo-Pvd and Pvd-metal absorbed with biphasic kinetics to FpvA: the bimolecular step (association of the ligand with the receptor) is followed by a slower step (t(1/2) values of 5 and 34 min for Pvd-metal and Pvd, respectively) that presumably leads to a more stable complex . The most likely explanation for this second step is that the binding of the ligand to the receptor induces a conformational change on FpvA, which may be different, depending on the loading status of Pvd . Analysis of the dissociation of metal-free Pvd from FpvA revealed an energy and a TonB dependency . The dissociation of iron-free Pvd from FpvA in the absence of the TonB protein occurs with slow kinetics in the range of hours, but it can be highly activated by the protonmotive force and TonB to reach a kinetic with a t(1/2) of 1 min . Apparently, under iron-limited conditions, TonB activates the FpvA receptor, resulting in a fast release of iron-free Pvd and generating an unloaded FpvA receptor, competent for binding extracellular Pvd-Fe.

Yi Chuan Xue Bao, 2004 Mar, 31(3), 311 - 6
{The study of optimal conditions of electroporation in Pseudomonas aeruginosa}; Shan ZY et al.; A P . aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain . Optimum conditions including growth stage of the strain, electroshock voltage, concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28 . It was showed that the highest transformation efficiency was up to 1.68 x 10(3) CFU/microgram DNA under the optimum conditions in which the competent cells were collected at logarithmic growth phase (OD(540) = 0.7-0.8) and concentrated to about 10(11) cells/ml, the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV . With this optimal condition, Mu transponson complexes have been successfully transformed into P . aeruginosa strain PA68 and the obtained efficiency was 2.47 x 10(4) CFU/microgram DNA . This is the first time to electroporate Mu transposon complexes into Pseudomonas spp . The artificial Mu transposons could integrate into bacterial genomes at a single site randomly . Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion . That will be very helpful for the study of genomic function of Pseudomonas spp.

Saudi Med J, 2004 Jun, 25(6), 780 - 4
Antimicrobial susceptibility pattern of clinical isolates of Pseudomonas aeruginosa; Al-Jasser AM et al.; OBJECTIVE: To determine the level of resistance to the widely used antipseudomonal antibiotics in clinical isolates of Pseudomonas aeruginosa (P . aeruginosa) . METHODS: The microbiology database of all clinical isolates of P . aeruginosa at the Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia, from January 1999 to December 1999 was reviewed . The antimicrobial susceptibilities were determined by a standardized method . RESULTS: Seven hundred and four P . aeruginosa isolates were tested . These strains were commonly isolated from surgical patients followed by intensive care units . Respiratory tract was the most common source of isolation . The antibiotic susceptibility rates were as follows: ciprofloxacin 92.2%, meropenem 91.6%, imipenem 90.2%, amikacin 85.8%, ceftazidime 81.8% piperacillin/tazobactam 81.3% and gentamicin 77.7% . Among 704 strains 6.4% were designated as being multidrug resistant . These were commonly isolated from respiratory tract specimens of patients in intensive care units . CONCLUSION: The clinical significance of these findings is important in the selection of appropriate empiric treatment of serious P . aeruginosa infections . It emphasizes the importance of a conservative approach to antibiotic therapy and continued antimicrobial susceptibility testing surveillance programs to curtail the problem of antibiotic resistance.

Pediatr Infect Dis J, 2004 Jun, 23(6), 504 - 10
Vaccination of cystic fibrosis patients against Pseudomonas aeruginosa reduces the proportion of patients infected and delays time to infection; Lang AB et al.; INTRODUCTION: Cystic fibrosis (CF) almost always leads to chronic airway infection with Pseudomonas aeruginosa . Despite advances in antibiotic therapy, after chronic infection rapid deterioration in lung function occurs, increasing morbidity and mortality . Prevention of infection by vaccination is desirable, but earlier trials produced disappointing results . The promising short term immunogenicity and safety of a new P . aeruginosa vaccine prompted us to evaluate its long term efficacy . We conducted a 10-year retrospective analysis of outcomes in a group of vaccinated patients . MATERIALS AND METHODS: In 1989-1990, 30 young children with CF, mean age 7 years, with no prior history of infection with P . aeruginosa, were vaccinated against P . aeruginosa with a polyvalent conjugate vaccine . We report the follow-up of 26 of these patients from 1989 to 2001 . The patients were given yearly vaccine boosters . Comparisons were made with a CF patient control group matched for gender, age and, where possible, genetic mutation . Vaccinated patients and controls were attending a single CF clinic and received the same clinical management throughout the study period . Main outcomes were time to infection, proportion of patients infected, development of P . aeruginosa mucoid phenotype, lung function and body weight . RESULTS: The time to infection with P . aeruginosa was longer in the vaccination group than in the control group, and fewer vaccinated patients than controls became chronically infected (32% versus 72%; P < 0.001) . The proportion of mucoid infections was higher in the control group (44%) than in the vaccinated group (25%) . Patients >/=18 years of age at the end of the study had a lower mean forced expiratory volume at 1 s (FEV1) than did those 13-17 years of age, but this difference was small in the vaccinated group (73.6% versus 83.7%) compared with the controls (48.0% versus 78.7%) . In the >/=18 year age category the mean FEV1% at 10 years was 73.6% (vaccinated) and 48.0% (controls) (P < 0.05) . In the vaccinated group only 11 (44%) of 25 patients were underweight at the 10-year follow-up compared with 18 (72%) of 25 at the beginning of the study . In the control group 17 (68%) of 25 patients were underweight at 10-year follow-up compared with 16 (64%) of 25 at the beginning of the study . CONCLUSION: Regular vaccination of young CF patients for a period of 10 years with a polyvalent conjugate vaccine reduced the frequency of chronic infection with P . aeruginosa . This was associated with better preservation of lung function . Vaccinated patients gained more weight during the study period, a possible indication of an improved overall health status.

Transplant Proc, 2004 May, 36(4), 958 - 60
Outcome of infections caused by multiple drug-resistant bacteria in liver transplant recipients; Gouvea EF et al.; OBJECTIVE: To evaluate the impact of infections caused by multiple-drug-resistant (MDR) bacteria on the clinical outcome of liver transplant recipients . METHODS: Retrospective study including all episodes of bacterial infection diagnosed in patients undergoing liver transplantation from January 19, 1999, to June 30, 2002 . The diagnosis of bacterial infection required microbiological documentation . Mortality associated with episodes of infection by MDR bacteria was compared to that observed after antibiotic-susceptible bacterial infections . RESULTS: Among 99 patients undergoing liver transplantation during the study period, there were 57 episodes of bacterial infections . Gram-negative bacilli were the predominant etiologic agents (76%) and Pseudomonas aeruginosa was the most frequent bacterial species found in these cases (23 isolates, 28%) . Thirty-six episodes of infection (63%) were caused by MDR bacteria . Mean time after transplantation to the diagnosis of infection was 17 days . Mortality associated with episodes of MDR bacterial infections (nine deaths, 25%) was not significantly different from that observed during episodes of antibiotic-susceptible bacteria (five deaths, 24%; P =.92) . CONCLUSION: These data suggest that resistance to multiple antimicrobial agents does not have an impact on the mortality associated to bacterial infections in liver transplant recipients.

Int J Antimicrob Agents, 2004 Jun, 23(6), 606 - 12
Reduction in the bactericidal activity of selected cathelicidin peptides by bovine calf serum or exogenous endotoxin; Bartlett KH et al.; Synthetic cathelicidin peptides exhibit enhanced antimicrobial action and avid binding to LPS, thereby detoxifying the action of endotoxin released from degrading bacteria . A series of cathelicidin antimicrobial peptide (CAP) and sheep myeloid antimicrobial peptide (SMAP) congeners were examined to determine whether LPS-binding could predict other beneficial characteristics of the peptides . The peptides were challenged in complex media with bovine calf serum or LPS, and their ability to kill the Gram negative pathogens Klebsiella pneumoniae (ATCC 43816) or Pseudomonas aeruginosa (PA103) was then assessed . LPS-binding efficiency was not correlated with antimicrobial activity in complex media . Additionally, LPS- and serum-binding may interfere with the antimicrobial activity of peptides in complex media .

Circ Res, 2004 Jul 23, 95(2), 196 - 203 Epub 2004 Jun 10.
Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY; Sayner SL et al.; Mammalian transmembrane adenylyl cyclases synthesize a restricted plasmalemmal cAMP pool that is intensely endothelial barrier protective . Bacteria have devised mechanisms of transferring eukaryotic factor-dependent adenylyl cyclases into mammalian cells . Pseudomonas aeruginosa ExoY is one such enzyme that catalyzes cytosolic cAMP synthesis, with unknown function . Pseudomonas aeruginosa genetically modified to introduce only the ExoY toxin elevated cAMP 800-fold in pulmonary microvascular endothelial cells over 4 hours, whereas a catalytically deficient (ExoY(K81M)) strain did not increase cAMP . ExoY-derived cAMP was localized to a cytosolic microdomain not regulated by phosphodiesterase activity . In contrast to the barrier-enhancing actions of plasmalemmal cAMP, the ExoY cytosolic cAMP pool induced endothelial gap formation and increased the filtration coefficient in the isolated perfused lung . These findings collectively illustrate a previously unrecognized mechanism of hyperpermeability induced by rises in cytosolic cAMP.

