Pseudomonas aeruginosa

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 102 - 6 Epub 2003 Jan 14.
Antimicrobial efficacy of a silver-zeolite matrix coating on stainless steel; Cowan MM et al.; A silver- and zinc-containing zeolite matrix (AgION) used as a coating for stainless steel was tested for antimicrobial efficacy against Escherichia coli 25922, Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, and Listeria monocytogenes 7644 . Assays were performed on flat coupon surfaces and in formed steel cups . AgION reduced microbial colony-forming units when compared to uncoated steel surfaces under all conditions tested . Percent reductions ranged from 84.536 to 99.999 after 4 h exposure, and from 99.992 to 100 after 24 h in all cases . The durability of the coatings declined most markedly when the coating had been applied with a wet process and scrubbed between uses with a test tube brush . Powder-coated surfaces cleaned with a towel retained a high degree of activity after five cycles of use.

Ann Med Interne (Paris), 2002 Dec, 153(8), 537 - 9
{Cutaneous and osteoarticular Scedosporium apiospermum infection}; Fays S et al.; Scedosporium apiospermum is a widely distributed fungus that can be found in the soil, manure and decaying vegetation . Human infection with this fungus is facilited by immunodepression . A 65-year-old man, who was taking oral methylprednisolone for rheumatoid polyarthritis had for a few months ulcerated or suppurative nodules whose incision discharged a thick honey-colored exudate . An ulceration over the first right metatarsophalangian articulation had left the bone exposed . The treatment for Pseudomonas aeruginosa, initially isolated in the exudate was unsuccessful . Other microbiology samples exhibited Scedosporium apiospermum, without bacteria . The pathogenic nature of the infection was proven on a skin and bone (head of the first metatarsian) biopsy showing numerous branching and septate hyphae . The patient was successfully treated by itraconazole . Scedosporium apiospermum is the cause of a growing number of human infections due to widespread use of immunosuppressors . Skin and lung localizations predominate . Osteoarticular infection is relatively rare, which contributes to the originality of this observation . Treatment is not well defined and essentially combines surgical drainage with antifungals like itraconazole . This emergent fungal infection, which has non specific clinical manifestations, must be considered in immunocompromised patients.

Biophys J, 2003 Mar, 84(3), 1765 - 72
Molecular basis for microbial adhesion to geochemical surfaces: computer simulation of Pseudomonas aeruginosa adhesion to goethite; Shroll RM et al.; The adhesion of Pseudomonas aeruginosa to the goethite mineral is investigated using classical molecular simulation . A fragment model for goethite has been integrated into a fully atomistic membrane model . Properties for the resulting system are evaluated for a 1.5-ns simulation in the isothermal-isobaric ensemble . The response of the membrane to the presence of the mineral is investigated . Radial distribution functions are used to present an average picture of the hydrogen bonding . Orientational vectors, assigned to the saccharide groups, reveal the extent of the mineral's perturbations on the membrane . Significant structural changes were observed for the outermost saccharide groups, several of which rotate to form hydrogen bonds with the mineral surface . The structure of the inner core, and the corresponding integrity of the membrane, is maintained . The mineral surface dehydrates slightly in the presence of the membrane as saccharide hydroxyl groups compete with water molecules for hydrogen-bonding sites on its surface.

FEBS Lett, 2003 Feb 27, 537(1-3), 182 - 6
Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS; Lebedev DV et al.; The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering . A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves . The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.

Cornea, 2003 Mar, 22(2), 131 - 4
Phenotype of Pseudomonas aeruginosa isolates causing corneal infection between 1997 and 2000; Cowell BA et al.; PURPOSE: To investigate the relationship between functional phenotype of and the associated human corneal infection . METHODS: This was an experimental pilot study of patients presenting with corneal infections at the Jules Stein Eye Institute with presumed infection during the period from 12/30/97 to 9/1/00 . Thirteen patients were admitted to the study based on positive identification of the causative pathogen as and patient consent . Data were collected (including bacterial cultures, lens wear schedule and care, gender and age, completed history questionnaire, clinical photographs) . Statistical analysis of possible correlations was performed . Phenotypes of were determined, and clinical factors associated with infection were explored . RESULTS: Both invasive and cytotoxic phenotypes of were isolated in equal proportion . Cytotoxic strains and invasive strains were found to be associated with patients younger than 50 years of age and older than 50 years of age, respectively . CONCLUSIONS: remains a significant pathogen in corneal infection, especially during contact lens wear . The age of the patient may influence the phenotype of causing infection . Since invasive and cytotoxic strains have different effects on corneal cells, treatment of the infection might require different approaches depending on this phenotype of the causative bacteria.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 1140 - 2
Postantibiotic effects of garenoxacin (BMS-284756) against 12 gram-positive or -negative organisms; Pankuch GA et al.; Conventional in vitro methods were used to determine the postantibiotic effects (PAEs), sub-MIC effects (SMEs), and postantibiotic sub-MIC effects (PA-SMEs) of garenoxacin for a range of organisms . The mean PAEs of garenoxacin for pneumococci, staphylococci, and enterococci were 0.3 to 2.2 h . For Escherichia coli and Pseudomonas aeruginosa, the PAEs were 0.9 to 1.6 h . The mean PA-SMEs (0.4 times the MIC) for pneumococci, staphylococci, and enterococci were 3.0 to >10 h, 1.8 to >10.7 h, and 5.8 h, respectively, while those for E . coli and P . aeruginosa were 7.6 and 4.4 h, respectively.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 1101 - 11
Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins; Jo JT et al.; The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including beta-lactams, chloramphenicol, macrolides, quinolones, and tetracycline . The MexX-MexY linker-pump combination has been shown to be involved in intrinsic resistance to aminoglycosides, but the identity of the cognate outer membrane channel component remains under debate . Fourteen uncharacterized OprM homologs identified in the genome of P . aeruginosa were examined as candidates for this role by assessing the minimum inhibitory concentrations (MICs) of aminoglycosides in P . aeruginosa strain PAK knockout mutants lacking the corresponding genes . Insertional inactivation of OpmG, OpmI, and OpmH resulted in decreases of various degrees in the MICs of streptomycin, kanamycin, and gentamicin . When reintroduced into P . aeruginosa on multicopy plasmids, OpmG was able to complement the susceptibility of an opmG::miniTn5 mutant; however, cloned opmH, the proposed ortholog of Escherichia coli tolC according to our phylogenetic analysis, was able to only partially complement the opmH::miniTn5 mutant . Mini-microarray hybridization analysis demonstrated that opmG disruption does not affect expression of OpmI or OpmH (ruling out such indirect effects on aminoglycoside resistance); however, opmH disruption did have possible effects on expression of OpmG and OpmI . Based on the data, we propose that OpmG is a major outer membrane efflux channel involved in aminoglycoside efflux in P . aeruginosa PAK and that OpmI, its most related paralog, may share an overlapping function.

Antimicrob Agents Chemother, 2003 Mar, 47(3), 972 - 8
Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1; Dean CR et al.; Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 micro g/ml) than many other bacteria (P . J . Petersen, N . V . Jacobus, W . J . Weiss, P . E . Sum, and R . T . Testa, Antimicrob . Agents Chemother . 43:738-744, 1999) . To elucidate the mechanism of resistance to tigecycline, P . aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline . Increased susceptibility to tigecycline (MIC, 0.5 to 1 micro g/ml) was specifically associated with loss of MexXY . Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline . To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 micro g/ml . Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment . One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump . The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion . Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM . The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively . Together, these data indicated drug efflux mediated by MexCD-OprJ . The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains . This suggests that glycylcyclines, although they are subject to efflux from P . aeruginosa, are generally inferior substrates for P . aeruginosa efflux pumps than are narrower-spectrum tetracyclines.

Biochem Biophys Res Commun, 2003 Mar 7, 302(2), 363 - 71
Chromosomal organization and transcription analysis of genes in the vicinity of Pseudomonas aeruginosa glmM gene encoding phosphoglucosamine mutase; Tavares IM et al.; A computer-aided analysis of the Pseudomonas aeruginosa PAO1 genome surrounding the glmM gene was carried out and the organization of this chromosomal region was compared with the equivalent regions in other gamma-proteobacteria species with the genome sequence available . glmM encodes the enzyme phosphoglucosamine mutase which catalyses the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate in the biosynthetic pathway leading to the synthesis of UDP-N-acetylglucosamine which is simultaneously a precursor for the biosynthesis of cell-wall peptidoglycan and outer membrane lipopolysaccharide . Northern blot analysis suggests that glmM may be a part of the five-cistron operonic structure composed by the Escherichia coli homologues ftsJ, ftsH, folP, glmM, and tpiA . The secG gene, downstream tpiA, does not make part of this polygenic organization, being actively transcribed as a monocistronic mRNA during transition to the stationary phase of growth . Differently, transcription of genes in the glmM operon is more active in the early exponential phase, decreasing with the increase of cell density during exponential growth and reaching negligible values in stationary phase cells.

Chem Biol Interact, 2003 Feb 1, 143-144, 149 - 58
Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L . leaves by disulfiram; Velasco-Garcia R et al.; Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents . As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P . aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs . DSF totally inactivated both enzymes in a time- and dose-dependent manner . In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M(-1) s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M(-1) s(-1) . At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1 x 10(3) M(-1) s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism . Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme . The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione . The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.

Chem Biol Interact, 2003 Feb 1, 143-144, 139 - 48
Monovalent cations requirements for the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, porcine kidney and amaranth leaves; Valenzuela-Soto EM et al.; Betaine aldehyde dehydrogenase from the human pathogen Pseudomonas aeruginosa requires K(+) ions for maintenance of its active conformation . In order to explore if this property is shared by other BADHs of different origins and to further understand the mechanism underlying the effects of these ions, we carried out a comparative study on the stability and quaternary structure of P . aeruginosa, porcine kidney and amaranth leaves BADHs in the absence of K(+) ions . At low enzyme concentrations, the bacterial and porcine enzymes were totally inactivated upon removal of K(+) following biphasic and monophasic kinetics, respectively, whereas the amaranth enzyme retained its activity . Inactivation of P . aeruginosa BADH was much faster than that of the porcine enzyme . The oxidized coenzyme protected both enzymes against inactivation by the absence of K(+), whereas betaine aldehyde afforded partial protection to the bacterial BADH and increased the inactivation rate of the porcine . Reactivation of the inactive enzymes, by adding back to the incubation medium K(+) ions, was dependent on enzyme concentration, suggesting that enzyme dissociation takes place in the absence of K(+) . In the bacterial enzyme, NH(4)(+) but not Na(+) ions could mimic the effects of K(+), whereas the three cations tested reactivated porcine BADH, indicating a requirement of this enzyme for high ionic strength rather than for a specific monovalent cation . Size exclusion chromatography of the inactivated enzymes confirmed that K(+) ions or other monovalent cations are required for the maintenance of the quaternary structure of these two BADHs . At pH 7.0, in the absence of K(+) in a buffer of low ionic strength, the active tetrameric form of P . aeruginosa BADH dissociated into inactive monomers and that of porcine kidney BADH into inactive dimers . Once reactivated, both enzymes reassociated into active tetramers.

Chem Biol Interact, 2003 Feb 1, 143-144, 129 - 37
Ligand-induced conformational changes of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L . leaves affecting the reactivity of the catalytic thiol; Munoz-Clares RA et al.; The reaction catalyzed by betaine aldehyde dehydrogenase (BADH) involves the nucleophilic attack of a catalytic cysteinyl residue on the aldehyde substrate . As a possible mechanism of regulation, we have studied the modulation by ligands of the reactivity and/or accessibility of the essential thiol of the enzyme from the human pathogen Pseudomonas aeruginosa and the leaves of the plant Amaranthus hypochondriacus (amaranth) . In the absence of ligands, the kinetics of inactivation by thiol modifying reagents of both enzymes were biphasic, suggesting the existence of two enzyme conformers differing in the reactivity of their catalytic thiolate . Preincubation of P . aeruginosa BADH with the coenzymes or the aldehyde prior to the chemical modification brought about active site rearrangements that resulted in an important decrease in the inactivation rate . Amaranth BADH responded similarly to the preincubation with NADH or betaine aldehyde but NAD(+) elicited opposite changes, increasing the rate of inactivation after prolonged preincubation . In amaranth BADH, the different behavior of both coenzymes, and the observed biphasic inactivation kinetics are consistent with the previously proposed iso kinetic mechanism, characterized by the existence of two interconvertible apoenzyme forms, one able to bind NAD(+) and the other NADH . Taken together, our results suggest that ligand-induced conformational changes in BADH from the two sources studied might be important for both proper enzyme function and protection against oxidation.

J Liposome Res, 2002, 12(3), 239 - 57
Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis; Fujimoto J et al.; Pseudomonas aeruginosa is a pathogen that frequently causes acute lung injury, bacteremia and sepsis in critically ill patients . As tissue macrophages are a major producer of inflammatory mediators that contribute to septic physiology, and are essential for eliminating bacteria from the circulation, we investigated the role of tissue macrophages in the generation of both inflammatory and anti-inflammatory cytokines in septic shock by using our mouse model of P . aeruginosa pneumonia . To see the effects of tissue macrophage depletion, we intravenously injected dichloromethylene-diphosphonate (Cl2MDP)-encapsulating liposomes in mice . Two days after the liposome injection, we instilled cytotoxic P . aeruginosa (PA103) into the lung that disseminates and causes septic shock . After the infection, we collected blood and bronchoalveolar lavage fluids . The samples were then analyzed for TNF-alpha, MIP-2, and IL-10 concentration . We compared these results to control mice that received either liposomes without Cl2MDP or phosphate buffered saline alone . Plasma TNF-alpha, MIP-2, and IL-10 levels were significantly decreased in the tissue macrophage-depleted mice compared to the control groups of mice . Although depletion of tissue macrophages by Cl2MDP-liposome administration did not affect the severity of bacteremia or the survival of infected mice, these results imply that tissue macrophages have a major role in the production of both proinflammatory and anti-inflammatory cytokines in the circulation and in the causing septic physiology associated with P . aeruginosa pneumonia.

East Mediterr Health J, 2001 Jan-Mar, 7(1-2), 38 - 45
Etiology of toe-web disease in Al-Ain, United Arab Emirates: bacteriological and mycological studies; Lestringant GG et al.; We examined and sampled 45 patients with toe-web intertrigo for bacteriological and mycological studies . Prominent isolated pathogens were the genus Candida (57.7%), genus Aspergillus (28.8%), Pseudomonas aeruginosa (26.7%) and coliforms (24.4%) . Dermatophytes scored 4.4% (Trichophyton rubrum) . There were 43 patents (95.5%) who presented with marked hyperkeratosis and maceration of the toe-webs involved . The tradition of the Emirati population of sitting cross-legged may, over time, induce in the toe-webs of overweight individuals a macerated pressure-reaction hyperkeratosis that is colonized by environmental germs . T . rubrum and T . mentagrophytes are uncommon in the Al-Ain environment and this may explain the rarity of dermatophytes in toe-web intertrigo in our study.

J Basic Microbiol, 2003, 43(1), 36 - 46
Identification of a major protein upon phosphate starvation of Pseudomonas aeruginosa PAO1; Madhusudhan KT et al.; To understand the physiology of non-differentiating bacteria exposed to nutrient deprivation and stress, various approaches have been employed in combination with detailed analysis of protein synthesis pattern . In this study, separation of proteins from clarified cell extracts of Pseudomonas aeruginosa PAO1 grown under phosphorus limiting conditions was achieved by high resolution two-dimensional gel electrophoresis (2-DE) . Limitation of phosphate in the growth medium revealed significant differences in the 2-DE pattern of proteins between phosphate starved cells and an unstarved control . A major protein identified as PstS, a phosphate binding protein of the pts operon was exclusively found on 2-DE gels of phosphate starved bacteria . The identity of protein was established based on the results of Edman degradation, amino acid analysis and mass spectrometry . PstS was also found in other pseudomonads, and therefore, it can be used as a landmark protein in proteomic studies . Additionally, we propose utilizing pstS of pseudomonads for testing bioavailable phosphate from soils and water streams.

Infect Immun, 2003 Mar, 71(3), 1590 - 5
DsbA of Pseudomonas aeruginosa is essential for multiple virulence factors; Ha UH et al.; DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds . In this study, we demonstrate that the dsbA gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of P . aeruginosa upon infection of HeLa cells, and twitching motility . The diverse phenotypes of the dsbA mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing . In light of its effect on various virulence factors, DsbA could be an important target for the control of P . aeruginosa infections.

Infect Immun, 2003 Mar, 71(3), 1453 - 61
Protection against fatal Pseudomonas aeruginosa pneumonia in mice after nasal immunization with a live, attenuated aroA deletion mutant; Priebe GP et al.; Studies of immunity to Pseudomonas aeruginosa have indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection . To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced an aroA deletion mutant of the P . aeruginosa serogroup O2/O5 strain PAO1, designated PAO1deltaaroA . Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits . Here, we assessed the protective efficacy of immunization with PAO1deltaaroA against acute fatal pneumonia in mice . After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose . Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains . The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup . In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera . However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses . These data suggest that a combination of humoral and cellular immunity is required for protection against P . aeruginosa lung infections, that such immunity can be elicited by using aroA deletion mutants, and that a multivalent P . aeruginosa vaccine composed of aroA deletion mutants of multiple serogroups holds significant promise.

Infect Immun, 2003 Mar, 71(3), 1328 - 36
Experimental Pseudomonas aeruginosa keratitis in interleukin-10 gene knockout mice; Cole N et al.; Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea . The host response to this infection is critical to the outcome . The cytokine interleukin-10 (IL-10) is thought to play an important role in modulating excessive inflammation and antimicrobial defenses . We have found that in IL-10(-/-) mice there is a significant decrease in bacterial load in corneas at 7 days postchallenge with P . aeruginosa . This decrease was accompanied by a reduction in neutrophil numbers in the cornea and changes in cytokine levels compared to those of wild-type mice . A characteristic increase in neovascularization in the cornea was found in the IL-10(-/-) mice . This increased angiogenesis correlated with an increased expression of KC, whereas the kinetics of macrophage inflammatory peptide 2 expression correlated with neutrophil numbers . This finding suggests that KC may play a role in corneal angiogenesis . The source of IL-10 in mouse corneas was identified as a subpopulation of infiltrating cells and keratocytes . This study demonstrates that IL-10 plays an important role in regulating the balance of inflammatory mediators during P . aeruginosa infection of the cornea.

Surg Infect (Larchmt), 2000, 1(1), 65 - 72
The role of quinolones in abdominal surgery; Nichols RL; The quinolone antibiotics have been a major advance for the treatment of various types of infections . These agents have generally good safety profiles, broad-spectrum activity, and favorable pharmacokinetics . In addition, several of these antibiotics are available in both intravenous and oral formulations, which allows for sequential therapy resulting in potential cost savings . However, patients can develop serious central nervous system side effects (seizures) and phototoxicity . In addition, the bioavailability of agents in this class can be reduced by coadministration with cations, such as magnesium, aluminum, calcium, and iron, which may make bioavailability unpredictable in patients . Although older quinolones such as ciprofloxacin were effective as prophylactic agents for biliary procedures and colorectal surgery and for the treatment of intra-abdominal infections, the use of these older quinolones was limited by the development of resistant organisms . In addition, because these agents had poor activity against anaerobes such as Bacteroides fragilis, the agents had to be combined with an antianaerobic agent, such as metronidazole, when anaerobic coverage was required . Recently, a new quinolone, trovafloxacin, has become available . Trovafloxacin has demonstrated increased activity against anaerobes in animal and human studies . However, the clinical profile of trovafloxacin for abdominal infections has not been fully demonstrated, and there is some concern that its activity against aerobic gram-negative bacilli, especially Pseudomonas aeruginosa, may not equal that of ciprofloxacin . Moreover, the safety profile of trovafloxacin is disadvantageous owing to reports of severe hepatic toxicity.

Microbes Infect, 2003 Jan, 5(1), 27 - 30
Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein detected in children with cystic fibrosis inhibit neutrophil-mediated killing of Pseudomonas aeruginosa; Sediva A et al.; Antineutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) were repeatedly found in cystic fibrosis (CF) patients . We analyzed the effect of BPI-ANCA in inhibiting neutrophil-mediated killing of Pseudomonas aeruginosa . The bactericidal effect expressed as percentage of killed bacteria after 1 h incubation with neutrophils was 55% when the neutrophils were pretreated with normal human serum, ranged from 49 to 63% with the sera from control BPI-ANCA-negative groups and sharply decreased to the mean 30.5% (range 8-51%) in the presence of BPI-ANCA . Furthermore, the effect mediated by BPI-ANCA was dose dependent and reflected the titer of BPI-ANCA in tested sera.

Surg Infect (Larchmt), 2002 Spring, 3(1), 29 - 33
Mycotic aneurysm of the descending thoracic aorta caused by Pseudomonas aeruginosa in a solid organ transplant recipient: case report and review; Feltis BA et al.; BACKGROUND: Pseudomonas aeruginosa is a rare cause of aortic mycotic aneurysms . Optimal treatment, including reconstructive graft material and appropriate length of antibiotic therapy, is being debated . METHODS: We describe a 26-year-old kidney-pancreas recipient who developed an aneurysm of the descending thoracic aorta caused by P . aeruginosa . RESULTS: After surgical debridement and cryopreserved allograft reconstruction, parenteral antibiotics were continued for 12 months, at which time the patient was converted to oral antibiotic therapy . Within 6 months, he redeveloped a thoracic aortic aneurysm, necessitating reoperation and lifelong parenteral antibiotic therapy . CONCLUSION: Herein we review and discuss the relevant literature concerning surgical and antibiotic treatment of mycotic thoracic aneurysms.

Indian J Pathol Microbiol, 2002 Jan, 45(1), 15 - 22
Study of fungal and bacterial infections of the diabetic foot; Chincholikar DA et al.; Microbiological study for aerobic organisms, anaerobic organisms and fungi from 105 cases of diabetic foot ulcers was carried out to determine the aetiological agents and their antibiograms . Out of 265 microbial isolates obtained, 160 were aerobes, 50 anaerobes and 55 fungal strains . Polymicrobial infection was observed in 73 (69.5%) cases . The most frequently isolated aerobic microorganisms were Staphylococcus aureus and Pseudomonas aeruginosa . Among the anaerobes Bacteroides melaninogenicus and Bacteroides fragilis were most common . Candida species were preponderant among the fungal isolates . Antimicrobial sensitivity pattern of the isolates is discussed in detail.

Clin Microbiol Infect, 2003 Feb, 9(2), 101 - 9
Infecton: a 99mTc-ciprofloxacin radiopharmaceutical for the detection of bone infection; Malamitsi J et al.; OBJECTIVE: To evaluate Infecton scintigraphy, with technetium-99m-radiolabeled ciprofloxacin, as a means to detect bone infection, in comparison with other conventional scintigraphic and radiologic methods . METHODS: Forty-five patients with known or suspected bone infection underwent 50 scans with Infecton . Almost all were also subjected to a three-phase 99mTc-methylene diphosphonate bone scan and most of them to a 99mTc-human polyclonal immunoglobulin scan as well as to a gallium-67-citrate scan, plus computerized tomography or magnetic resonance imaging or both . Clinical laboratory criteria for the presence of osteomyelitis were based on the definitions of the Centers for Disease Control and Prevention . RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa were the most frequently isolated pathogens . Based on the CDC clinical laboratory criteria as well as on conventional scan results, Infecton was characterized in 35 studies as 'true positive', in eight as 'true negative', in two as 'false positive', in one as 'false negative', and in four as 'indeterminate' . The sensitivity and specificity of Infecton scintigraphy were found to be 97.2% and 80%, respectively, with positive and negative predictive values of 94.6% and 88.9% . CONCLUSIONS: It is concluded that Infecton is a very sensitive and quite specific marker of bone infection, but care must be taken in cases of excessive new bone formation and primary bone tumors, where false-positive results may be obtained.

Ulus Travma Derg, 2003 Jan, 9(1), 34 - 6
{Evaluation of severe burns managed in intensive care unit}; Kurtoulu M et al.; BACKGROUND: This study designed to evaluate the results of the patients with severe burns treated in the intensive care unit . METHODS: Between May 1997 and May 2002, fifty-four patients who had thermal and electrical burns managed at the Intensive Care Unit of General Surgery of Istanbul University Medical Faculty . RESULTS: Forty-five of patients were males and nine were females with mean age of 39 years . The mean hospital stay was 26 days . The mean total body surface area burns was 60% . Pseudomonas aeruginosa was detected in wound cultures at the 68% of patients . 83% of cases died . Mortality was due to sepsis in 66% of patients . CONCLUSION: Besides early debridmant, early enteral feeding, woundcare and intensive care unit support, establishing of specific burncenters may reduce morbidity and mortality rates in severe burns.