Proteomics, 2004 May, 4(5), 1241 - 6
Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates; Arevalo-Ferro C et al.; Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients . Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not . Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins . About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity . This percentage reflects a relatively high degree of intraclonal variability . Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors . It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Cell Microbiol, 2004 Jul, 6(7), 639 - 50
Differential post-transcriptional activation of human phagocytes by different Pseudomonas aeruginosa isolates; Pollard AJ et al.; Pseudomonas aeruginosa is a pulmonary pathogen in individuals with impaired mucociliary clearance such as cystic fibrosis or mechanical ventilation . Non-opsonic phagocytosis of P . aeruginosa can be mediated by either CR3 or CD14 and different strains appear to have a bias towards one or the other receptor . Strain Fc808 is ingested through CD14 whereas P1 (Fc194) uses CR3 . In an in vitro culture system, the inflammatory response of macrophages to these two different strains of P . aeruginosa was divergent at the protein level, with higher IL-6 and tumour necrosis factor (TNF)-alpha production generated in response to strain P1 and higher IL-1 beta production in response to strain Fc808 . Interaction of macrophages with these two bacterial strains induced distinct gene expression patterns as detected by gene array analysis, with prominence of genes encoding pro-inflammatory cytokines, surface receptors, transcription factors and proteins involved in phagocytosis . However, comparison of gene expression data and cytokine response data with the two bacterial strains indicated that production of IL-1 beta, IL-6 and TNF-alpha was under differential post-transcriptional control . Interestingly, this effect did not correlate with receptor bias but instead was related to the different LPSs of the two strains . The use of specific mitogen-activated protein kinase (MAPK) inhibitors suggested a role for extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the differential cytokine production by strains P1 and Fc808 . These results indicate that strains of the same species of bacteria may induce differential macrophage phagocytic and inflammatory responses with likely consequence for bacterial clearance and host injury.

J Am Chem Soc, 2004 Jun 16, 126(23), 7244 - 56
The selenocysteine-substituted blue copper center: spectroscopic investigations of Cys112SeCys Pseudomonas aeruginosa azurin; Ralle M et al.; Azurin is a small electron-transfer protein belonging to the cupredoxin family . The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions . A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out . Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A(||), a decrease in g-values, and a large increase in rhombicity of the g-tensor . The Cu-Se and Se-Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 A, respectively, 0.14 A longer than the Cu-S distance of the WT protein . Unexpectedly, the Cu-Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the "rack" hypothesis to the Se-substituted protein.

J Agric Food Chem, 2004 Jun 16, 52(12), 3915 - 9
Essential oil of Valeriana officinalis L . cultivars and their antimicrobial activity as influenced by harvesting time under commercial organic cultivation; Letchamo W et al.; The essential oil content and the composition of subterranean parts of two valerian (Valeriana officinalis, L.) cultivars Select and Anthose, from certified commercial organic fields, were determined by hydrodistillation, followed by gas chromatography (GC) and GC/mass spectrometry analysis . Eight and fourteen month old cv . Select had 0.67 and 0.87% essential oil, while similar aged cv . Anthose contained 0.97 and 1.1% essential oil . Forty-three and fifty-three components from cv . Select and cv . Anthose oils were detected, respectively . The oil composition significantly varied due to the cultivar type, plant age, and/or harvesting time . The major components for cv . Select were valerenal, bornyl acetate, 15-acetoxy valeranone, valerenic acid, and camphene, while cv . Anthose had valerenal, (-)-bornyl acetate, alpha-humulene, camphene, 15-acetoxy valeranone, and valerenic acid . With further aging of the plants, the valerenal, valerenic acid, and alpha-humulene contents increased . The oil of cv . Select had a strong antimicrobial effect against Aspergillus niger, Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae, while cv . Anthose showed low or no activity against all test microbes, including Pseudomonas aeruginosa, suggesting that the inhibitory activity of valerian oil depends on the cultivar and its developmental stage . The oil profile of our cultivars did not match the literature proposed chemotype profiles.

Microbiology, 2004 Jun, 150(Pt 6), 1851 - 7
AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933; Gliese N et al.; The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933 . Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol . After complementation with the intact agmR gene, growth on ethanol was restored . Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c(550) and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon . Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control . These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner . Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

Microbiology, 2004 Jun, 150(Pt 6), 1671 - 80
FpvB, an alternative type I ferripyoverdine receptor of Pseudomonas aeruginosa; Ghysels B et al.; Under conditions of iron limitation, Pseudomonas aeruginosa secretes a high-affinity siderophore pyoverdine to scavenge Fe(III) in the extracellular environment and shuttle it into the cell . Uptake of the pyoverdine-Fe(III) complex is mediated by a specific outer-membrane receptor protein, FpvA (ferripyoverdine receptor) . Three P . aeruginosa siderovars can be distinguished, each producing a different pyoverdine (type I-III) and a cognate FpvA receptor . Growth of an fpvA mutant of P . aeruginosa PAO1 (type I) under iron-limiting conditions can still be stimulated by its cognate pyoverdine, suggesting the presence of an alternative uptake route for type I ferripyoverdine . In silico analysis of the PAO1 genome revealed that the product of gene PA4168 has a high similarity with FpvA . Inactivation of PA4168 (termed fpvB) in an fpvA mutant totally abolished the capacity to utilize type I pyoverdine . The expression of fpvB is induced by iron limitation in Casamino acids (CAA) and in M9-glucose medium, but, unlike fpvA, not in a complex deferrated medium containing glycerol as carbon source . The fpvB gene was also detected in other P . aeruginosa isolates, including strains producing type II and type III pyoverdines . Inactivation of the fpvB homologues in these strains impaired their capacity to utilize type I ferripyoverdine as a source of iron . Accordingly, introduction of fpvB in trans restored the capacity to utilize type I ferripyoverdine.

J Med Microbiol, 2004 Jul, 53(Pt 7), 609 - 15
Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales; Scott FW et al.; Most past studies of cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients in the UK suggest that it is a rare occurrence . However, two recent reports of highly transmissible strains in patients in regional centres in England (Liverpool and Manchester) have raised questions as to the extent of the problem and prompted a nationwide survey to establish the distribution of P . aeruginosa strain genotypes among these patients . Isolates of P . aeruginosa were requested from over 120 hospitals in England and Wales and a sample size of approximately 20% of the CF patient population in each centre was recommended . In total, 1225 isolates were received from 31 centres (range 1 to 330) . Single patient isolates were typed by SpeI macrorestriction and PFGE . A panel of strains of the common genotypes including representatives of reported transmissible strains was assembled and further characterized by fluorescent amplified fragment length polymorphism (FAFLP) genotyping . At least 72% of all patients harboured strains with unique genotypes . Small clusters of related strains were evident in some centres, presumably indicating limited transmission of local strains . The most prevalent strain was indistinguishable from that previously described as the 'Liverpool' genotype, and accounted for approximately 11% of patient isolates from 15 centres in England and Wales . The second most common genotype (termed Midlands 1) was recovered from 86 patients in nine centres and the third genotype, which matched closely the PFGE profile of Clone C, a genotype originally described in Germany, was found in eight centres and was isolated from 15 patients . A fourth genotype, identical to the published Manchester strain, was found in three centres . FAFLP analysis revealed some microheterogeneity among strains of the Liverpool genotype but all isolates of this genotype were positive by PCR for a strain-specific marker . These data suggest that cross-infection with P . aeruginosa has occurred both within and widely between CF centres in England and Wales . The two most common genotypes accounted for more than one-fifth of patients' isolates examined and transmissible genotypes were found in all but three centres studied . These results emphasize the need for continued surveillance of P . aeruginosa genotypes in CF patients to provide informed infection control policy in treatment centres.

J Clin Microbiol, 2004 Jun, 42(6), 2806 - 9
Survey of ferroxidase expression and siderophore production in clinical isolates of Pseudomonas aeruginosa; Huston WM et al.; Ferroxidase (encoded by the mco gene), a component of a ferrous iron uptake pathway in Pseudomonas aeruginosa, was detected in all of the 35 respiratory clinical isolates surveyed; in contrast, considerable variation in siderophore expression was observed . The ubiquitous expression of this periplasmic ferroxidase suggests that it plays a key role in iron uptake in this opportunistic pathogen.

J Clin Microbiol, 2004 Jun, 42(6), 2523 - 9
Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase in Northern Italy; Pagani L et al.; Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum beta-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of bla(PER) ESBL determinants . Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired bla(PER-1) gene . PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin . An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed . The bla(PER-1) gene was not transferable and appeared to be chromosomally located . An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the bla(PER-1) locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing bla(PER-1) revealed a conserved structure in representatives of the various clonal lineages . The present findings indicate that MDR P . aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.

Am J Respir Crit Care Med, 2004 Sep 15, 170(6), 633 - 40 Epub 2004 Jun 07.
Heme oxygenase-1 expression in human lungs with cystic fibrosis and cytoprotective effects against Pseudomonas aeruginosa in vitro; Zhou H et al.; Inflammation and oxidative stress play important roles in cystic fibrosis (CF) lung disease . Inflammatory/oxidant-mediated induction of heme oxygenase-1 (HO-1) is believed to be a cytoprotective response . This study examined HO-1 expression in lung samples from patients with CF using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction . In addition, we evaluated myeloperoxidase staining as a marker of acute inflammation and potentially an increase in oxidant stress and Prussian blue and ferritin staining to assess iron status of the lung . Macrophage HO-1 staining was increased in diseased lungs as compared with normal control subjects and correlated with myeloperoxidase staining . Quantitative reverse transcription-polymerase chain reaction further supported an increase in HO-1 expression in CF lung disease . Although iron staining was minimal, ferritin staining was increased in diseased lungs in concert with HO-1 staining . To determine whether HO-1 induction was cytoprotective, we evaluated a CF airway epithelial cell line, IB3.1, in response to Pseudomonas aeruginosa-induced injury/apoptosis in cells overexpressing HO-1 by either transient or stable transfection of pcDNA3.1/HO-1 construct . Overexpression of HO-1 resulted in protection against P . aeruginosa-induced injury/apoptosis . This suggests that the induction of HO-1 in patients with CF is a cytoprotective event and that augmenting its expression is a potential therapy against bacterial injury.