Int Immunopharmacol, 2003 Feb, 3(2), 247 - 56
A Limulus anti-LPS factor-derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice; Vallespi MG et al.; Sepsis in experimental animals and humans has been associated with perturbed immune response . A major event contributing to the decrease in immune functions in septic disorders seems to be the inadequate balance of cytokines mediating the interactions between the innate and adaptive immune systems . We previously observed that a cyclic peptide derived from the Limulus anti-LPS factor (LALF), which partially protect mice from endotoxic shock lethality, has the ability to modulate cytokine secretion in vitro . We herein examined the effects of the LALF(31-52) peptide in an experimental model of Gram-negative peritoneal sepsis and analyzed the cytokine gene expression in the spleen and liver of peptide-treated mice . The prophylactic administration of LALF(31-52) abrogated the systemic TNF-alpha response, reduced organ damage and increased the survival of infected mice . Histological examination of spleen and liver in peptide-treated mice showed prevention of tissue damage induced by the high dose of Pseudomonas aeruginosa . This treatment modulates the cytokine gene expression in these tissues, stimulating IL-2, IL-12 and IL-13 mRNA synthesis, while IL-4 and IL-10 mRNA expression was not modified . This cytokine profile induced by the LALF-derived peptide seems to be favorable for host resistance against Gram-negative bacteria acute infection . In addition, peptide treatment was effective after the initiation of the systemic inflammatory response, promoting a significant increase in mice survival . These results further demonstrate the immunomodulatory potential of LALF(31-52) and are relevant for the design of prophylactic and therapeutic strategies for acute bacteria infection and sepsis, especially for preventing or ameliorating host immunity defects in these disorders .

Dev Cell, 2003 Feb, 4(2), 253 - 63
Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway; Wolfgang MC et al.; Type III secretion systems (TTSSs) are utilized by numerous bacterial pathogens to inject effector proteins directly into host cells . Using a whole-genome microarray, we investigated the conditions and regulatory factors that control the expression of the Pseudomonas aeruginosa TTSS . The transcriptional response of known TTSS genes indicates a hierarchical pattern of expression in which a set of secretion apparatus and regulatory genes is constitutively expressed . Further analysis of genes coordinately regulated with those encoding the TTSS led to the identification of a signaling pathway that originates from a membrane-associated adenylate cyclase and controls TTSS gene expression . Transcriptome analysis of mutants lacking the ability to synthesize cAMP or the cAMP binding protein Vfr implicated this pathway in the global regulation of host-directed virulence determinants, including the TTSS.

Yao Xue Xue Bao, 2001 Jun, 36(6), 419 - 22
{Synthesis and antibacterial activity of 5-amino-6, 8-difluoro-1-(5-fluoro-2-pyridyl)-7-(3-methyl-1-piperazinyl)-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid and its analogues}; Liu JY et al.; AIM: To design and synthesize 1-(5-fluoro-2-pyridyl) quinolone derivatives, and to study their in vitro antibacterial activities . METHODS: Eight new compounds of 5-amino-6,8-difluoro-1-(5-fluoro-2-pyridyl)-7-(3-methyl-1-piperazinyl)-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid and its analogues were synthesized from ethyl 6-nitro-2, 3, 4, 5-terafluorobenzoylacetate and ethyl 3-methoxy-2, 4, 5-trifluorobenzoylacetate through 5 or 6 steps . RESULTS: Fifteen new compounds (1-15) were obtained including 8 desired compounds (8-15) . The structures of the compounds were determined by 1HNMR, MS . CONCLUSION: In vitro antibacterial activities of the compounds (8-15) against Staphylococcus aureus-16, Escherichia coli-26 and Pseudomonas aeruginosa-17 were lower than control of ciprofloxacin.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 85 - 92
Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach; Thompson C et al.; The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin . A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A . From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain . The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein . In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity . These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 51 - 7
Optimisation of AP-PCR fingerprinting discriminatory power for clinical isolates of Pseudomonas aeruginosa; Dabrowski W et al.; Recently methods based on analysis of arbitrarily amplified target sites of microorganism genomes have been extensively applied in microbiological studies . The range of their applications is limited by problems with discrimination and reproducibility resulting from lack of standardised and reliable methods of optimisation . By orthogonal-array optimisation most advantageous and optimal parameters for highly discriminatory primers (CagA2+CMVin2) were selected and efficient AP-PCR (arbitrarily primed-polymerase chain reaction) fingerprinting conditions for Pseudomonas aeruginosa isolates were set up . Stable and multiplex amplicon profiles obtained in this study revealed high level of intraspecies DNA polymorphism among 20 analysed clinical strains of P . aeruginosa proving optimised AP-PCR fingerprinting to be useful in epidemiological typing of the species.

Intern Med, 2003 Jan, 42(1), 25 - 32
Clinical analysis of patients requiring long-term mechanical ventilation of over three months: ventilator-associated pneumonia as a primary complication; Kobashi Y et al.; OBJECTIVE: To evaluate the clinical features, etiology, and prognosis of patients who required long-term mechanical ventilation (LMV) of over three months for respiratory failure following underlying disease, and observation of their clinical course until death . PATIENTS: Thirty-seven patients (27 males, 10 females) treated in the internal and medical intensive care unit at Kawasaki Medical School Kawasaki Hospital over the 16-year period from April 1985 to March 2001 were retrospectively studied . RESULTS: Many of these patients were elderly males with respiratory disease such as pulmonary emphysema or old pulmonary tuberculosis, which had developed into acute respiratory failure resulting in respiratory tract infection and initiation of mechanical ventilation . The survival rates of one year, three years and five years after the start of mechanical ventilation were 60%, 30%, and 16%, retrospectively, and the prognoses were poor . Respiratory tract infection was the most common and serious complication . Specifically, ventilator-associated pneumonia (VAP) was a complication in 21 patients and also the main-cause of death . VAP was observed 2.3 years after the initiation of mechanical ventilation with significant differences in the following risk factors being observed between VAP (+) and VAP (-) groups: chronic obstructive pulmonary disease, duration of mechanical ventilation, prior antibiotics, aspiration of gastric contents and use of histamine-type II receptor antagonist . The causative pathogens of VAP were Pseudomonas aeruginosa and Staphylococcus aureus, which were frequently isolated from tracheal aspirates . All patients with VAP caused by MRSA died shortly after contracting the infection . CONCLUSIONS: This study has demonstrated that appropriate treatment for respiratory tract infections such as VAP and the prevention of nasocomial infection due to MRSA is of paramount importance for patients requiring long-term mechanical ventilation of over three months.

J Chemother, 2002 Dec, 14(6), 579 - 83
The integration of four major determinants of antibiotic action: bactericidal activity, postantibiotic effect, susceptibility, and pharmacokinetics; Li RC et al.; A functional pharmacokinetic/pharmacodynamic (PK/PD) index that could simultaneously describe three controlling PD variables, i.e., bactericidal activity, postantibiotic effect (PAE), and susceptibility, in relation to pharmacokinetics, was designed using an in vitro kinetic model . Tobramycin was tested against one standard and five clinical strains of Pseudomonas aeruginosa . The organisms showed minimum inhibitory concentrations (MICs) ranging between 1 and >1000 microg/ml . The model allowed antibiotic concentrations to be reduced exponentially from initial concentrations at fixed multiples of MIC . Antibiotic removal was performed when the decreasing concentrations hit the MIC of individual strain to provide a wide range of AUC(>MIC) within an identical frame of AUC(>MIC)/MIC (AUIC) values . Viable counts were measured at antibiotic addition and before/after its removal for bactericidal activity and PAE assessments . A linear relationship was observed between PAE and bactericidal rate constants, though the pattern varied among different strains . Characterization of the exposure (AUC(>MIC))-effect relationships using the Emax model revealed that the less susceptible strains displayed lower Emax and higher EC50 for both antimicrobial effects . By employing the AUIC as a common frame of reference, regression analysis showed a significant linear correlation (p < 0.05) between the mean PAE and bactericidal rate data and, thereby simultaneously defining the four contributing factors of the PK/PD system . It appears that the AUIC, by conveying the pharmacokinetic and susceptibility information, could serve as a PK/PD index in bridging the interdependency of PAE and bactericidal activity . More importantly, the collective assessment of these four factors would allow more optimal evaluation of dosage regimens.

J Chemother, 2002 Dec, 14(6), 568 - 73
A survey of antimicrobial drug resistance in respiratory tract pathogens, isolated in a northern Italian teaching hospital between 1990 and 1999; Ferrara A et al.; Drug susceptibility test results of respiratory tract pathogens, isolated from patients admitted to the Clinic of Respiratory Diseases of the IRCCS San Matteo Hospital, University of Pavia (Italy) between 1990 and 1999, were retrospectively evaluated . A total of 1366 bacterial isolates were collected, including 499 gram-positive and 867 gram-negative strains . In comparison to methicillin-susceptible Staphylococcus aureus, the methicillin-resistant strains (MRSA) showed high levels of resistance to many selected antibiotics, except for glycopeptides . Resistance rates to beta-lactams were high in both Pseudomonas aeruginosa and in the other gram-negative isolates, while aminoglycoside and ciprofloxacin resistance was less than 20% . Some pathogens became more resistant to selected antimicrobials during the observation period, including staphylococci to methicillin, MRSA to ciprofloxacin, P . aeruginosa isolates to imipenem and ciprofloxacin, and the other gram-negative strains to almost all drugs considered, with the exception of cefotaxime and cotrimoxazole.

Mol Microbiol, 2003 Feb, 47(4), 1123 - 33
The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa; Jain S et al.; Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype . Alginate is a linear polymer of d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate (G) . AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate to the mixed M+G sequence of alginate . To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG . Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK . High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK . Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm . AlgK appears to share the second role . AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE) . To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function . The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.

Mol Microbiol, 2003 Feb, 47(4), 903 - 15
Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator; Pohl E et al.; Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals . Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration . In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur) . Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A . Here, we present the first crystal structure of Fur from P . aeruginosa in complex with Zn2+ determined at a resolution of 1.8 A . Furthermore, X-ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution . The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1949 - 54 Epub 2003 Feb 10.
Hypersusceptibility of cystic fibrosis mice to chronic Pseudomonas aeruginosa oropharyngeal colonization and lung infection; Coleman FT et al.; No transgenic cystic fibrosis (CF) mouse model developed to date mimics the major clinical phenotype found in humans with CF, chronic Pseudomonas aeruginosa lung infection . In a transgenic CF transmembrane conductance regulator (cftr) mouse colony, we found WT, heterozygous, and homozygous CF mice housed in the same cage became chronically colonized in the oropharynx with environmental P . aeruginosa when the bacterium was present in drinking water . Elimination of P . aeruginosa from drinking water resulted in clearance in most WT and CF heterozygous, but not homozygous mice . For experimental evaluation, a combination of specific animal husbandry techniques and an oral infection route showed cftr(-/-) mice but not WT mice can be chronically colonized by P . aeruginosa with subsequent lung translocation, yielding a pathologic picture indicative of chronic lung infection . In some instances, mucoid isolates of P . aeruginosa were recovered from lungs, indicating conditions were present for conversion to mucoidy . Overexpression of human CFTR in the lungs of WT mice markedly accelerated the clearance rate of P . aeruginosa, demonstrating that lung levels of CFTR play an important role in defense against infection . P . aeruginosa mutants unable to express the surface polysaccharide alginate or the global regulator GacA were deficient in their ability to colonize the mice . CF mice made potent immune responses to P . aeruginosa outer membrane antigens . Overall, we found that under the proper conditions, transgenic CF mice are hypersusceptible to P . aeruginosa colonization and infection and can be used for evaluations of lung pathophysiology, bacterial virulence, and development of therapies aimed at treating CF lung disease.

Biochemistry, 2003 Feb 18, 42(6), 1673 - 83
Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-beta-lactamases; Siemann S et al.; The inhibitory effect of a variety of thiol compounds on the function of binuclear metallo-beta-lactamases, with a particular focus on IMP-1 from Pseudomonas aeruginosa, has been investigated . Thiol inhibitors, depending on their structural features, fall into two categories, one in which inhibition at neutral pH was instantaneous and the other in which inhibition was time-dependent . While mercaptans with anionic substituents in the vicinity of their SH groups exhibited the former type of inhibition, neutral thiols appear to induce a slow, time-dependent isomerization of the initially formed EI complex to a tighter EI complex . Kinetic parameters describing the latter process were obtained by fitting progress curves of substrate hydrolysis using standard and numerical procedures . The failure of charged thiols to exhibit slow binding is suggested to be due to a rapid isomerization of the initial EI complex . Slow binding in the case of neutral thiols was observed only below pH 8 . Studies on the pH dependence of catalysis by IMP-1 revealed that (i) enzyme inactivation at low pH is a slow process with presumably two groups with a pK(a) of approximately 5.2 in the protein being responsible for the loss of activity, (ii) inhibition by thiols is independent of pH between pH 5 and 9, and (iii) an apparent enhancement of the catalytic activity of IMP-1 by thiols occurs at pH <5 . The last mentioned phenomenon is explained by a model in which mercaptans retard the proton-dependent isomerization of the enzyme . Studies on the thiol-mediated inhibition of the binuclear forms of Bacteroides fragilis (CcrA) and Bacillus cereus (BcII strain 5/B/6) metallo-beta-lactamase have revealed that while CcrA was instantaneously albeit moderately inhibited by mercaptans, BcII mimicked IMP-1 in its interaction with thiols . These differences are proposed to be due partly to the structural divergence of these proteins in the vicinity of Zn2.

New Microbiol, 2003 Jan, 26(1), 109 - 14
Typing of Pseudomonas aeruginosa strains in Turkish cystic fibrosis patients; Yagci A et al.; The majority of cystic fibrosis (CF) patients suffer from chronic respiratory infection with the opportunistic bacterial pathogen Pseudomonas aeruginosa . The virulence of P . aeruginosa is associated with the presence of various extracellular factors, like alginate, elastase, alkaline protease which contribute tissue destruction and assist bacterial invasion . Virulence factor production of P . aeruginosa strains isolated from 46 CF patients followed in two cities in Turkey was detected . Strains were compared genotypically by arbitrarily primed PCR . Antimicrobial susceptibilities to 12 antibiotics were determined by broth microdilution method . Evaluation of virulence factor results revealed that 95.8% of the strains were alginate, 71.7% elastase and 52.1% alkaline protease producers . AP-PCR analysis revealed 35 genotypes indicated almost a complete discrepancy among the strains . The most effective drugs were penems and quinolones . Among aminoglycosides amikacin was the most effective one and a high level resistance to beta lactams was observed . Alginate is the most important virulence factor in the chronic colonisation of CF patients with P . aeruginosa . No evidence for cross infection between patients and for relationship between phenotypes and genotypes of the strains was found.

Shock, 2003 Feb, 19(2), 113 - 6
Impaired production of interferon-gamma and tumor necrosis factor-alpha but not of interleukin 10 in whole blood of patients with sepsis; Rigato O et al.; It has been demonstrated that lipopolysaccharide (LPS)-induced cytokine response in patients with sepsis differ from the normal host, yet this has not been controlled for the presence of underlying disease . We studied the ability of LPS and killed gram-negative bacteria (GNB) to induce tumor necrosis factor (TNF)-alpha and interleukin (IL) 10, and of phytohemagglutinin (PHA) to induce interferon (IFN)-gamma, in whole blood from patients with sepsis (SP, n = 20), patients with matched underlying disease and without sepsis (control patients, n = 20), and healthy volunteers (HV, n = 20) . LPS-induced TNF-alpha production was lower in SP (median = 638 pg/mL) compared with control patients (4060 pg/mL; P= 0.003), and control patients production was lower compared with HV (5329 pg/mL; P < 0.001) . Pseudomonas aeruginosa-induced TNF-alpha production was lower in SP (1443 pg/mL) than in control patients (7319 pg/mL; P < 0.05), and was not different between control patients and HV (6612 pg/mL; P = 0.6) . IFNy production was lower in SP (948 pg/mL) compared with control patients (5516 pg/mL; P < 0.001), and the control patients production was lower compared with HV (11,282 pg/mL; P < 0.001) . IL-10 production was not different among the three groups . Down-regulation of TNF-alpha production in patients with sepsis, although not restricted to them, was more pronounced with LPS than with GNB . Although the presence of underlying disease may be involved in the regulatory mechanisms of host response, the use of controls with matched underlying diseases provides strong evidence for the septic condition in the down-regulation of inflammatory response in patients with sepsis.

J Clin Microbiol, 2003 Feb, 41(2), 822 - 5
Spread of integron-associated VIM-type metallo-beta-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals; Giakkoupi P et al.; Fifty-eight imipenem-nonsusceptible (MIC >or= 8 microg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied . Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR . In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes . DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures . Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains . Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains.

Chem Biol, 2003 Jan, 10(1), 1 - 2
Molecular radio jamming: autoinducer analogs; Fast W; Synthesis of an N-acyl-homoserine lactone (N-acyl-HSL) analog library yields agonists and antagonists of the Pseudomonas aeruginosa quorum-sensing system . Active compounds also reveal heterogeneity in the lactone ring binding pockets of N-acyl-HSL-activated transcription factors.

Zhong Yao Cai, 1997 Jan, 20(1), 38 - 40
{Preliminary study on the pharmacological action spicatus}; Gai H et al.; In this report the pharmacological action of Spicatus was studied . The results insicated that it had diureric, antibiotic and anti-inflammatory effects, yet had Iittle toxic side-effect . It had significant inhibitory effect on crofon oil-caused mice ear swell . It also had marked diuretic effect in orcinary rats, but had Iittie effect on uric pH the rats . It exhibited certain inhibition of Staphycoloccus aureus, Eschrichia coli and Pseudomonas aeruginosa in vitro . The maximum tolerable dose test in mice showed no marked toxic effect, LD50 > 80 g/kg.

Heart Lung, 2003 Jan-Feb, 32(1), 59 - 64
Current management of bronchiectasis: review and 3 case studies; Silverman E et al.; Bronchiectasis is the abnormal, irreversible dilatation of diseased bronchi . Permanently dilated airways, usually in the medium-sized bronchi, are inflamed and often obstructed with thick, purulent secretions . Known causative factors include postinfection bronchial damage, postinhalation injury, hypersensitivity reactions, and congenital airway obstructive disorders . Typical symptoms include sputum overproduction, fever, pleurisy, dyspnea, and chronic cough . Diagnosis involves radiographic studies and pulmonary function testing . Treatment includes oral, aerosolized, or intravenous antibiotic therapy according to the severity of the exacerbation, and mucus clearance by means of bronchial hygiene assistive devices, chest physiotherapy, postural drainage, and high-frequency chest compression . We present a review of bronchiectasis and offer 3 case studies illustrating current management of different presentations, including use of aerosolized antibiotics for patients infected with Pseudomonas aeruginosa . Although an adjunctive program of pulmonary rehabilitation may be useful for patients with bronchiectasis, no confirming studies have been performed to date, and additional research in this area is warranted.

Am J Respir Crit Care Med, 2003 Jun 1, 167(11), 1478 - 82 Epub 2003 Jan 31.
Role of the quorum-sensing system in experimental pneumonia due to Pseudomonas aeruginosa in rats; Lesprit P et al.; The virulence of Pseudomonas aeruginosa is partly controlled by the las quorum-sensing system . A rat model of acute pneumonia was used to investigate the pathophysiological impact of this system by comparing the virulence of the wild-type virulent laboratory strain PAO1 with that of its lasR-deleted mutant PAOR . In comparison with PAO1, PAOR was avirulent after an instillation of 106 cfu (mortality rates, 72 versus 0%, respectively; p < 0.0001) . A ten-fold higher inoculum slightly increased the mortality rate induced by PAOR (25%), which remained lower than that induced by PAO1 (75%, p = 0.0001) . In addition, with both inocula lung and bronchoalveolar lavage bacterial counts were significantly lower in rats infected with PAOR than with PAO1 (p </= 0.01) . Histopathological analysis showed that PAO1 induced a drastic vascular congestion and neutrophil infiltration of the lungs, whereas lung injury in rats infected with PAOR was mild with predominantly macrophage infiltration . This study adds evidence that the quorum-sensing system has an important role in the pathophysiology of P . aeruginosa pulmonary infection.

Int J Biol Macromol, 2003 Jan 15, 31(4-5), 195 - 205
Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation; Tsuge T et al.; The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria . PhaJ provides monomer units for PHA synthesis from the fatty acid ss-oxidation cycle . Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa . This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P . aeruginosa through a genomic database search . The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein {(R)-3HA-ACP} dehydrases, FabA(Pa) and FabZ(Pa), to supply monomers from enoyl-CoA substrates for PHA synthesis were determined . The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short- (C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively . Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4(Pa) were performed using purified samples . Kinetic analysis revealed that only PhaJ4(Pa) exhibits almost constant maximum reaction rates (V(max)) irrespective of the chain length of the substrates . The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity . These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.

J Am Chem Soc, 2003 Feb 12, 125(6), 1575 - 86
High-throughput catch-and-release synthesis of oxazoline hydroxamates . Structure-activity relationships in novel inhibitors of Escherichia coli LpxC: in vitro enzyme inhibition and antibacterial properties; Pirrung MC et al.; LpxC is a zinc amidase that catalyses the second step of lipid A biosynthesis in Gram-negative bacteria . Oxazolines incorporating a hydroxamic acid, which is believed to coordinate to the single essential zinc ion, at the 4-position are known inhibitors of this enzyme . Some of these enzyme inhibitors exhibit antibacterial activity through their inhibition of LpxC . We recently developed a method for the synthesis of oxazolines using resin capture and ring-forming release that eliminates traditional purification steps and can be used in high-throughput synthesis . Using our method, oxazoline hydroxamates with diverse 2-substituents were prepared in library form as candidate inhibitors for LpxC . Two conventional methods for oxazoline synthesis were also applied to generate more than 70 compounds . The groups at the 2-position included a wide variety of substituted aromatic rings and a limited selection of alkyl groups . These compounds were screened against wild-type and LpxC inhibitor-sensitive strains of Escherichia coli, as well as wild-type Pseudomonas aeruginosa . Inhibition of the E . coli LpxC enzyme was also investigated . A broad correlation between enzyme inhibitory and antibacterial activity was observed, and novel compounds were discovered that exhibit antibacterial activity but fall outside earlier-known structural classes.

Eur Surg Res, 2003 Jan-Feb, 35(1), 35 - 40
Influence of omentectomy on peritoneal defense mechanisms in an experimental model of intra-abdominal infection; Agca B et al.; BACKGROUND/AIM: The omentum has an important role as part of peritoneal defense mechanisms . The aim of this study is to show the bactericidal activity of peritoneal fluid and the role of the omentum as a peritoneal defense mechanism in experimental animals with intra-abdominal infections . METHODS: 40 male Spraque-Dawley rats weighing between 250 and 300 g were used in this study . The rats were randomly divided into four groups consisting of 10 animals . The operative procedures were done under sterile conditions . In group I sham laparotomy was done . In group II, the distal part of the cecum was ligated, and cecum perforation was performed . In group III, total omentectomy was performed after cecal ligation and perforation . In group IV only omentectomy was performed . Baseline and 2- and 4-hour peritoneal fluid samples were taken using a Pasteur pipette during laparotomy under anesthesia . Total peritoneal cells counts, bactericidal activity of peritoneal fluid, and types of phagocytic cells in the peritoneal fluid were assessed . RESULTS: As compared with baseline values, the total peritoneal cell counts were increased at the 2nd and 4th h in all groups (p < 0.05) . A significant increase was observed after 4 h as compared with 2 h in sham laparotomy, cecal ligation+perforation+omentectomy, and omentectomy groups (p < 0.05) . A significant increase in the cell counts after 2 h was found in the other groups when compared to the sham laparotomy group (p = 0.0001) . After 4 h, there was a significant difference between the groups, but especially prominent in the cecum ligation+perforation+omentectomy group (p = 0.0001) . Proliferating colony counts of Escherichia coli and Pseudomonas Aeruginosa decreased after 2 h, and there was no proliferation in the subsequent cultures . It was observed that the macrophage counts significantly increased after 2 and 4 h as compared with baseline in intragroup assessments (p = 0.0001) . In the intergroup assessment, an increase was observed in the macrophage counts at baseline and after 2 and 4 h, and this was significant in the cecal ligation+perforation+omentectomy group (p = 0.0001) . In the omentectomy group, a significant decrease was observed in the macrophage counts between the 2nd and 4th h (p = 0.0001) . CONCLUSION: Removal of the omentum in the presence of intra-abdominal infections causes the peripherally derived macrophages to take over the defensive role of macrophages of peritoneal origin as a compensatory mechanism, thus the peritoneal bactericidal activity against E . COLI, the major pathogen in intra-abdominal infections, does not change after omentectomy .