J Hosp Infect, 2004 Jun, 57(2), 112 - 8
Risk factors and clinical outcomes of nosocomial multi-drug resistant Pseudomonas aeruginosa infections; Cao B et al.; Risk factors for multi-drug resistant Pseudomonas aeruginosa (MDRP) infections were investigated using a case-control study design involving MDRP patients (N = 44) and controls (N = 68) . A retrospective cohort study was performed to study the predictive factors of clinical outcome in MDRP patients . Multivariate analysis demonstrated that previous exposure to imipenem/meropenem {odds ratio (OR), 44.8} and mechanical ventilation (OR 8.2) were risk factors for nosocomial infections of MDRP . Of 44 cases of MDRP infections, 20 patients died directly from P . aeruginosa infections . Pulsed-field gel electrophoresis (PFGE) analysis on serial isolates from three patients showed that profiles of isolates from the same patient were closely related or indistinguishable . Multivariate analysis demonstrated that patients with adverse clinical outcomes were more likely to have been treated with mechanical ventilation (OR 12.8), and more likely to have MDRP resistance patterns that did not change during treatments (OR 26.5) . We concluded that mechanical ventilation and previous exposure to imipenem/meropenem were independent risk factors for MDRP infections, while mechanical ventilation and antibiotic resistance switch were predictive factors of outcomes of MDRP infections .

J Hosp Infect, 2004 Jun, 57(2), 105 - 11
Surveillance of multi-drug resistant Pseudomonas aeruginosa in an urban tertiary-care teaching hospital; Jung R et al.; Antimicrobial resistance in Pseudomonas aeruginosa is a serious clinical problem . To determine the incidence of multi-drug resistant P . aeruginosa, resistance rates of P . aeruginosa clinical isolates against commonly used antibiotics were evaluated for the period 1998 to 2002 . Multi-drug resistance was defined as resistance to at least three of the four drugs, ceftazidime, imipenem, ciprofloxacin, and tobramycin . Resistance to most anti-pseudomonal agents has increased by >20% over the five-year period, with dramatic increases observed with fluoroquinolones, tobramycin, and imipenem (resistance increased by 34-37%) . In 1998, 78% of isolates were susceptible to all four anti-pseudomonal agents and no isolate was considered multi-drug resistant . However, in 2002, only 27% of isolates were sensitive to all four of the drugs and 32% were considered multi-drug resistant . Multi-drug resistance in individual institutions may be significantly higher than rates reported in nationwide surveillance studies and may more accurately reflect the true magnitude of local resistance problems . On-going surveillance within individual institutions is critical for the selection of appropriate empiric antimicrobial therapy .

Int J Tuberc Lung Dis, 2004 Jun, 8(6), 691 - 702
Bronchiectasis: not an orphan disease in the East; Tsang KW et al.; Bronchiectasis is a common disease in the developing world . While the aetiology of bronchiectasis is diverse, many patients suffer from idiopathic disease . Although the pathogenesis of bronchiectasis is poorly understood, there are three distinct pathogenic elements, namely infection, inflammation and enzymatic actions . These interact to perpetuate airway destruction in many cases . There are four patient stereotypes: rapidly progressive, slowly progressive, indolent disease and haemoptysis-predominant . The diagnosis of bronchiectasis is best made with high resolution computed tomography, which should be followed by delineation of aetiology and evaluation of disease severity . Management of bronchiectasis is unsatisfactory and there are no disease-modifying drugs or treatment guidelines . Specific therapy to correct an underlying defect should be instituted whenever possible, although established disease often continues to deteriorate relentlessly . Treatment with prolonged, high-dose antibiotics is useful for exacerbations and probably also for some severely affected patients with frequent exacerbations who habour Pseudomonas aeruginosa in their airways . Commencement of long-term nebulised aminoglycoside, elective in-patient intravenous antibiotic therapy, long-term oral antibiotic or low-dose macrolide therapy requires special considerations . Inhaled corticosteroid therapy reduces chemokine expression in bronchiectasis in vivo, and may be useful for some patients . For severely affected patients, the use of non-invasive positive-pressure ventilation with supplementary oxygen sometimes helps . The lack of enthusiasm about bronchiectasis has already resulted in a lack of research in the treatment of this frustrating disease, and such research needs to be encouraged.

South Med J, 2004 May, 97(5), 465 - 71
Declining susceptibility to neomycin and polymyxin B of pathogens recovered in otitis externa clinical trials; Cantrell HF et al.; BACKGROUND: Otitis externa is usually treated empirically with topical neomycin/polymyxin B/hydrocortisone . The predominant pathogens associated with this infection are Pseudomonas aeruginosa and Staphylococcus aureus . METHODS: Two multicenter clinical trials (one in adults and adolescents, and one in children), conducted between 1995 and 1996, compared neomycin/polymyxin B/hydrocortisone with ofloxacin for the treatment of otitis externa; two similar trials were conducted between 1999 and 2000 . Assessments included the minimum inhibitory concentrations (MICs) of each antimicrobial drug for the major pathogens, bacterial eradication, and clinical efficacy . RESULTS: The MICs of all bacterial isolates (including P . aeruginosa) for neomycin and polymyxin B increased markedly in the 1999 to 2000 studies compared with the 1995 to 1996 studies . In the later studies, mean MICs for all major pathogens tested had increased above the breakpoint for polymyxin B (> or = 4 microg/ml) . In contrast, MICs of all isolates for ofloxacin remained similar between the two study periods and were within the susceptible range for this drug . CONCLUSIONS: Although the bacterial eradication rates for both treatments in each study were equivalent, the clinical cure rate for neomycin/polymyxin B/hydrocortisone was lower (87%) than for ofloxacin (93%) . Therefore, the organisms most often causing otitis externa appear to be developing resistance to neomycin and polymyxin B but not to ofloxacin.

Protein Expr Purif, 2004 Jul, 36(1), 11 - 8
A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli; Rao XC et al.; Peptide antibiotics are often hard to express in engineered bacteria at high level . According to the properties of peptide antibiotics, a heterologous protein PaP3.30, encoded by ORF30 of Pseudomonas aeruginosa bacteriophage PaP3, was selected as a carrier molecule . The gene of the carrier molecule was constructed into the plasmid pQE-32 to give rise to the vector pQE-PaP30 for expression of peptide antibiotics in Escherichia coli . A his-tagged fusion protein was genetically constructed with a peptide antibiotic at its carboxy terminus . The novel carrier molecule was used for high-level expression of six peptide antibiotics with different sizes and isoelectric points in E . coli, which are hPAB-beta, MSI-78, Melletin, hBD-1, Cecropin A, and an ovine anion peptide . And further, one of six peptide antibiotics, hPAB-beta (an analog of a human peptide antibiotic), was taken as an example for studies of recovery of interesting products from the fusion partner, purification and antimicrobial activity evaluation . The results indicated that the expressed fusion protein existed as an inclusion body in the cytoplasm and the expression amounts of six peptide antibiotic fusions are all higher than 34% of the total cell protein . The expression products could be easily purified by Ni-NTA chromatography . Cyanogen bromide was used to cut at the methionine linker between the carrier and hPAB-beta peptide . hPAB-beta was recovered from the fusion partner and purified to homogeneity with High S cation-exchange and Bio-gel P6 gel chromatography . The bactericidal activities of the purified recombinant hPAB-beta against P . aeruginosa are 31-64 microg/ml, and against Staphylococcus aureus are > or = 128 microg/ml, being comparable to that of the chemical synthesis peptide . These results show that the carrier molecule can result in high-level expression of peptide antibiotics, and expression products can be easily recovered from their fusion partner and retain their bioactivity.

Biotechnol Prog, 2004 May-Jun, 20(3), 670 - 8
Modeling the quorum sensing regulatory network of human-pathogenic Pseudomonas aeruginosa; Viretta AU et al.; The biochemical network underlying quorum sensing in human-pathogenic Pseudomonas aeruginosa is one of the best characterized . Mathematical modeling is required to untangle the complexity of its architecture and dynamics . We present a qualitative model of the P . aeruginosa quorum-sensing network including interactions between the las and rhl modules, the signaling molecule PQS and the regulatory proteins Mvfr and VfR . Simulations exemplify the model to reproduce natural network behavior and suggest quorum-sensing responses to pharmacological interference.

Eur Respir J, 2004 May, 23(5), 679 - 84
Genotype/phenotype correlation of the G85E mutation in a large cohort of cystic fibrosis patients; Decaestecker K et al.; In this European study, the phenotype in 68 patients, homozygous or compound heterozygous for the G85E mutation, was investigated . Each index case was compared with two cystic fibrosis (CF) patients from the same clinic, matched for age and sex: one with pancreatic sufficiency (PS) and one with pancreatic insufficiency (PI) . When comparing 31 G85E/F508del and F508del/F508del patients, there were no differences in median age at diagnosis, mean sweat chloride value, most recent weight for height, most recent forced expiratory volume in one second % predicted, prevalence of chronic Pseudomonas aeruginosa colonisation and typical CF complications . However, PI was less frequent in the G85E/F508del group . Comparison of 55 G85E patients (with second mutation known and not classified as mild) with PS controls (n=44) showed that the G85E patients had a significantly higher sweat chloride, more often failure to thrive at diagnosis, higher prevalence of PI, worse current weight for height, higher prevalence of chronic P . aeruginosa colonisation and liver cirrhosis . Pulse-chase experiments revealed that G85E cystic fibrosis transmembrane conductance regulator failed to mature on a M470 as well as on a V470 background . Therefore, G85E is a class II mutation . Although there is variability in its clinical presentation, G85E mutation results in a severe phenotype.