Exp Eye Res, 2003 Feb, 76(2), 221 - 31
Role and regulation of CXC-chemokines in acute experimental keratitis; Xue ML et al.; The aim of this study was to elucidate the expression of chemokines, their role and regulation in bacterial corneal infection using three bacterial strains (Pseudomonas . aeruginosa- invasive, cytotoxic and contact lens induced acute red eye strains) which have been shown to produce three distinct patterns of corneal disease in the mouse . The predominant chemokine expressed in response to all three strains was MIP-2 . Prolonged expression of high levels of MIP-2 was associated with increased severity of corneal inflammation . Significantly reduced disease severity upon administration of anti-MIP-2 antibodies suggested that MIP-2 may play an important role in the pathogenesis of Pseudomonas keratitis at least in part by being a major chemoattractant for polymorphonuclear leukocytes (PMN) recruitment . Interestingly, the numbers of bacteria in eyes with neutralized MIP-2 activity did not decrease even though the severity of the disease was decreased . This implies PMNs as the major destructive factor in microbial keratitis . Further, neutralization of IL-1beta activity alone using monoclonal antibodies resulted in significant reduction of both MIP-2 and KC activity indicating that chemokine levels were regulated by IL-1beta . These studies demonstrate that the regulation of MIP-2 activity may be beneficial in reducing corneal damage during microbial keratitis in rodents and perhaps that regulation of the human homologue of MIP-2, IL-8, may be useful for controlling keratitis in humans .

Nat Med, 2003 Mar, 9(3), 322 - 30 Epub 2003 Feb 03.
Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts; Grassme H et al.; Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals . Here we show that P . aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts . Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P . aeruginosa, induce apoptosis and regulate the cytokine response in infected cells . Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice . Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

J Bacteriol, 2003 Feb, 185(4), 1316 - 25
Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa; Spencer DH et al.; Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1 . The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent . Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone . While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity . We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing . A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype . Overall, we conclude that most of the PAO1 genome represents a core P . aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes . Approximately half of these additional sequences are novel.

J Antimicrob Chemother, 2003 Feb, 51(2), 423 - 6
Ex vivo synergy of arachidonate-enriched serum with ceftazidime and amikacin on multidrug-resistant Pseudomonas aeruginosa; Giamarellos-Bourboulis EJ et al.; Three multidrug-resistant strains of Pseudomonas aeruginosa were incubated ex vivo with sera sampled after a 10 min intravenous infusion of 25 mg/kg of arachidonic acid (AA) in 10 rabbits in the presence of ceftazidime and amikacin . Lipid peroxidation was assessed during bacterial growth . A statistically significant decrease in bacterial cells was found by the interaction of antimicrobials and serum sampled in the middle of infusion and 15 and 30 min after infusion of AA and was accompanied by elevated levels of malonodialdehyde . This effect of AA is probably attributed to lipid peroxidation and raises the possibility of its application in experimental infections.

J Antimicrob Chemother, 2003 Feb, 51(2), 353 - 9
Antimicrobial-induced release of endotoxin from Pseudomonas aeruginosa: comparison of in vitro and animal models; Tsuji M et al.; This study was designed to compare the amount of lipopolysaccharide (LPS) induced following exposure to doripenem, imipenem/cilastatin, meropenem and ceftazidime in an in vitro computerized-simulation system (simulating the drug concentration pattern in human plasma after administration of a drug), with that induced by exposure to a drug at a constant concentration . When Pseudomonas aeruginosa was exposed to the test drugs at constant concentrations of 0.1 x, 1 x and 10 x MIC, differential relative induction of LPS was observed as follows: ceftazidime > meropenem, doripenem > imipenem/cilastatin . In the computerized-simulation system, however, the amount of LPS induced by treatment with ceftazidime (1 g) was similar to that by doripenem (250 mg), imipenem/cilastatin (500 mg) and meropenem (500 mg) . In a rat model of P . aeruginosa bacteraemia, rates of eradication of bacteria from the blood were similar for carbapenems and ceftazidime except for 1 h post-administration of ceftazidime . Serum LPS levels induced by treatment with doripenem (30 mg/kg), imipenem/cilastatin (30 mg/kg), meropenem/cilastatin (30 mg/kg) and ceftazidime (50 mg/kg) were almost the same at 3 h after administration of each drug . Data obtained from computerized-simulation systems might be more applicable than those obtained from organisms exposed to constant drug concentrations for estimating the amount of LPS in the plasma of human patients infected with Gram-negative bacteria.

J Antimicrob Chemother, 2003 Feb, 51(2), 347 - 52
Multicentre surveillance of Pseudomonas aeruginosa susceptibility patterns in nosocomial infections; Van Eldere J; OBJECTIVES: To determine susceptibility rates and patterns in Pseudomonas aeruginosa strains isolated from nosocomial infections . METHODS: Seven hundred and sixteen P . aeruginosa isolates from 40 different hospitals in Belgium and the Grand Duchy of Luxembourg were collected in 1999 . RESULTS: Resistance rates varied significantly between hospitals . Of the fluoroquinolones, ciprofloxacin showed least resistance (24%), levofloxacin showed 27.5% resistance and ofloxacin 37.5% . Of the aminoglycosides, amikacin was the most potent antibiotic (10.5% resistance), followed by isepamicin (12%), tobramycin (19.5%) and gentamicin (23.5%) . Of the beta-lactam antibiotics, meropenem was the most active (9.5% resistance); piperacillin and piperacillin/tazobactam had, respectively, 24% and 17.5% resistance, ceftazidime 28.5%, cefepime 29.5%, ticarcillin/clavulanic acid 37% and aztreonam 55.5% . MIC distribution curves show the presence of significant subpopulations, with MICs just below breakpoint for many antibiotics . CONCLUSION: Resistance of P . aeruginosa to penicillins, cephalosporins, fluoroquinolones and aminoglycosides varies between hospitals, but is increasing.

J Antimicrob Chemother, 2003 Feb, 51(2), 257 - 66
On functional and structural heterogeneity of VIM-type metallo-beta-lactamases; Docquier JD et al.; The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens . In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded approximately 80 mg of protein per litre of culture . The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency . Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K(m) values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems . Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound . These data indicated that at least some of the amino acid differences between the two proteins have functional significance . Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.

Biomaterials, 2003 Apr, 24(9), 1663 - 70
Multiple surface properties of worn RGP lenses and adhesion of Pseudomonas aeruginosa; Bruinsma GM et al.; The aim of this study is to determine rigid gas permeable (RGP) lens surface properties prior to and after wear that are influential on adhesion of Pseudomonas aeruginosa . After 10 and 50 days of wear and after end-stage use, lenses were collected for determination of physico-chemical surface properties and bacterial adhesion in a parallel plate flow chamber . Water contact angles on unused RGP lenses amounted 47+/-13 degrees and were affected by wear . In addition, %O at the lens surfaces, as determined by X-ray photoelectron spectroscopy increased after use for 10 and 50 days, but decreased after end-stage wear . The %N hardly increased after wear and, in line, SDS-PAGE did not indicate adsorbed proteins . The surface roughness of the lenses, as measured by atomic force microscopy amounted 9 nm after 10 and 50 days of use, but end-stage lenses were significantly rougher (48+/-23 nm) . Moreover, initial deposition of P . aeruginosa #3 increased with increasing roughness for end-stage lenses . Multiple regression analysis, however, revealed that both physical and chemical surface properties were predictive for initial bacterial deposition to lens surfaces . After 10 days of wear, bacterial deposition was governed by the water contact angle, surface roughness, %O, %N, and %Si, while after 50 days of wear the surface roughness, %N, and %Si were found predictive for bacterial deposition . Initial bacterial deposition to end-stage lenses was solely dependent on the surface roughness . Summarizing, physico-chemical surface properties of RGP lenses change slightly during the first 10-50 days of wear, but end-stage lenses all had increased surface roughness, concurrent with increased bacterial adhesion.

Am J Respir Crit Care Med, 2003 Feb 1, 167(3), 384 - 9
End-organ dysfunction in cystic fibrosis: association with angiotensin I converting enzyme and cytokine gene polymorphisms; Arkwright PD et al.; The clinical course of patients with cystic fibrosis (CF) with functionally similar mutations in the CF transmembrane conductance regulator gene is variable and must therefore relate to secondary genetic and environmental factors . We examined the hypothesis that polymorphisms of certain inflammatory mediator and regulatory genes affect clinical outcome by influencing the degree of end-organ damage . By studying the possible association between clinical outcome and angiotensin I-converting enzyme (ACE) and cytokine genotypes by amplification refractory mutation system-polymerase chain reaction, using stored DNA from 261 white patients with CF, we found that ultrasound features of cirrhosis occurred more frequently in patients with the high-producer (DD) rather than the low-producer (II) ACE genotype (odds ratio {95% confidence interval}, 3.7 {1.2 to 12}) . Moreover, significant pulmonary dysfunction (age at which FEV1 < 50%) was associated with the high-producer ACE genotype (2.3 {1.2 to 4.5}) and transforming growth factor-beta1 genotype (2.6 {1.0 to 6.8}) as well as with age at first colonization with Pseudomonas aeruginosa (9.1 {1.1 to 72}) . We conclude that the high-producer ACE genotype predicts patients with CF who have an increased chance of developing portal hypertension; and high-producer ACE and TGF-beta1 genotypes are secondary genetic factors contributing to pulmonary dysfunction in these patients.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 1999 Dec, 16(4), 406 - 10
{Study of bacterial adhesion to prosthetic valve materials in vitro}; Huang Y et al.; This article reports a study of the bacterial adhesion to prosthetic valve materials in vitro . The method for assessing the degree of bacterial adhesion to prosthetic valve materials was established primarily . The capacities of staphylococcus aureus(SA), staphylococcus epidermidis(SE), Escherichia Coli(EC) and Pseudomonas aeruginosa(PA) for adhesion to Dacron, Pyrolytic carbon and polytetrofluoroethylene (PTFE) were quantitatively determined by the plate counting and Gamma-ray counting of 125I radiolabeled bacteria in vitro . The results showed that the capacities of four types of bacteria for adhesion to Dacron, Pyrolytic carbon and PTFE coincided with the bacterial-growth curve . The capacities of four bacteria for adhering to Dacron were stronger . The adhesion of SE to Pyrolytic carbon was the strongest . The adhesion of PA Kept up a high level . The capacities of EC and PA for adhering to PTFE were the strongest . The results indicate that the capacities of different types of bacteria for adhesion to the same prosthetic valve material are different, and the capacities of one type of bacteria for adhesion to different materials are also different.

Se Pu, 1999 Jan, 17(1), 46 - 8
{Purification of outer membrane proteins in Pseudomonas aeruginosa by high performance ion-exchange liquid chromatography}; Zuo L et al.; Pseudomonas aeruginosa PAO1 was used in this study . Isolation of outer membrane was accomplished by treating the cell envelope with EDTA and lysozyme, followed by centrifugation . The outer membrane (10 mg of protein) was mixed with 34 mmol/L octyl beta-glucoside-5 mmol/L EDTA-10 mmol/L Tris-HCl (pH 8.0) and subjected to supersonic oscillation for 2 min . The centrifuged supernatant (100 kgf for 30 min at 20 degrees C) was applied onto a DEAE ion-exchange high performance liquid chromatographic column (TSK gel-DEAE-5PW column, 0.75 cm x 7.5 cm i.d.) that was equilibrated with a solution of 10 mmol/L Tris-HCl buffer (pH 8.0) containing 2.5 mmol/L beta-C12E8 and 1 mmol/L EDTA . The column was washed with the same solution and eluted with a linear gradient of 0-0.5 mol/L NaCl in the same solution and fractions A, B, C were collected . Proteins in these fractions were analyzed by SDS-polyacrylamide gel electrophoresis and quantified by the method of Lowry et al . Protein E(Mr 43,000), G(Mr 25,000) and H (Mr 19,000) flowed through the column without adsorption in fraction A . Protein C(Mr 70,000), D(Mr 46,000) and a small amount of F (Mr 34,000) were eluted in fraction B . Fraction A was concentrated with ultrafiltration and applied again onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L beta-C12E8 and 1 mmol/L MgCl2 . Fraction B was subjected to DEAE ion-exchange HPLC column in the presence of EDTA . This fraction was then applied onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L octylglucoside and 1 mmol/L EDTA . By these procedures protein C, D and E were purified to apparent homogeneity as judged by SDS-PAGE . In this work, we purified the outer membrane proteins of Pseudomonas aeruginosa, and used a new technique selectively solubilizing the cytoplasmic membrane with sodium lauryl sarcosinate for isolating the outer membrane proteins of Pseudomonas aeruginosa because of its relative simplicity.

DNA Repair (Amst), 2003 Mar 1, 2(3), 273 - 84
recX, a new SOS gene that is co-transcribed with the recA gene in Escherichia coli; Pages V et al.; recX is a small open reading frame located downstream of recA that is conserved in many bacteria . In Escherichia coli, the recX gene (also named oraA) is a 501 bp open reading frame that encodes a predicted basic protein . Transcriptional analysis by Northern blots showed that in E . coli the recX gene is SOS-regulated . Primer extension data and transcriptional fusions indicate that recX transcription is down regulated with respect to recA by an intrinsic transcription terminator that is located between the recA and recX coding sequences . Despite the presence of this terminator, a recA-recX message resulting from transcriptional readthrough is detected at a level of 5-10% of the recA message . In addition, transcriptional/translational fusion experiments show that recX expression is further down regulated at the translational level reaching an estimated protein level about 500-fold lower than RecA . Strains in which the recX gene was disrupted were constructed by insertion of an antibiotic resistance cassette . The survival after UV irradiation, the spontaneous and UV-induced mutation rates were not significantly different in these recX strains compared to the corresponding wild type strain . Overexpression of RecA was shown to be lethal in a recX deletion strain in Pseudomonas aeruginosa {J . Bacteriol . 175 (1993) 2451}, Mycobacterium tuberculosis {Mol . Microbiol . 30 (1998) 525} and Streptomyces lividans {J . Bacteriol . 182 (2000) 4005} suggesting that the recX gene may act as a regulator of recA . In contrast in E . coli, in a recX deletion strain, RecA overexpression is neither toxic nor is the expression of the recA(+) gene affected in a recX deletion strain at the basal level or after UV induction.

J Antibiot (Tokyo), 2002 Nov, 55(11), 975 - 92
S-3578, a new broad spectrum parenteral cephalosporin exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa . Synthesis and structure-activity relationships; Yoshizawa H et al.; A series of 7-aminothiadiazolylcephalosporins having a 1-(substituted)-1H-imidazo{4,5-b}pyridinium group at the C-3' position of the cephem nucleus were synthesized and evaluated for in vitro antibacterial activities . Among the cephalosporins prepared in this study, 7beta-{2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-ethoxyiminoacetamido}-3-{1-(3-methylaminopropyl)-1H-imidazo{4,5-b}pyridinium-4-yl}methyl-3-cephem-4-carboxylate sulfate (S-3578) showed extremely potent broad spectrum activity against both gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and gram-negative bacteria including Pseudomonas aeruginosa, and good water solubility.

Hua Xi Yi Ke Da Xue Xue Bao, 2000 Sep, 31(3), 334 - 7
{A clinical study on PA-MSHA vaccine used for adjuvant therapy of lymphoma and lung cancer}; Li Z et al.; The objective of this study was to evaluate the effectiveness and safety of pseudomonas aeruginosa MSHA vaccine (PA-MSHA vaccine) used as an immune modulator . The study was carried out after the design of a non-blind, randomized controlled trial . The malignant lymphoma patients were divided into experiment group (45 cases) and control group (43 cases) . Likewise were distributed the lung cancer patients: 44 cases in experiment group and 45 cases in control group . The two experiment groups received chemotherapy + PA-MSHA vaccine . The two control groups were given chemotherapy alone . The results showed that the clinical efficacy rate was 95.56% for the exp . group and 69.77% (P < 0.01) for the control group of the malignant lymphoma cases; the clinical efficacy rate was 59.09% for the exp . group and 42.22% (P < 0.01) for the control group of the lung cancer cases . The infection rate was 17.78% for the exp . group and 37.21% (P < 0.05) for the control group of the malignant lymphoma cases; the infection rate was 15.91% for the exp . group and 40.00% (P < 0.05) for the control group of the lung cancer cases . The comprehensive immune effectiveness rates for the exp . group and control group of the malignant lymphoma cases were 77.78% and 23.26% (P < 0.01) respectively . The comprehensive immune effectiveness rates for the exp . group and control group of the lung cancer cases were 84.09% and 35.56% (P < 0.01) respectively . Adverse reactions were found in 5 cases, of which, one was given medical care while the others automatically recovered . These results indicate that PA-MSHA vaccine, as a new kind of immune modulator, can improve the effectiveness of treatment for tumor with low adverse reaction rate.

Trans Am Ophthalmol Soc, 2002, 100, 243 - 71
The use of antimicrobial peptides in ophthalmology: an experimental study in corneal preservation and the management of bacterial keratitis; Mannis MJ; PURPOSE: Bacterial keratitis is an ocular infection with the potential to cause significant visual impairment . Increasing patterns of antibiotic resistance have necessitated the development of new antimicrobial agents for use in bacterial keratitis and other serious ocular infections . With a view to exploring the use of novel antimicrobial peptides in the management of ocular infection, we performed a series of experiments using synthetic antimicrobial peptides designed for the eradication of common and serious ophthalmic pathogens . METHODS: Experiments were performed with three clinical ocular isolates--Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis--in three experimental settings: (1) in vitro in a controlled system of 10 mM sodium phosphate buffer, (2) in vitro in modified chondroitin sulfate-based corneal preservation media (Optisol), and (3) in an in vivo animal model (rabbit) simulating bacterial keratitis . In all cases, outcomes were measured by quantitative microbiological techniques . RESULTS: The candidate peptides (CCI A, B, and C and COL-1) produced a total reduction of the test pathogens in phosphate buffered saline . In modified Optisol, the peptides were effective against S epidermidis at all temperatures, demonstrated augmented activity at 23 degrees C against the gram-positive organisms, but were ineffective against P aeruginosa . The addition of EDTA to the medium augmented the killing of P aeruginosa but made no difference in the reduction of gram-positive organisms . In an in vivo rabbit model of Pseudomonas keratitis, COL-1 demonstrated neither clinical nor microbicidal efficacy and appeared to have a very narrow dosage range, outside of which it appeared to be toxic to the ocular surface . CONCLUSION: Our data indicate that the antimicrobial peptides we tested were effective in vitro but not in vivo . In an age of increasing antibiotic resistance, antimicrobial peptides, developed over millions of years as innate defense mechanisms by plants and animals, may have significant potential for development as topical agents for the management of severe bacterial keratitis . However, modifications of the peptides, the drug delivery systems, or both, will be necessary for effective clinical application.

Antimicrob Agents Chemother, 2003 Feb, 47(2), 582 - 7
Biochemical characterization of the acquired metallo-beta-lactamase SPM-1 from Pseudomonas aeruginosa; Murphy TA et al.; SPM-1 is a new metallo-beta-lactamase recently identified in Pseudomonas aeruginosa strain 48-1997A, isolated in Sao Paulo, Brazil . Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of beta-lactam antibiotics . Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins . The highest k(cat/)K(m) ratios (in micromolar per second) overall were observed for this subgroup . The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-beta-lactamase IMP-1, yet for the most part, SPM-1 has k(cat)/K(m) values higher than those of IMP-1 . Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1 . A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-beta-lactamases and which provides an explanation for the kinetic profiles observed.

Burns, 2003 Feb, 29(1), 43 - 8
Effects of dietary arginine supplementation on antibody production and antioxidant enzyme activity in burned mice; Shang HF et al.; This study investigated the effect of arginine (Arg) supplementation on specific antibody production and antioxidant enzyme activities in burned mice vaccinated with detoxified Pseudomonas exotoxin A linked with the outer membrane proteins I and F, named PEIF . Also, the survival rate of burned mice complicated with Pseudomonas aeruginosa was evaluated . Experiment 1: Thirty BALB/c mice were assigned to two groups . One group was fed a control diet with casein as the protein source, while the other group was supplemented with 2% Arg in addition to casein . The two groups were isonitrogenous . The mice were immunized twice with PEIF, and the production of specific antibodies against PEIF was measured every week . After 8 weeks, all mice received a 30% body surface area burn injury . Mice were sacrificed 24h after the burn . The antioxidant enzyme activities and lipid peroxides in the tissues as well as the specific antibody production were analyzed . Experiment 2: Twenty-eight mice were divided into two groups and vaccinated as described in experiment 1 . After the burn the mice were infected with P . aeruginosa, and the survival rate was observed for 8 days . The results demonstrated that antioxidant enzyme activities and lipid peroxides in tissues were significantly lower in the Arg group than in the control group after the burn . The production of specific antibodies against P . aeruginosa significantly increased in the Arg group at 4 and 7 weeks after immunization, and 24h after the burn . The survival rates of vaccinated burned mice after bacterial infection did not significantly differ between the two groups . These results suggest that vaccinating mice with Arg supplementation may enhance humoral immunity and attenuate the oxidative stress induced by burn injury . However, Arg supplementation did not improve survival in vaccinated mice complicated with P . aeruginosa infection.

Intensive Care Med, 2003 Mar, 29(3), 396 - 402 Epub 2003 Jan 23.
Bactericidal activity response of blood neutrophils from critically ill patients to in vitro granulocyte colony-stimulating factor stimulation; Yang K et al.; OBJECTIVE: Neutrophil function impairment is common in nonneutropenic critically ill patients . Whether granulocyte colony-stimulating factor (G-CSF) may be useful for preventing nosocomial infection in these patients is debated . The response of blood neutrophils from critically ill patients to G-CSF was investigated in vitro . DESIGN AND SETTING: Prospective study, laboratory investigation in two intensive care units . PATIENTS: 52 critically ill patients without immunosuppression . MEASUREMENTS: Neutrophils obtained from 52 patients on the 5th day of their intensive care unit stay were incubated with and without G-CSF (1, 10, 100 ng/ml) . Reactive oxygen species (ROS) release and bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa were evaluated . Plasma cytokines (interleukin 10, tumor necrosis factor alpha, and G-CSF) were measured . RESULTS: Median values (25th-75th percentiles) indicated no stimulatory effect of G-CSF on neutrophil bactericidal activity against either organism: S . aureus, 100% (95-109) of the unstimulated condition with 1 ng/ml G-CSF, and P . aeruginosa, 102% (98-109) with 1 ng/ml G-CSF . However, wide interindividual variability was found, ranging from marked inhibition to marked stimulation . Similar variability was found for ROS release . No correlations were found between ROS release and bactericidal activities against either bacterial strain . Inhibition of neutrophil bactericidal activity by G-CSF was associated with significantly higher plasma interleukin 10 concentrations . Plasma G-CSF levels were significantly higher in patients whose neutrophil bactericidal activity was unresponsive to G-CSF, suggesting G-CSF receptor downregulation . CONCLUSIONS: The effect of G-CSF on in vitro neutrophil bactericidal activity varied widely, depending on endogenous levels of G-CSF and was not predictable based on severity scores.