Salud Publica Mex, 2004 Mar-Apr, 46(2), 149 - 57
{RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients}; Ortiz-Herrera M et al.; OBJECTIVE: To characterize P . aeruginosa strains isolated from bronchoalveolar lavage fluid of cystic fibrosis (CF) patients over a 3 year period . MATERIAL AND METHODS: A prospective follow-up study was carried out in a population of cystic fibrosis patients . The random amplified polymorphic DNA (RAP.D) technique was used to amplify DNA of P . aeruginosa strains isolated from bronchoalveolar lavage fluid samples of five CF patients from the Servicio de Neumologia y Cirugia del Torax del Instituto Nacional de Pediatria (Mexico City Chest Clinic of the National Pediatrics Institute) in Mexico City, between June 1996 and June 2002 . Amplification patterns were established for each isolate to accurately identify all strains and to carry out an epidemiological analysis of P . aeruginosa among the selected CF patients . RESULTS: Eighteen different DNA amplification patterns were defined and used to identify each P . aeruginosa strain isolated from the different bronchoalveolar lavage samples . No correlation was observed between the different P . aeruginosa strain genotypes and mucoid or non-mucoid phenotypes, as strains with different phenotypes showed similar amplification patterns . Several strains with different amplification patterns were identified in samples obtained from the same patient, suggesting coinfection with ore than one P . aeruginosa strain . Two siblings with CF shared similargenotypes, suggesting the occurrence of cross- contamination . Similar genotypes of P . aeruginosa strains were isolated throughout the study period . CONCLUSION: Genotypic characterization of P . aeruginosa strains in CF patients allows more accurate epidemiological analyses of this important host-agent relationship.

Antibiot Khimioter, 2003, 48(12), 18 - 21
{Efficacy and safety of cefoperazone/sulbactam in the treatment of children with mucoviscidosis during exacerbation of the bronchopulmonary process}; Semykin SIu et al.; The use of cefoperazone/sulbactam in combination with amikacin in the treatment of 20 patients with mucoviscidosis and exacerbation of bronchopulmonary pathological process resulted in marked positive dynamics of the clinical and functional indices of the lungs state . The bacteriological effect with respect to the main pathogens in the cases of mucoviscidosis was strain-dependent: eradication of 10 (83.4%) out of 12 Staphylococcus aureus strains and only 3 (15.8%) out of 19 Pseudomonas aeruginosa strains . The most frequent adverse reaction was diarrhea (5 children) successfully corrigated by loperamide . Discontinuation of the drug use was required in 3 patients because of macrohematuria (1 child) and allergic eruption.

J Bacteriol, 2004 Jun, 186(12), 4046 - 50
Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa; Firoved AM et al.; The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp . NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.

J Bacteriol, 2004 Jun, 186(12), 3855 - 61
Transcriptome analysis of the ArgR regulon in Pseudomonas aeruginosa; Lu CD et al.; Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration . We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P . aeruginosa . We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine . Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions . The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter . Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system . The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR . Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis . These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds.

J Bacteriol, 2004 Jun, 186(12), 3848 - 54
The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa; Hashim S et al.; The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions . ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate . The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism . Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant . The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments . Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression . Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes . DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site . In silica analysis of genomic information for P . fluorescens, P . putida, and P . stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P . aeruginosa likely apply to other pseudomonads.

Genetika, 2004 Apr, 40(4), 462 - 8
{Comparison of genomes of new gigantic Pseudomonas aeruginosa phages from native populations from different regions}; Krylov VN et al.; To study the genome diversity of bacteriophages from geographically distant natural populations, new giant phi KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (phi KZ, Lin68, EL) . A broad spectrum of lytic activity was demonstrated for all phi KZ-like phages . Phages of the phi KZ species proved to be common in natural populations of various regions, while IL- and Lin68-related phages were extremely rare . Most phi KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments . The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species . Phages isolated in different geographical regions showed no substantial difference . Some phages only slightly differing in DNA restriction pattern from phi KZ may be used to study the origin of phi KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).

Exp Parasitol, 2004 Mar-Apr, 106(3-4), 75 - 84
Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3; Choudhury A et al.; This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie . In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm . Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain . Experiments on larval survivability and viable cell count revealed peak immune response on third day . Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set . The haemolymph of A . assama larvae was analyzed for proteins associated with bacterial infection . Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae . Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae . Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts . General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.

Med Clin (Barc), 2004 May 15, 122(18), 698 - 700
{Patients with cystic fibrosis managed at the cystic fibrosis units of Madrid: cross-sectional study of 387 subjects}; Garcia Hernandez G et al.; BACKGROUND AND OBJECTIVE: Our objective was to describe the clinical and genetics features of patients with cystic fibrosis (CF) attended in Madrid . PATIENTS AND METHOD: This was a descriptive, cross-sectional study of CF patients attended during 2001 . Demographic, genetic, anthropometric, pancreatic insufficiency, diabetes mellitus, Pseudomonas aeruginosa colonization, lung function and body mass index (BMI) (z score) data were recorded and compared with the American CF Registry . RESULTS: 387 patients were included, most of them living in Madrid (n = 247 {63%}), 209 males (54%), with a mean age (SD) of 15.15 (10.42) years (younger than 18 years, 248, 64.24%) . F508del was the most common mutation (52.8% of chromosomes) with 104 homozygous patients . Pancreatic insufficiency was present in 310 subjects (80.1%), diabetes mellitus in 30 (7.8%) and P . aeruginosa colonization in 126 (33.1%) . Lung function was measured in 309 patients: mean of FEV1 and FVC predicted values (SD) was 82.5 (27.11) and 89.32 (21.89), respectively . The mean BMI z score was 0.0796 (1.18) . CONCLUSIONS: CF patients from Madrid have a good nutritional status, less P . aeruginosa colonization, less pancreatic insufficiency and better lung function than those of the American CF Registry . The lower prevalence of homozygous F508del in our population may explain, at least partly, our findings.

Nippon Shokakibyo Gakkai Zasshi, 2004 May, 101(5), 502 - 9
{Strategy for bacterial translocation in acute pancreatitis}; Furuya T et al.; Although bacterial translocation (BT) is thought to be a main cause of secondary pancreatic infection, the clinical significance is still unclear . Therefore, we investigated relationship between pancreatic infection and BT, analyzing the results of treatments such as continuous regional arterial infusion of protease inhibitor and antibiotics (CRAI), selective digestive decontamination, and early enteral nutrition (SDD/EN) in 45 severe acute pancreatitis patients . The infection rate of 17 cases without CRAI, SDD/EN was 58.8%, and mortality was 23.5% . Antibiotics-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) was often proven in infected pancreatic tissue . Whereas, in 16 cases who underwent both therapy, only one patient died of multi-organ failure (6.3%) . Pseudomonas aeruginosa and K . pneumoniae were proven from his feces before pancreatic infection coincidence, and, in autopsy, these bacteria and MRSA were proved from necrotic pancreatic tissue . BT must be large cause of secondary pancreas infection, because CRAI and SDD/ EN prevent secondary pancreatic infection . But we must consider other infection routes, and development of antibiotics resistant bacteria.

J Mass Spectrom, 2004 May, 39(5), 505 - 13
Lipid A components from Pseudomonas aeruginosa PAO1 (serotype O5) and mutant strains investigated by electrospray ionization ion-trap mass spectrometry; Bedoux G et al.; The lipid A components of the Pseudomonas aeruginosa strains PAO1 (wild-type) and derived mutants PAO1 algC::tet and PAO1 PDO100 were isolated after mild acetic acid hydrolysis of LPS . Their structural heterogeneities were characterized using electrospray ionization (ESI) ion-trap mass spectrometry (MS) with direct infusion in the negative ion mode without prior derivatization . The ESI-mass spectra revealed monophosphorylated molecules corresponding to known tetra-, penta- and hexaacylated structures of P . aeruginosa lipid A . The MS/MS fragmentation patterns allowed the location of fatty acyl chains on the disaccharide backbone of lipid A . In addition, a hexaacylated lipid A containing a hexadecanoyl chain was detected for the first time in strain P . aeruginosa PAO1 . With multiple stages of fragmentation (MS(n)), the position of this hexadecanoyl chain O-linked to the decanoyl chain at the C-3(') position of the glucosamine backbone was determined . This sensitive method is suitable to reveal lipid A heterogeneity, i.e . the nature, number and distribution of acyl chains, without prior lipopolysaccharide purification . The lipid A from mutant strains were also characterized and significant differences were shown in the abundance of monophosphorylated lipid A components between the wild-type and the mutant strains .

Antibiot Khimioter, 2004, 49(1), 26 - 9
{3-Year experience of use of cefepime (Maxipime) for the treatment of hospital infections in a specialized surgical hospital}; Mitrokhin SD et al.; Microbiological and clinical monitoring for 3 years (from 2001 to 2003) confirmed high clinical and microbiological efficacy of cefepime (Maxipime, Bristol-Myers-Squibb) in the treatment of infectious complications in patients with solid tumors in an oncologic hospital . It should be noted, however, that high efficacy of cefepime and wide ranges of the indications to its use do not allow to consider it as an agent for the treatment of all possible complications in such patients . The drug is not active against enterococci, not always clinically sufficiently effective in the treatment of Pseudomonas aeruginosa infections, it is impossible to use cefepime in monotherapy of suspected anaerobic infections . Therefore, widespread uncontrolled use of cefepime should be prohibited . It should be prescribed strictly by the indications with the account of the pathogen susceptibility, the infection severity and the recommended doses and regimens . The use of cefepime is undoubtedly valid when other antimicrobials fail or when empirical antimicrobial therapy of severe cases is required, including those under intensive care.

Arch Med Res, 2004 May-Jun, 35(3), 251 - 7
Relationship between clinical and environmental isolates of Pseudomonas aeruginosa in a hospital setting; Ruiz L et al.; BACKGROUND: Populations of Pseudomonas aeruginosa have been extensively studied, although there is no general agreement concerning their genetic structure . It has been proposed that P . aeruginosa is a very homogeneous species with 90% of individuals within the same clonal group; nonetheless, other results suggested that Pseudomonas populations are panmictic . Here we compared P . aeruginosa populations from clinical and environmental samples, both isolated from the Bellvitge Hospital of the University of Barcelona in Spain . METHODS: Antibiotic susceptibility determination as well as whole cell and outer membrane protein denaturing gel electrophoresis, pulsed-field electrophoresis, and random amplified polymorphic DNA analysis were performed . RESULTS: Environmental isolates were much more susceptible to antibiotics than those isolated from clinical specimens . The remainder of the analyses revealed high degree of diversity . CONCLUSIONS: Whole-cell proteins, outer-membrane proteins, and pulsed field electrophoresis did not support a close relationship between clinical and environmental isolates . Random amplified polymorphic DNA (RAPD) confirmed the distance between isolates from both sources . This suggests that the origin of hospital infections by P . aeruginosa is due mainly to growth of bacterial strains acquired by patients prior to hospital admission or from patient-to-patient through healthcare workers (HCWs).

Invest Ophthalmol Vis Sci, 2004 Jun, 45(6), 1897 - 903
Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection; Zhu H et al.; PURPOSE: To understand the importance of Pseudomonas aeruginosa quorum-sensing systems in the development of corneal infection, the genotypic characteristics and pathogenesis of seven ocular isolates with low-protease and acyl homoserine lactone (AHL) activity and quorum-sensing mutants of PAO1 deficient in lasI, lasR, or rhlR were investigated in the study . METHODS: The possession of the quorum-sensing genes lasI, lasR, rhlI, rhlR, and the quorum-sensing controlled genes lasB, aprA, and rhlAB in the clinical isolates were determined by polymerase chain reaction and Southern blot hybridization . Elastinolytic activity, controlled by the las system, was assayed using elastin Congo red and rhamnolipid production controlled by the rhl system was assessed using agar plates containing methylene blue/cetyltrimethyl ammonium bromide . Induction of keratitis was examined in a scarified inbred BALB/c mouse model . RESULTS: The clinical isolates Paer1 and -3 were lasI and lasR negative, and the isolates Paer2 and -4 were rhlR and rhlAB negative . The isolates Paer17, Paer26, 6294 and 6206 possessed all the genes examined . There was no rhamnolipid production in clinical isolates Paer2 and -4 . The isolates Paer1 and -3 were virtually avirulent in the scarified mouse corneas . Using isogenic PAO1 mutants, strain lasI showed a markedly reduced virulence in the corneal infection model . The remainder of the clinical isolates and the lasR or rhlR mutant strains caused severe keratitis . CONCLUSIONS: These results indicate that quorum-sensing deficiency may occur naturally in clinical isolates, and the possession of lasI and hence a functional Las quorum-sensing system may be important in development of corneal infection.

J Dermatol, 2004 Feb, 31(2), 116 - 8
Swan-neck deformity and paresthesia following giant orf; Uzel M et al.; Orf is a zoonotic infection caused by a parapoxvirus that primarily infects sheep and goats . Human orf infection can take place when abraded skin comes into contact with infected animals . It occurs most commonly on the index finger . The characteristic lesion resembles a tumor and resolves spontaneously, usually without any complications . However, rare complications such as lymphangitis, adenitis, erythema multiforme, erysipelas, papulovesicular eruption, Pseudomonas aeruginosa infection, and bullous pemphigoid have been reported . Herein, we report a case of giant orf causing swan-neck deformity and paresthesia . These complications have not been previously reported in the literature.

Microbiol Res, 2004, 159(1), 51 - 7
Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication; Guerin-Mechin L et al.; To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared . Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride . The outer membrane acts as a barrier to prevent these disinfectants from entering the cell . It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate . For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule . Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts . This suggests that the inner membrane could also play a role in resistance to DDAB.

J Infect Chemother, 2004 Apr, 10(2), 76 - 85
Optimization of dose and dose regimen of biapenem based on pharmacokinetic and pharmacodynamic analysis; Takata T et al.; Pharmacokinetic and pharmacodynamic (PK/PD) parameters, which are important indices of the therapeutic efficacy of antimicrobials, and the minimum inhibitory concentration (MIC) predictive of clinical efficacy at common clinical doses, were examined for biapenem (BIPM; 300 mg b.i.d.), imipenem/cilastatin (IPM/CS; 500 mg/500 mg b.i.d.), meropenem (MEPM; 500 mg b.i.d.), and ceftazidime (CAZ; 1000 mg b.i.d.), using a mouse model of thigh infection caused by Pseudomonas aeruginosa . The PK/PD parameter that most closely correlated with the therapeutic efficacy of all these antimicrobials was time above MIC (T > MIC) . The values of T > MIC predictive of clinical efficacy against P . aeruginosa infection varied among antimicrobials and were >/=17%, >/=17%, >/=23%, and >/=33% for BIPM, IPM/CS, MEPM, and CAZ, respectively . From these values and the known plasma concentrations of the antimicrobials in humans after administration at the common clinical doses, the MIC for bacterial strains at which clinical efficacy can be expected was estimated to be </=4.4 microg/ml for BIPM, </=6.1 microg/ml for IPM/CS, </=2.2 microg/ml for MEPM, and </=13.6 microg/ml for CAZ . These MICs nearly coincided with the MIC(80) of the antimicrobials for 104 clinical isolates of P . aeruginosa strains . These results indicate that, even at a low dose, of 300 mg b.i.d., the clinical efficacy of BIPM against P . aeruginosa infection can be expected to be comparable to that of IPM/CS, MEPM, and CAZ.

Ecotoxicol Environ Saf, 2004 Jun, 58(2), 277 - 83
Suboptimal chlorine treatment of drinking water leads to selection of multidrug-resistant Pseudomonas aeruginosa; Shrivastava R et al.; The present study was undertaken to investigate the spectrum of bacteria present in the River Gomti water before and after chlorination for drinking purposes . We observed that the strains of Pseudomonas aeruginosa that survived chlorination on three out of seven occasions were resistant to almost all the antibiotics tested . The chlorine-resistant bacteria had mucoid colonies and grew better at 24 degrees C . All attempts to isolate the plasmid responsible for chlorine resistance were unsuccessful . Laboratory experiments using different strains of the P . aeruginosa in distilled water showed that only the resistant strain survived chlorine treatment at a dose of < or =500 microg/L . Similar results were obtained when water collected from seven different sites on the River Gomti was treated with graded doses of chlorine . At the higher dose of chlorine, all the bacteria died in 30 min, whereas with lower doses all the bacteria survived . The present study underscores the importance of measuring water chlorine concentrations to assure they are sufficiently high to remove pathogenic bacteria from drinking water . To our knowledge, this is the first report in the literature of the selection of multidrug-resistant bacteria by suboptimal chlorine treatment of water.

Ann Vasc Surg, 2004 Jul, 18(4), 497 - 501
In vitro activity and in vivo efficacy of antimicrobial-coated vascular grafts; Darouiche RO et al.; The serious medical consequences and costly management of infections associated with vascular grafts have prompted an expanding interest in examining the preventive efficacy of antimicrobial-coated vascular grafts . The purpose of antimicrobial coating of vascular grafts is to reduce bacterial colonization of the device and, hopefully, the occurrence of clinical infection . In this study we demonstrated that expanded-polytetrafluoroethylene vascular grafts coated with minocycline and rifampin provide broad-spectrum antimicrobial activity in vitro, as reflected by zones of inhibition, against Staphylococcus epidermidis, S . aureus, Enterococcus faecium, and Pseudomonas aeruginosa . We also showed in a rabbit model that subcutaneously placed minocycline/rifampin-coated vascular grafts have lower rates of staphylococcal device colonization (1/24 = 4% vs . 8/30 = 27%, p = 0.033) and device-related infection (0/24 = 0% vs . 6/30 = 20%, p = 0.028) than uncoated grafts . These promising results encourage the clinical evaluation of vascular grafts coated with minocycline and rifampin.

Methods Mol Biol, 2004, 268, 293 - 5
Microbiological analysis of cosmetics; Herrera AG; Cosmetics are products of chemical or natural origin dedicated specifically for use in skin and mucosa . The constant development of the cosmetic industry has generated the necessity to carry out microbiological analysis on the raw materials used in the industrial production of cosmetics as well as the final products, with the purpose of obtaining products of good microbiological quality.Cosmetic products are recognized to be substrates for the survival and development of a large variety of microorganisms, since they possess some of the nutrients that facilitate growth such as: lipids, polysaccharides, alcohol, proteins, amino acids, glucosides, esteroids, peptides, and vitamins . Also, the conditions of readiness (oxygenation, pH, temperature, osmotic degree, superficial activity, perfume, and essential oils) present in the cosmetic products favor microbial multiplication.Routine analyses to determine the microbiological quality of a cosmetic product include the following: Count of mesophilic aerobic microorganisms . Most probable number (MPN) of total coliforms . Count of molds and yeasts . Absence/presence of Staphylococcus aureus probe . Absence/presence of Pseudomonas aeruginosa probe.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2069 - 74
Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates; Yamane K et al.; Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997 . These strains harbor the newly identified 16S rRNA methylase gene (rmtA) . When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed . The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared . In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na(+)/H(+) antiporter gene-like sequence (sequence 2) was identified downstream of rmtA . This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp kappagamma elements . Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus . In strain AR-11, the rmtA gene and sequence 2 were found, but the 5' end of the orfA gene was truncated and replaced with IS6100 . An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11 . The G+C content of the rmtA gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2043 - 8
Biochemical characterization of the naturally occurring oxacillinase OXA-50 of Pseudomonas aeruginosa; Girlich D et al.; The bla(OXA-50) gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1 . By using a mutant strain of P . aeruginosa PAO1 that had an inactivated bla(AmpC) cephalosporinase gene, the bla(OXA-50) gene was shown to be expressed constitutively in P . aeruginosa . This beta-lactamase gene was cloned onto a multicopy plasmid and expressed in P . aeruginosa and Escherichia coli . It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P . aeruginosa but not in E . coli . Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 beta-lactamase . It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level . Very similar oxacillinase genes were identified in all P . aeruginosa isolates from various geographical origins tested . The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring bla(AmpC) cephalosporinase gene of P . aeruginosa . The study indicated that P . aeruginosa harbors two naturally encoded beta-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.