Am J Respir Cell Mol Biol, 2003 Feb, 28(2), 249 - 56
Surfactant protein A and D differently regulate the immune response to nonmucoid Pseudomonas aeruginosa and its lipopolysaccharide; Bufler P et al.; We investigated the role of the surfactant proteins (SPs) A and D in the pulmonary immune defense of nonmucoid strains of Pseudomonas aeruginosa, the most etiologic agents of nosocomial Pseudomonas pneumonia . We first examined the interactions of recombinant human SP-D dodecamers and purified natural or recombinant human SP-A with two smooth, and two rough, clinical isolates of nonmucoid P . aeruginosa . SP-D bound to all four isolates, but agglutinated only one rough and one smooth strain . SP-D functioned as an opsonin to enhance the uptake of all four strains by the human monocytic cell line Mono Mac 6 (MM6) . SP-D also enhanced tumor necrosis factor-alpha secretion by MM6 cells in response to purified lipopolysaccharide (LPS) isolated from the rough, but not the smooth, strains . Although SP-A bound to all four strains, it did not cause bacterial aggregation or enhance uptake . It showed small but statistically significant inhibitory effects on the cytokine response of MM6 cells to one strain of smooth organisms, but did not significantly alter the response to purified LPS . This study in combination with previously published data strongly suggests that SP-D may play important roles in the local innate pulmonary defense against nonmucoid P . aeruginosa of diverse LPS phenotypes, and preferentially augments the cellular response to rough P . aeruginosa endotoxin.

Clin Infect Dis, 2003 Feb 1, 36(3), e55 - 7 Epub 2003 Jan 20.
Hot tub-associated necrotizing pneumonia due to Pseudomonas aeruginosa; Crnich CJ et al.; We describe a case of severe necrotizing pneumonia due to community-acquired Pseudomonas aeruginosa . Cultures of fluid obtained from the filter of the patient's hot tub grew the same P . aeruginosa strain as that grown from culture of the patient's sputum . Centers for Disease Control and Prevention guidelines should be strictly followed for hot tub maintenance to prevent P . aeruginosa overgrowth: the range of free chlorine levels in the water should be kept at 1-3 mg/L, and the pH should be kept at 7.2-7.8.

Pediatr Res, 2003 Feb, 53(2), 313 - 9
IL-10 controls Aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis; Casaulta C et al.; Up to 90% of patients with cystic fibrosis (CF) are chronically colonized with Pseudomonas aeruginosa, and 10% to 50% of CF patients are colonized with Aspergillus fumigatus . Despite an extensive inflammatory reaction, patients cannot eliminate the microorganisms . The present study demonstrates that an IL-10 mediated T-cell tolerance to major infectious agents A . fumigatus and P . aeruginosa plays an important role in the control of T-cell-mediated inflammatory responses in CF . Peripheral blood mononuclear cells of CF patients secreted significantly higher amounts of IL-10 . T-cell response against recombinant A . fumigatus antigens rAsp f 3, rAsp f 4, rAsp f 6, and heat-inactivated P . aeruginosa was controlled by IL-10 . Proliferation and interferon-gamma production was significantly increased when endogenous IL-10 was blocked in aspergillus and pseudomonas antigen-stimulated cells of CF patients . The role of IL-10 was further documented by increased spontaneous proliferation of peripheral blood mononuclear cells of CF patients after preincubation with antisense oligonucleotides blocking the synthesis of IL-10 receptor-associated kinases janus tyrosine kinase 1 and tyrosine kinase 2 . Together, these data demonstrate an important role of IL-10-mediated peripheral T-cell tolerance to P . aeruginosa and A . fumigatus in the control of the intensity of the inflammatory T-cell response in CF.

Environ Microbiol, 2002 Dec, 4(12), 883 - 97
Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core; Daniels C et al.; Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised patients and trauma victims and causes fatal lung infections in people with cystic fibrosis . This microorganism produces a number of virulence factors, one of which is lipopolysaccharide (LPS), which has been shown to mediate many biological effects including resistance to serum killing and phagocytosis . These biological activities have been correlated to the length of the O-polysaccharide and its distribution on the outer membrane . Wzz is responsible for regulation of the size distribution of the O-antigen . Wzz has been found to participate solely in the Wzy-dependent pathway for LPS biosynthesis, which produces heteropolymeric O-polysaccharide such as the B-band LPS of P . aeruginosa . Our laboratory has previously reported characterization of a Wzz protein encoded in the B-band O-antigen biosynthesis cluster of PAO1 . The availability of the genome sequence of P . aeruginosa PAO1 has made it possible to identify a second functional Wzz protein (PA0938, Wzz2) . Gene replacement was used to generate an unmarked wzz2delta knock-out and a wzz2delta/wzz1::Gm double knock-out . As expected, the wzz2delta strain produced LPS with modal length imparted by Wzz1, and the wzz2delta/wzz1::Gm strain produced LPS O-antigen with a non-modal (random) length . Both wzz1 and wzz2 from P . aeruginosa PAO1 were cloned and expressed with an N-terminal His6 tag . His6-Wzz1 and His6-Wzz2 were purified to near homogeneity by immobilized metal affinity chromatography (IMAC) . These preparations were used to develop specific polyclonal antibodies against each of the proteins . In vivo protein cross-linking followed by Western immunoblotting indicated that Wzz1 forms dimers whereas Wzz2 forms octamers . By generation of a wzz2delta/rmlC double mutant and analysis of the LPS, we have made the novel observation that polymerization of modal chain length-distributed O-antigen occurred before ligation to the lipid A core . We have shown an association between the Wzz proteins and O-antigen polymer chains using immunoprecipitation with anti-O5 O-antigen monoclonal antibody MF15-4 . Both Wzz1 and Wzz2 could be co-precipitated with O5 polymer.

Environ Microbiol, 2002 Dec, 4(12), 872 - 82
Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates; Pirnay JP et al.; Genomes are constantly evolving . Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance . Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa . The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed . A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported . An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before . A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains . We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.

J Bacteriol, 2003 Feb, 185(3), 1071 - 81
Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa; Firoved AM et al.; Pseudomonas aeruginosa is the dominant pathogen causing chronic respiratory infections in cystic fibrosis (CF) . After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of P . aeruginosa to the mucoid, exopolysaccharide alginate-overproducing phenotype . The emergence of mucoid P . aeruginosa in CF is associated with respiratory decline and poor prognosis . The switch to mucoidy in most CF isolates is caused by mutations in the mucA gene encoding an anti-sigma factor . The mutations in mucA result in the activation of the alternative sigma factor AlgU, the P . aeruginosa ortholog of Escherichia coli extreme stress sigma factor sigma(E) . Because of the global nature of the regulators of mucoidy, we have hypothesized that other genes, in addition to those specific for alginate production, must be induced upon conversion to mucoidy, and their production may contribute to the pathogenesis in CF . Here we applied microarray analysis to identify on the whole-genome scale those genes that are coinduced with the AlgU sigmulon upon conversion to mucoidy . Gene expression profiles of AlgU-dependent conversion to mucoidy revealed coinduction of a specific subset of known virulence determinants (the major protease elastase gene, alkaline metalloproteinase gene aprA, and the protease secretion factor genes aprE and aprF) or toxic factors (cyanide synthase) that may have implications for disease in CF . Analysis of promoter regions of the most highly induced genes (>40-fold, P < or = 10(-4)) revealed a previously unrecognized, putative AlgU promoter upstream of the osmotically inducible gene osmE . This newly identified AlgU-dependent promoter of osmE was confirmed by mapping the mRNA 5' end by primer extension . The recognition of genes induced in mucoid P . aeruginosa, other than those associated with alginate biosynthesis, reported here revealed the identity of previously unappreciated factors potentially contributing to the morbidity and mortality caused by mucoid P . aeruginosa in CF.

Biol Reprod, 2003 Feb, 68(2), 509 - 15
Messenger RNA (mRNA) expression for the antimicrobial peptides beta-defensin-1 and beta-defensin-2 in the male rat reproductive tract: beta-defensin-1 mRNA in initial segment and caput epididymidis is regulated by androgens and not bacterial lipopolysaccharides; Palladino MA et al.; Mechanisms of antimicrobial protection in male reproductive organs are poorly understood . Defensins are antimicrobial peptides produced by many epithelial tissues . The goals of the present study were 1) . to test the hypothesis that adult rat male reproductive organs express mRNA for rat beta-defensin (RBD)-1 and RBD-2, 2) . to examine if defensin mRNA expression in the testis and epididymis is induced by bacterial lipopolysaccharides (LPS), and 3) . to investigate the effects of androgens on defensin mRNA expression in the epididymis . Total RNA from reproductive organs was analyzed by relative reverse transcription-polymerase chain reaction analysis . RBD-1 mRNA was detected in the testis . All segments of epididymis expressed equal levels of RBD-1 mRNA with higher expression than in the testis, whereas accessory sex glands showed expression equal to that in the testis . Expression of RBD-2 mRNA was primarily restricted to the penis . Effects of inflammation on defensin mRNA expression were examined in rats administered a unilateral injection of LPS from Pseudomonas aeruginosa or Escherichia coli . Expression of RBD-1 mRNA in the testis and epididymis was unaffected by LPS . To test the hypothesis that circulating androgens regulate RBD-1 mRNA expression in the epididymis, rats were subjected to bilateral orchiectomy (orch) or to orch plus a 3.5-cm implant containing testosterone . Expression of RBD-1 mRNA in the initial segment and caput was unchanged following 1-day orch but showed androgen-sensitive expression after 5 and 15 days . Expression of RBD-1 mRNA in corpus and cauda was not affected by orch . Results of this study suggest that RBD-1 may play an antimicrobial role in the testis and epididymis.

J Microbiol Methods, 2003 Mar, 52(3), 379 - 88
A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity; Hoefel D et al.; Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria . Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters . In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product . Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not . CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation . We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state . This study shows that CFDA/SE is a poor marker of bacterial activity.

J Microbiol Methods, 2003 Mar, 52(3), 361 - 6
Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay; Swanson B et al.; Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis and mechanically ventilated patients by binding to specific carbohydrate residues on the surface of lung epithelial cells . Studies have shown that blocking this interaction may have therapeutic effects in vivo . To test compounds that may have an effect on the binding of P . aeruginosa to epithelial cells, we have developed a pseudomonal adhesion assay that is compatible with high throughput technology . This assay utilizes a 96-well culture plate assay and P . aeruginosa strains that have been modified to bioluminesce . This method has proven to be a rapid, sensitive and reproducible system for screening agents that inhibit bacterial adhesion.

J Enzyme Inhib Med Chem, 2002 Aug, 17(4), 261 - 6
Unsymmetrical 1,1'-disubstituted ferrocenes: synthesis of Co(ii), Cu(ii), Ni(ii) and Zn(ii) chelates of ferrocenyl -1-thiadiazolo-1'-tetrazole, -1-thiadiazolo-1'-triazole and -1-tetrazolo-1'-triazole with antimicrobial properties; Chohan ZH et al.; Condensation reactions of 1,1''-diacetylferrocene with different heteroaromatic amines such as, 2-amino-1,3,4-thiadiazole, 5-aminotetrazole and 3-amino-1,2,4-triazole to form unsymmetrically 1,1'-disubstituted ferrocenes have been studied . The obtained compounds have been further investigated for their liganding and biological properties upon chelation with Co(II), Cu(II), Ni(II) and Zn(II) metal ions . The synthesized compounds have been characterized by physical, spectral and analytical data and have been screened against pathogenic bacterial strains e.g., Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, showing moderate activity as antibacterials in vitro.

J Enzyme Inhib Med Chem, 2002 Aug, 17(4), 235 - 46
Characterization of competitive inhibitors for the transferase activity of Pseudomonas aeruginosa exotoxin A; Armstrong S et al.; A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A . The IC50 values for the compounds tested ranged from 87 nM to 484 microM for NAP and CMP12, respectively . It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD+ substrate with a Ki of 45 +/- 5 nM, which was in good agreement with the dissociation constant determined independently (KD = 56 +/- 6 nM) . The IC50 value for NAP was 87 +/- 12 nM, which strongly correlated with the Ki and KD values . Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry . Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site . This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.