J Am Acad Dermatol, 2004 Jun, 50(6), 845 - 9
Microbiologic evaluation of skin wounds: alarming trend toward antibiotic resistance in an inpatient dermatology service during a 10-year period; Valencia IC et al.; BACKGROUND: Increasing resistance to commonly used antibiotics has been seen for patients with superficial skin wounds and leg ulcers . OBJECTIVES: We sought to evaluate bacterial isolates from leg ulcers and superficial wounds for resistance to commonly used antibiotics and to compare current data with previous data . METHODS: We performed a chart review for patients admitted to a tertiary care dermatology inpatient unit from January to December 2001 . Comparison was made with 2 previous surveys of the same inpatient service from 1992 and 1996 . RESULTS: Bacterial isolates were cultured from 148 patients, 84% (72 of 86) with leg ulcers and 38% (76 of 202) with superficial wounds . Staphylococcus aureus and Pseudomonas aeruginosa were the most common bacterial isolates in both groups . For patients with leg ulcers, S aureus grew in 67% of isolates (48/72) of which 75% (36/48) were methicillin-resistant (MRSA) . Of leg ulcers, 35% (25/72) grew P aeruginosa, which was resistant to quinolones in 56% of cultures (14/25) . For patients with superficial wounds, S aureus was isolated in 75% (57/76) and 44% were MRSA (25/57) . P aeruginosa grew in 17% of isolates (13/76) and was resistant to quinolones in 18% . We found a marked increase in antibiotic resistance for both leg ulcers and superficial wounds . Over time, MRSA increased in leg ulcers from 26% in 1992 to 75% in 2001 . For superficial wounds, MRSA increased from 7% in 1992 to 44% in 2001 . P aeruginosa resistance to quinolones in leg ulcers increased from 19% in 1992 to 56% in 2001, whereas for superficial wounds there was no resistance in 1992 and 18% resistance in 2001 . CONCLUSION: Rapid emergence of antibiotic-resistant bacteria continues and is a problem of increasing significance in dermatology . Common pathogenic bacteria, S aureus and P aeruginosa, showed increased resistance to commonly used antibiotics . Selection of antibiotics should be on the basis of local surveillance programs.

Med Clin (Barc), 2004 May 8, 122(17), 648 - 52
{Home intravenous antibiotic treatments in cystic fibrosis units of Madrid}; Giron RM et al.; BACKGROUND AND OBJECTIVE: A descriptive study of the home intravenous antibiotic treatment (HIVAT) course in cystic fibrosis (CF) units of Madrid in an 18 month period . Different patient features were recorded, antibiotherapy, intravenous access, complications and their resolutions . We accessed the improvement of the pulmonary function, forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) at the end of the treatment . PATIENTS AND METHOD: For an 18 months period (January 2002-June 2003) the patients with CF who received HIVAT and fulfilled the previously fixed criteria were included . The next clinic variables were collected: age, sex, bacterial colonization of respiratory tree, pancreatic function and pulmonary function in steady phase, before and after HIAT . RESULTS: 56 patients, 31 male and 25 female, were given 90 courses of HIAT (34 patients received only one course) . Mean age was 20.06 (8.07) years . 57.1% of the patients were colonized by Pseudomonas aeruginosa . The most frequently used antibiotics were ceftazidime and tobramycin . Courses of treatment lasted a mean of 4.08 (5.09) days inpatient and 11.89 (4.96) at home . In 87.7% of the course were used the intravenous cannulae, Port-a-cath in 6.7% and Venocath in 7.8% . The intravenous access was replaced, in the 64.4% of the cases, by the nurse of the CF Unit, in the 21.4% in the emergency room of the nearest hospital and in 11.9% in primary care center . Three occasions skin reactions were reported . The parameters of pulmonary function improved significatively after HIVAT . CONCLUSIONS: The HIVAT is a therapeutic option that, following predetermined inclusion criteria, has a low complication rate and improves pulmonary function.

Protein Sci, 2004 Jun, 13(6), 1547 - 56
The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol; Levin I et al.; Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme . We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 A, using the molecular replacement method . The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry . The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria . The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (k(cat) = 4.5 sec(-1); Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate . The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path . The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.

J Bacteriol, 2004 Jun, 186(11), 3653 - 5
The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv . phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK; Hernandez-Flores JL et al.; In Pseudomonas syringae pv . phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa . However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.

Int J Med Microbiol, 2004 Apr, 293(7-8), 479 - 82
The multi-talented bacterial adenylate cyclases; Lory S et al.; Bacterial pathogens produce a variety of toxins capable of altering the levels of cAMP in the cells of infected hosts . Moreover, cAMP is an important signaling molecule in many bacterial species, involved in regulation of gene expression in response to a variety of environmental stimuli . The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes three adenylate cyclases . One of these is exoenzyme Y, which is translocated into the host cell via a type III secretion system (TTSS) . The other two cyclases are CyaA and CyaB, that generate cAMP for intracellular signaling, and together with the cognate cAMP-binding protein Vfr, control the expression of the TTSS and several virulence factors . Using a mouse infection model, it was shown that CyaB, a membrane-bound class III adenylate cyclase plays a more prominent role in regulation of TTSS-encoding genes than CyaA . Given the wide distribution of the class III adenylate cyclases among bacteria, cAMP-dependent regulation of gene expression may have evolved as a conserved mechanism for sensing environmental signals ranging from nutritional content of the surrounding media to the presence of host tissues.

Chang Gung Med J, 2004 Mar, 27(3), 182 - 7
Pseudomonas aeruginosa corneal ulcer related to overnight orthokeratology; Hsiao CH et al.; BACKGROUND: Overnight orthokeratology was thought to be a safe and non-invasive alternative for low-grade myopia and astigmatism correction . We assessed histories, clinical courses, and visual outcomes of the patients with pseudomonal keratitis related to overnight orthokeratology . METHODS: The records of six patients with pseudomonal keratitis related to overnight orthokeratology were reviewed from January 2001 through December 2002 . RESULTS: The average age of the patients was 13 years . The average period between the time that the patient started the overnight orthokeratology program and the onset of infectious keratitis was 17 months . All patients presented with painful red eyes . The area of the corneal ulcer was central in three, and paracentral in three eyes . The corneal infiltrate was small in one eye, and medium in five eyes . The corneal scrapings from these six patients revealed Pseudomonas aeruginosa . All patients responded well to topical antibiotic treatment . Two of six eyes had a final visual acuity within two lines of the pre-infection vision at the last follow-up . Four of the eyes examined lost their best-corrected visual acuity due to central corneal scar or irregular astigmatism . CONCLUSIONS: Overnight orthokeratology contact lens wear has the potential complication of pseudomonal keratitis and may cause significant visual impairment.

Methods Mol Biol, 2004, 266, 289 - 304
Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies; Lehoux DE et al.; Microbial pathogens possess a repertoire of virulence determinants that make unique contributions to bacterial fitness during infection . In this chapter, we focus on the recent progress and adaptations of signature-tagged mutagenesis (STM) by PCR instead of hybridization . This is a PCR-based STM mutation-based screening method using a population of bacterial mutants for the simultaneous identification of multiple virulence genes in microbial pathogens by negative selection . Modifications of STM developed in our laboratory have been applied to Pseudomonas aeruginosa PAO1 . Screening of a collection of 6912 STM mutants in the rat chronic lung model of infection identified 214 P . aeruginosa STM mutants defective in virulence . For further studies, and to illustrate better the strategies that need to be utilized, we present detailed analysis of nine selected STM mutants . The data obtained indicate that in vivo, defects in virulence give a wide variety of phenotypes: defects in known virulence factors have been found, thereby validating the method; defects have also been found in orthologs with predicted functions, and in some genes whose functions cannot be predicted from databases . A general strategy and a simple scenario is discussed using the nine STM mutants selected for further characterization . PCR-based STM represent a genomics-based method for in vivo high-throughput screening of new virulence factors.

J Biol Chem, 2004 Jul 30, 279(31), 32100 - 5 Epub 2004 May 17.
The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro; Friedman YE et al.; The Fur protein represses transcription of iron-responsive genes in bacteria . The discovery that Fur is a zinc metalloprotein and the use of surrogate metals for Fe(2+) for in vitro studies question whether Fur is a direct iron sensor . In the present study, we show that the affinity of Fur from Bradyrhizobium japonicum (BjFur) for its target DNA increases 30-fold in the presence of metal, with a K(d) value of about 2 nM . DNase I footprinting experiments showed that BjFur protected its binding site within the irr gene promoter in the presence of Fe(2+) but not in the absence of metal, showing that DNA binding is Fe(2+)-dependent . BjFur did not inhibit in vitro transcription from the irr promoter using purified components in the absence of metal, but BjFur repressed transcription in the presence of Fe(2+) . Thus, BjFur is an iron-responsive transcriptional repressor in vitro . A regulatory Fe(2+)-binding site (site 1) and a structural Zn(2+)-binding site (site 2) inferred from the recent crystal structure of Fur from Pseudomonas aeruginosa are composed of amino acids highly conserved in many Fur proteins, including BjFur . BjFur mutants containing substitutions in site 1 (BjFurS1) or site 2 (BjFurS2) bound DNA with high affinity and repressed transcription in vitro in an Fe(2+)-dependent manner . Interestingly, only a single dimer of BjFurS2 occupied the irr promoter, whereas the wild type and BjFurS1 displayed one- or two-dimer occupancy . We suggest that the putative functions for metal-binding sites deduced from the structure of P . aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole.

J Clin Invest, 2004 May, 113(10), 1482 - 9
TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells; Soong G et al.; Toll-like receptors (TLRs) mediate host responses to bacterial gene products . As the airway epithelium is potentially exposed to many diverse inhaled bacteria, TLRs involved in defense of the airways must be broadly responsive, available at the exposed apical surface of the cells, and highly regulated to prevent activation following trivial encounters with bacteria . We demonstrate that TLR2 is enriched in caveolin-1-associated lipid raft microdomains presented on the apical surface of airway epithelial cells after bacterial infection . These receptor complexes include myeloid differentiation protein (MyD88), interleukin-1 receptor-activated kinase-1, and TNF receptor-associated factor 6 . The signaling capabilities of TLR2 are amplified through its association with the asialoganglioside gangliotetraosylceramide (Gal beta 1,2GalNAc beta 1,4Gal beta 1,4Glc beta 1,1Cer), which has receptor function itself for many pulmonary pathogens . Ligation of either TLR2 or asialoGM1 by ligands with specificity for either receptor, by Pseudomonas aeruginosa, or by Staphylococcus aureus stimulates IL-8 production through activation of NF-kappa B, as mediated by TLR2 and MyD88 . Thus, TLR2 in association with asialo-glycolipids presented within the context of lipid rafts provides a broadly responsive signaling complex at the apical surfaces of airway cells to initiate the host response to potential bacterial infection.

Int J Food Microbiol, 2004 May 1, 92(3), 365 - 73
Heterotrophic plate count and consumer's health under special consideration of water softeners; Hambsch B et al.; The phenomenon of bacterial growth in water softeners is well known since years . To upgrade the hygienic safety of water softeners, the German DIN Standard 19636 was developed, to assure that the distribution system could not be contaminated by these devices and that the drinking water to be used in the household still meets the microbiological standards according to the German drinking water guidelines, i.e . among others heterotrophic plate count (HPC) below 100 CFU/ml . Moreover, the standard for the water softeners includes a test for contamination with Pseudomonas aeruginosa which has to be disinfected during the regeneration phase . This is possible by sanitizing the resin bed during regeneration by producing chlorine . The results of the last 10 years of tests of water softeners according to DIN 19636 showed that it is possible to produce water softeners that comply with that standard . Approximately 60% of the tested models were accepted . P . aeruginosa is used as an indicator for potentially pathogenic bacteria being able to grow also in low nutrient conditions which normally prevail in drinking water . Like other heterotrophs, the numbers of P . aeruginosa increase rapidly as stagnation occurs . Normally P . aeruginosa is not present in the distributed drinking water . However, under certain conditions, P . aeruginosa can be introduced into the drinking water distribution system, for instance, during construction work . The occurrence of P . aeruginosa is shown in different cases in treatment plants, public drinking water systems and in-house installations . The compliance with DIN 19636 provides assurance that a water softener will not be a constant source of contamination, even if it is once inoculated with a potentially pathogenic bacterium like P . aeruginosa .

Burns, 2004 Jun, 30(4), 357 - 61
Changes of microbial flora and wound colonization in burned patients; Erol S et al.; To determine time related changes of microbial colonization of burn wounds and body flora of burned patients, a prospective study was carried out . Fifty-one patients who were hospitalized at least 3 weeks were enrolled in the study . Periodic swabs were taken from burn wound, nasal, axillary, inguinal, and umbilical regions of the patients on admission and 7th, 14th, and 21st days of hospitalization . The mean body surface area burned was 22.9% . 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 . There was a gradual decrease in the number of isolates of CNS and a marked increase in the numbers of S . aureus and Pseudomonas aeruginosa from admission to 21st day . At the 21st day, the most frequent organisms were S . aureus (37.6%), CNS (34.7%), and P . aeruginosa (16.2%) . Methicillin resistance of staphylococci strains were increased constantly in study period . While 35.3% of burn wounds were sterile on admission, microbial colonization reached 86.3% within the first week . Nasal carriage of methicillin resistant S . aureus increased from 3.9% to 62.7% at 21st day . The nature of microbial wound colonization and flora changes should be taken into consideration in empirical antimicrobial therapy of burned patients.

J Am Soc Mass Spectrom, 2004 Jun, 15(6), 862 - 9
Electrospray/mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines (HAQs) produced by Pseudomonas aeruginosa; Lepine F et al.; The opportunistic pathogen Pseudomonas aeruginosa produces a large array of 4-hydroxy-2-alkylquinolines (HAQs) . These compounds were analyzed by LC/MS, using positive electrospray ionization, in the culture supernatant of strain PA14 . Fifty-six HAQs and related compounds were detected and their {M + H}(+) ions were further analyzed by collision induced dissociation (CID) . These HAQs were grouped into five different series based on the presence of an hydrogen or hydroxyl group at the 3 position, an N-oxide group in place of the quinoline nitrogen, and an unsaturation on their alkyl side chain . Two new analogs of 3,4-dihydroxy-2 heptylquinoline, the Pseudomonas quinolone signal (PQS), were found with an alkyl chain longer by one and two methylene groups . Moreover, two additional series of compounds were identified in which a saturated or unsaturated alkyl side chain is located at the 3 position along with an hydroxyl group at the 3 position and a ketone at the 2 position . No HAQ N-oxides, nor any compounds from the latter two series, were detected in a pqsL mutant derivative of PA14, indicating that this gene is involved in the biosynthesis of these compounds . This work demonstrates the large repertoire of HAQ and HAQ-related compounds produced by P . aeruginosa, and provides insight into N-oxides biosynthesis and confirm the hypothesis that N-oxides are the precursors of compounds from Series 6 and 7.

J Mol Recognit, 2004 May-Jun, 17(3), 167 - 73
The tunnelling conductance of molecularly ordered metalloprotein arrays; Davis JJ et al.; Metalloproteins can be self-assembled in molecularly ordered, electrochemically addressable arrays . We report here on a study of the transport characteristics of the blue copper protein, azurin, from Pseudomonas aeruginosa, by a combination of electrochemical and scanning probe techniques (scanning tunnelling microscopy and conducting atomic force microscopy) . Redox-switchable chemisorbed molecular arrays can be formed from both wild-type and mutant proteins using the strong affinity of cysteine residue thiolates for pristine gold surfaces . The molecular transconductance of single protein molecules within these arrays has been studied under controllable conditions where it has been additionally possible to resolve the effects of protein mechanical perturbation . Although tunnelling appears to be non-resonant and adequately explained through the use of a square barrier model, under some conditions the contribution of the redox-active copper centre to conductance is resolvable .

Diagn Microbiol Infect Dis, 2004 May, 49(1), 67 - 70
Effect of antibiotic sequence on combination regimens against Pseudomonas aeruginosa in a multiple-dose, in vitro infection model; Zelenitsky SA et al.; The goal of this study was to investigate the effect of antibiotic sequence on combination regimens against Pseudomonas aeruginosa in an in vitro infection model . Ceftazidime plus ciprofloxacin and ceftazidime plus tobramycin were dosed every 12 h for 48 h using simultaneous or staggered administration . Simultaneous dosing and ceftazidime followed by ciprofloxacin or tobramycin were significantly more active at both 24 h (p = 0.03) and 48 h (p < 0.0001) than ciprofloxacin or tobramycin followed by ceftazidime . Final bacterial kill was sixfold greater with the former regimens . This study showed that antibiotic sequence had a significant and class dependent effect on antibacterial response . The clinical relevance of these observations warrants further investigations in animal models.

Protein Expr Purif, 2004 Jun, 35(2), 373 - 80
Isolation of aminoglycoside nucleotidyltransferase (2'')-Ia from inclusion bodies as active, monomeric enzyme; Wright E et al.; Aminoglycoside nucleotidyltransferase( 2'')-Ia (ANT( 2'') confers resistance to pathogenic bacteria against several aminoglycoside antibiotics including gentamicin, kanamycin, and tobramycin . The gene for this aminoglycoside-modifying enzyme has been cloned from a clinical isolate of Pseudomonas aeruginosa . This gene was inserted into an overexpression vector, the vector was then transformed into Escherichia coli BL21(DE3), and the protein has been isolated in the form of inclusion bodies . Optimal refolding conditions have been determined to be direct dilution of solubilized inclusion bodies into 0.1M Tris-HCl, pH 8.5, 0.2M KCl, 0.4M l-arginine, and 5mM reduced glutathione at 4 degrees C . The refolded enzyme is monomeric in solution and has similar kinetic properties and substrate selectivity to the enzyme isolated in soluble form.

Free Radic Biol Med, 2004 Jun 1, 36(11), 1448 - 59
Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide; Reszka KJ et al.; Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis . Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs . We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite . The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore . Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself . That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2 . In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation . This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal . This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.

Cas Lek Cesk, 2004, 143(3), 187 - 90
{Evaluation of three dosage regimens of amikacin using pharmacokinetic models in patients with cystic fibrosis}; Halacova M et al.; BACKGROUND: Once-daily administration of aminoglykosides is routinely used, but comparative efficacy data for patients with cystic fibrosis are not available . METHODS AND RESULTS: The aim of the this study was to compare the predicted pharmacodynamic (PD) activity of amikacin at 28 mg/kg/den administered every 24 hod.(q24h), q12h, and q8h . Pharmacokinetic (PK) data were derived from analysis of the amikacin serum concentration from 42 CF children patients . Individual pharmacokinetics values were used to construct serum concentration--versus time curves and to determine various indices (c peak/MIC ratio and time during the concentration was less than the MIC--T < MIC) for all three dose regimens described above . MIC (minimal inhibitory concentration) for Pseudomonas aeruginosa was 4 mg/l . Significantly lower c peak/MIC but shorter T < MIC were noted when regimens of q8h versus q12h (p < 0.001), q8h vs . q24h (p < 0.001) and q12h vs . q24h (p < 0.001) were compared . This analysis suggests that the potential advantage of achieving a greater c peak/MIC with once-daily aminoglycoside administration may be neutralized by the significantly greater T < MIC in CF patients compared with that achieved with multiple-daily-dosing regimens . CONCLUSIONS: Routine use of once daily amikacin administration could not be recommended until the clinical data confirming efficiency of this dose modality are available.

Microbiology, 2004 May, 150(Pt 5), 1327 - 38
Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa; Smania AM et al.; MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity . In this work it is shown that the disruption of the P . aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies . Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization . One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming . Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event . It is also shown that these P . aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P . aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells . It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants . By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations . Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P . aeruginosa CF isolates is discussed.

J Clin Microbiol, 2004 May, 42(5), 2074 - 9
PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients; Spilker T et al.; Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients . Much of CF patient management depends on accurate identification of P . aeruginosa from sputum culture . However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species . To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification . Both assays yielded DNA fragments of the predicted size . We tested 42 culture collection strains (including 14 P . aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum . Based on these 85 strains, the specificity and sensitivity of both assays were 100% . To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates . The results indicated that preliminary phenotypic testing had misidentified several isolates . The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays . Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P . aeruginosa . Both assays show 100% sensitivity and specificity.

J Antimicrob Chemother, 2004 Jun, 53(6), 1098 - 100 Epub 2004 May 05.
Adjunctive efficacy of granulocyte colony-stimulating factor on treatment of Pseudomonas aeruginosa pneumonia in neutropenic and non-neutropenic hosts; Babalola CP et al.; OBJECTIVES: Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation of neutrophils and enhances their phagocytic and microcidal activity . Increasing resistance to existing antibacterials and the dearth of new alternatives have complicated the treatment of Gram-negative infections . The aim of this study was to evaluate the efficacy of G-CSF in the treatment of Pseudomonas aeruginosa pneumonia when administered in combination with ceftazidime in both neutropenic and non-neutropenic hosts . METHODS: A group of mice were rendered neutropenic with cyclophosphamide . Pneumonia was induced by intratracheal instillation of approximately 5 x 10(7) cfu/mL and approximately 5 x 10(9) cfu/mL (LD(100)) of the organism to neutropenic and non-neutropenic mice, respectively . Two hours after inoculation, the mice received normal saline and 5% dextrose, G-CSF (300 micro g/kg per day x 3 days), ceftazidime (2000 mg/kg x 2 doses) or a combination of G-CSF and ceftazidime . Survival was monitored at different time points for 5 days . RESULTS: Treatment with G-CSF showed a dose-dependent increase in survival from 50 to 300 micro g/kg . In neutropenic mice, survival was markedly better in the G-CSF + ceftazidime group compared with controls (P = 0.0001), G-CSF (P = 0.0002) or ceftazidime (P = 0.0172) . In non-neutropenic mice, survival in the G-CSF + ceftazidime group (20%) was significantly higher than in the control and G-CSF groups (P = 0.0001) but not significantly higher than ceftazidime alone (9%) (P > 0.05) . CONCLUSIONS: G-CSF administered in combination with antibiotic after onset of severe P . aeruginosa pneumonia may improve therapeutic outcome and this suggests a new treatment option in the management of pneumonia especially in neutropenic patients.

Laryngoscope, 2004 May, 114(5), 850 - 4
Acute otitis externa: efficacy and tolerability of N-chlorotaurine, a novel endogenous antiseptic agent; Neher A et al.; OBJECTIVE: The study's objective was to test the tolerability and efficacy of the endogenous antiseptic N-chlorotaurine (NCT) in comparison with a standard clinical treatment according to a phase IIb clinical trial protocol . STUDY DESIGN: The antimicrobial agent NCT was compared with the antibiotic component drops Otosporin (containing neomycin, polymyxin B, and hydrocortisone) for topical treatment of acute otitis externa in a randomized and rater-blinded clinical study . METHODS: Fifty patients suffering from acute otitis externa were divided into two groups according to a randomized list . The test group was treated with 1 mL of 1% aqueous NCT solution, the reference group with 1 mL of Otosporin . The substances were applied to the external ear canal at one daily session until the signs of infection disappeared . Efficacy and tolerability were evaluated daily by visual analogue scale and a six-step infection score . In addition, smears were analyzed to identify the causative pathogens . RESULTS: Both medications were equally well tolerated by the patients . The treatment was successful for all patients of the NCT group, whereas in one patient from the reference group, the infection did not disappear . The inflammation score improved more rapidly in the NCT group, which resulted in an earlier termination of the therapy . This difference became highly significant on days 4 to 7 (P <.01 each) . Time needed for disappearance of inflammation (score 0) was 5.6 +/- 1.6 (mean +/- SD, range 3-9) days in the NCT group and 7.4 +/- 1.6 (range 4-10) days in the Otosporin group (P <.001) . As expected, microbiologic cultures from ear swabs revealed Pseudomonas aeruginosa (58%) followed by Staphylococcus aureus (18%) as the main causative pathogens . CONCLUSIONS: NCT appears to be well tolerated and more effective than the therapy using antibiotic component drops . Because of its endogenous nature and its higher efficacy, NCT appears to be a good choice for topical treatment of acute otitis externa.

J Bacteriol, 2004 May, 186(10), 3270 - 3
Isolation and characterization of a generalized transducing phage for Pseudomonas aeruginosa strains PAO1 and PA14; Budzik JM et al.; A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa . A clear-plaque variant of this bacteriophage was isolated . DMS3 is capable of mediating generalized transduction within and between P . aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P . aeruginosa.

J Bacteriol, 2004 May, 186(10), 2973 - 83
Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB; Nehme D et al.; The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P . aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer . A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization . Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein . Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization) . In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C . A . Elkins and H . Nikaido, J . Bacteriol . 185:5349-5356, 2003) . Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB . Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.

J Bacteriol, 2004 May, 186(10), 2936 - 45
Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1; Heurlier K et al.; In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules . RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P . aeruginosa . An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P . aeruginosa . A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P . aeruginosa . Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation . Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

J Am Chem Soc, 2004 May 12, 126(18), 5859 - 66
X-ray magnetic circular dichroism of Pseudomonas aeruginosa nickel(II) azurin; Funk T et al.; We show that X-ray magnetic circular dichroism (XMCD) can be employed to probe the oxidation states and other electronic structural features of nickel active sites in proteins . As a calibration standard, we have measured XMCD and X-ray absorption (XAS) spectra for the nickel(II) derivative of Pseudomonas aeruginosa azurin (NiAz) . Our analysis of these spectra confirms that the electronic ground state of NiAz is high-spin (S = 1); we also find that the L(3)-centroid energy is 853.1(1) eV, the branching ratio is 0.722(4), and the magnetic moment is 1.9(4) mu(B) . Density functional theory (DFT) calculations on model NiAz structures establish that orbitals 3d(x2-y2) and 3d(z2) are the two valence holes in the high-spin Ni(II) ground state, and in accord with the experimentally determined orbital magnetic moment, the DFT results also demonstrate that both holes are highly delocalized, with 3d(x2-y2) having much greater ligand character.

J Biol Chem, 2004 Jul 16, 279(29), 30871 - 9 Epub 2004 May 03.
Degradation of pulmonary surfactant protein D by Pseudomonas aeruginosa elastase abrogates innate immune function; Alcorn JF et al.; The alveolar epithelium is lined by surfactant, a lipoprotein complex that both reduces surface tension and mediates several innate immune functions including bacterial aggregation, alteration of alveolar macrophage function, and regulation of bacterial clearance . Surfactant protein-D (SP-D) participates in several of these immune functions, and specifically it enhances the clearance of the pulmonary pathogen Pseudomonas aeruginosa, a common cause of morbidity and mortality in cystic fibrosis (CF) patients . P . aeruginosa secretes a variety of virulence factors including elastase, a zinc-metalloprotease, which degrades both SP-A and SP-D . Here we show that SP-D is cleaved by elastase to produce a stable 35-kDa fragment in a time-, temperature-, and dose-dependent manner . Degradation is inhibited by divalent metal cations, a metal chelator, and the elastase inhibitor, phosphoramidon . Sequencing the SP-D degradation products localized the major cleavage sites to the C-terminal lectin domain . The SP-D fragment fails to bind or aggregate bacteria that are aggregated by intact SP-D . SP-D fragment is observed when normal rat bronchoalveolar lavage (BAL) is treated with Pseudomonas aeruginosa elastase, and SP-D fragments are present in the BAL of CF lung allograft patients . These data show that degradation of SP-D occurs in the BAL environment and that degradation eliminates many normal immune functions of SP-D.

J Biol Chem, 2004 Jul 9, 279(28), 29351 - 8 Epub 2004 May 03.
Conserved amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization and enzyme activity of neutral ceramidase; Tani M et al.; Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling . Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH(2)-terminal anchoring region (Tani, M., Iida, H., and Ito, M . (2003) J . Biol . Chem . 278, 10523-10530) . We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase . Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells . Point mutation analysis revealed that Ile(758), the 4(th) amino acid residue from the COOH terminus, and Phe(756) are essential for the enzyme to function . The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus . Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome . It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution.

Ann Pharmacother, 2004 Jun, 38(6), 992 - 5 Epub 2004 Apr 30.
Intrathecal amikacin for the treatment of pseudomonal meningitis; Corpus KA et al.; OBJECTIVE: To report a case of gram-negative bacillary meningitis (GNBM) secondary to multidrug-resistant Pseudomonas aeruginosa that was treated with intravenous meropenem and intrathecal and intravenous amikacin . CASE SUMMARY: A 76-year-old Arabic woman with previous placement of an extraventricular device developed meningitis secondary to P . aeruginosa as a result of a previous pneumonia . The patient was treated with intravenous meropenem and amikacin, with the addition of intrathecal amikacin, until cerebrospinal cultures remained negative for 18 days . She did not experience any adverse effects as a result of the administration of the intrathecal amikacin . Although the meningitis subsequently resolved, the patient eventually died due to Candida glabrata fungemia . DISCUSSION: Dual therapy is recommended for patients with P . aeruginosa meningitis . In our patient, the increasing resistance to imipenem and resistance to all other potential antibiotics resulted in the use of an alternative administration technique that has not been well documented in recent literature . CONCLUSIONS: In patients who have GNBM due to P . aeruginosa, the combination of intrathecal and intravenous amikacin may be an option for therapy, especially when clinical options are limited by resistance, severity of illness, and location of the infection . More information is required and further study is needed on this topicPublication Types:

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