N Engl J Med, 2003 Jan 16, 348(3), 221 - 7
An outbreak of Pseudomonas aeruginosa infections associated with flexible bronchoscopes; Srinivasan A et al.; BACKGROUND: Endoscopes, including bronchoscopes, are the medical devices most frequently associated with outbreaks of nosocomial infections . We investigated an outbreak of Pseudomonas aeruginosa infections after bronchoscopic procedures . METHODS: Microbiologic results were reviewed to determine the rates of recovery of P . aeruginosa from bronchoalveolar-lavage specimens . Environmental samples from endoscopes and the endoscopy suite were cultured . Medical records were reviewed to identify infections in the 14 days after a bronchoscopy . RESULTS: The rate of recovery of P . aeruginosa from bronchoalveolar-lavage specimens obtained with use of endoscopy-suite bronchoscopes increased from 10.4 percent at base line to 31.0 percent during the outbreak (relative risk, 2.97; 95 percent confidence interval, 2.28 to 3.90) . Cultures of samples from three bronchoscopes grew P . aeruginosa, whereas cultures of samples from the environment, instrument-cleaning machines, and gastrointestinal endoscopes did not . The three bronchoscopes had been part of a nationwide recall . A total of 414 patients underwent bronchoscopy during the outbreak, and there were 48 respiratory tract and bloodstream infections among 39 of these patients (9.4 percent) . In 32 infections (66.7 percent), P . aeruginosa was confirmed as a potentially causative organism . Exposure to a potentially contaminated bronchoscope may have had a role in the death of three patients . The rate of recovery of P . aeruginosa returned to base line after the instruments were removed from service . CONCLUSIONS: This large outbreak of P . aeruginosa infections related to bronchoscopy was apparently caused by a loose biopsy-port cap in the bronchoscopes . Instrument safety and surveillance methods for bronchoscopy must be improved, and better recall procedures are needed for medical devices .

Nucleic Acids Res, 2003 Jan 15, 31(2), 602 - 7
Human RNase 7: a new cationic ribonuclease of the RNase A superfamily; Zhang J et al.; Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence . The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart . Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine-lysine- histidine triad which are signature motifs of this superfamily . The protein is atypically cationic with an isoelectric point (pI) of 10.5 . Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4) . An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus . While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40% . Of particular interest, ECP/RNase 3's cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine . This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.

Biomaterials, 2003 Apr, 24(8), 1507 - 18
Adhesion of Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly(vinyl chloride) (PVC) from endotracheal intubation devices; Triandafillu K et al.; Pseudomonas aeruginosa pneumonia is a life threatening complication in mechanically ventilated patients that requires the ability of the bacteria to adhere to, and colonize the endotracheal intubation device . New strategies to prevent or reduce these nosocomial infections are greatly needed . We report here the study of a set of P . aeruginosa clinical isolates, together with specific mutants, regarding their adhesion on native and chemically modified poly(vinyl chloride) (PVC) surfaces from endotracheal intubation devices . The adhesion of the different strains to untreated PVC varied widely, correlating with several physico-chemical characteristics known to influence the attachment of bacteria to inert surfaces . The adhesion patterns were compared to the calculations obtained with the DLVO theory of colloidal stability . These results illustrate the importance of testing different clinical isolates when investigating bacterial adhesion . Oxygen plasma treatment of the PVC pieces yielded a hydrophilic surface and reduced the number of adhering bacteria by as much as 70% . This reduction is however unlikely to be sufficient to prevent P . aeruginosa colonization of endotracheal intubation devices .

Biochim Biophys Acta, 2003 Jan 20, 1619(2), 213 - 9
Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin; Stoitsova SR et al.; The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis . Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence . The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer . Interactions between PA-I and adenine were examined by equilibrium dialysis using {3H} adenine . The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively . These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement . The titration curve confirmed the existence of two classes of adenine-binding sites . Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered . This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.

Rhinology, 2002 Dec, 40(4), 226 - 8
Recurrent facial pain due to Pseudomonas aeruginosa sinusitis; Danielides V et al.; Pseudomonas (P) aeruginosa has been rarely reported as a causative agent of chronic sinusitis in otherwise healthy individuals, mostly as part of polymicrobial infections, while it has been frequently described among immunocompromised patients . We report a case of chronic maxillary sinustitis due to P . aeruginosa presenting as recurrent facial pain in a previously healthy middle-aged woman . Bacteriological diagnosis was established by tissue cultures and definitive treatment was achieved by surgical intervention and postoperative antibiotic treatment along with topical care.

J Infect Chemother, 2002 Dec, 8(4), 371 - 3
Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: substrate specificities of the efflux systems; Okamoto K et al.; Overproduction of multidrug efflux systems MexAB-OprM, MexCD-OprJ, and MexXY/OprM of Pseudomonas aeruginosa caused reduction of susceptibility of the mutant, which lacked AmpC and all three systems to panipenem, meropenem, S4661 and DU6681a; meropenem, S4661 and DU6681a; and BO2727, panipenem, meropenem, S4661 and DU6681a, respectively, but not reduction of the susceptibility to imipenem and biapenem . Thus, we determined substrate specificities of these efflux systems to carbapenems.

Zhongguo Zhong Yao Za Zhi, 2001 Apr, 26(4), 223 - 6
{Screening of anti-bacteria activity of extracts of Gaultheria leucocarpa var . yunnanensis}; Ma XJ et al.; OBJECTIVE: To further develop Gaultheria leucocarpa var . yunnanensis, anti-bacteria constituents in it were screened . METHOD: The constituents were extracted by chromatographic process . The anti-bacteria test was made with regulatory method of analysis . RESULT AND CONCLUSION: Anti-bacteria test with extracts of water, acetic ester and n-butanol showed that 3 extracts from 22 samples had anti-Staphylococcus aureus action, and the extracts from root and stem showed the same result . 2 extracts could kill Escherichia coli and Pseudomonas aeruginosa . The lower the concentrate, the less the anti-bacteria action was . These results suggested that not only essential oil but other ingredients from G . leucocarpa var.yunnanensis have anti-bacteria activity . Anti-fungi test of the same extracts didn't indicate remarkable action.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Sep-Oct, (5), 67 - 8
{Cultivation of Pseudomonas aeruginosa in media with chlorella fermentative hydrolysate as nutrient base}; Akhapkina IG et al.; The specific features of the growth of P . aeruginosa in media prepared with the use of Chlorella fermentative hydrolysate as nutrient basis were studied . The accumulation of microbial biomass was shown to depend on increased content of amino nitrogen in the media, while the effectiveness of the pyocyanin formation was linked with decreased content of amino nitrogen and a rise in the amount of sodium chloride.

J Biotechnol, 2003 Feb 27, 101(1), 11 - 8
Evaluation of bacterial aerotaxis for its potential use in detecting the toxicity of chemicals to microorganisms; Shitashiro M et al.; Bacterial aerotaxis (the movement of a cell toward oxygen) was evaluated for its potential use in detecting the toxicity of chemicals to microorganisms . The level of toxicity was determined by the concentration of test chemicals resulting in a 50% inhibition of aerotaxis of Pseudomonas aeruginosa PAO1 after 40 min of exposure . The aerotactic responses of P . aeruginosa were measured by using chemotaxis well chambers . Each clear acrylic chamber had a lower and upper well separated by a polycarbonate filter with a uniform pore size of 8.0 microm . To automatically detect bacterial cells that crossed the filter in response to a gradient of oxygen, P . aeruginosa PAO1 was marked with green fluorescent protein (GFP), and the GFP fluorescence intensity in the upper well was continuously monitored by using a fluorescence spectrometer . By using this technique, volatile chlorinated aliphatic compounds, including trichloroethylene (TCE), trichloroethane, and tetrachloroethylene, were found to be inhibitory to bacterial aerotaxis, suggesting their possible toxicity to microorganisms . We also examined more than 20 potential toxicants for their ability to inhibit the aerotaxis of P . aeruginosa . Based on these experimental results, we concluded that bacterial aerotaxis has potential for use as a fast and reliable indicator in assessing the toxicity of chemicals to microorganisms.

Can Respir J, 2002 Nov-Dec, 9(6), 401 - 6
Additive effect of dornase alfa and Nacystelyn on transportability and viscoelasticity of cystic fibrosis sputum; Sun F et al.; OBJECTIVE: To investigate the effect of dornase alfa (DA), Nacystelyn (NAL) and their combination on mucociliary transportability and mucus viscoelasticity of cystic fibrosis (CF) sputum, and to assess whether the combination possesses an additive effect . DESIGN: Determination of transportability in frog palate and viscoelasticity in vitro . SETTING: Research laboratory at a medical centre . PATIENTS: Sputa from 15 patients with CF, chronically infected with Pseudomonas aeruginosa, were studied . INTERVENTIONS: Sputum samples were incubated without any drug solution as a control, and with normal saline, DA, NAL and a mixture of DA and NAL in concentrations approximating those achieved in clinical practice . RESULTS: Normal saline (10% volume) by itself had a small effect on CF sputum transportability with a mean increase of 9%, and on viscoelasticity with a mean of decrease of 0.22 log units, respectively, compared with control (incubation without saline) . DA (200 nM) further increased the transportability by a mean of 35% versus saline and decreased viscoelasticity by a mean of 0.30 log units . NAL (100 M) increased the transportability by a mean of 32% and decreased viscoelasticity by a mean of 0.22 log units from the levels achieved with saline . The mixture of DA plus NAL at one-half of the above concentration of each agent produced an additional increase in the transportability, by a mean of 18%, and a further decrease in viscoelasticity, by a mean of 0.25 log units, compared with DA or NAL as a single treatment . CONCLUSIONS: The combination of DA and NAL exhibits an additive effect for both the viscoelasticity and transportability of CF sputum samples . The two agents appear to act well together in breaking down the bonding due to extracellular DNA and mucins . Clinical studies should be undertaken to see whether the additive combination at lower concentration produces the anticipated benefits of improved airway clearance and fewer side effects.

Nucleic Acids Res, 2003 Jan 1, 31(1), 266 - 9
PRODORIC: prokaryotic database of gene regulation; Munch R et al.; The database PRODORIC aims to systematically organize information on prokaryotic gene expression, and to integrate this information into regulatory networks . The present version focuses on pathogenic bacteria such as Pseudomonas aeruginosa . PRODORIC links data on environmental stimuli with trans-acting transcription factors, cis-acting promoter elements and regulon definition . Interactive graphical representations of operon, gene and promoter structures including regulator-binding sites, transcriptional and translational start sites, supplemented with information on regulatory proteins are available at varying levels of detail . The data collection provided is based on exhaustive analyses of scientific literature and computational sequence prediction . Included within PRODORIC are tools to define and predict regulator binding sites . It is accessible at http://prodoric.tu-bs.de.

Hunan Yi Ke Da Xue Xue Bao, 2000 Dec 28, 25(6), 567 - 9
{Distribution of bacteria and analysis of their sensibility to antibiotics in patients with hospital-acquired pneumonia}; Cheng WC et al.; OBJECTIVES: This study was explore the distribution of the bacteria and their sensibility to antibiotics in hospital-acquired pneumonia . METHODS: One hundred and ninety-six bacterium species were collected in patients with the hospital-acquired pneumonia to make sputum culture . The sensibility of the bacteria to antibiotics were examined by KB paper method and the minimal-inhibitory-concentration by gel double multiple dilute method . RESULTS: Most of the G- bacteria were pseudomonas aeruginosa (30%) and klebsiella bacillus (22%) . Most of the G+ bacteria were staphylococcus epidermidis (14%) and staphylococcus aureus (12%) . G- bacteria were sensitive to impienem(98%), cefoperazone(90%), ceftriaxone(90%), leftazidime(92%), ciprofloxacin(90%), and amikacin(89%) . The sensibility of vancomycin to G+ bacteria was 100% . CONCLUSIONS: The pseudomonas aeruginosa, klebsiella bacillus, staphylococcus epidermidis, and staphylococcus aureus are the most important bacteria in patients with hospital-acquired pneumonia . Imipenem, cefoperazone, ceftriazone, leftazidime, ciprofloxacin, amikacin, and vancomycin are effective antibiotics for treating hospital-acquired pneumonia.

J Reprod Med, 2002 Dec, 47(12), 1035 - 7
Pseudomonas aeruginosa-infected IUD associated with pelvic inflammatory disease . A case report; King JA et al.; BACKGROUND: While pelvic infection is known to be an infrequent complication of intrauterine device (IUD) use, infections are usually related to microorganisms introduced at the time of insertion or by sexual contact . CASE: We diagnosed a 35-year-old woman with an IUD for 6 years with pelvic inflammatory disease (PID) and implemented antibiotic therapy . Her clinical course worsened, and exploratory surgery revealed a right tuboovarian abscess with multiple loculated pelvic abscesses . Culture of the IUD found heavy growth of Pseudomonas aeruginosa . CONCLUSION: P aeruginosa has not previously been described in association with infections of the upper female genital tract . Double coverage with appropriate antipseudomonal agents is essential for proper treatment of pseudomonal infections.

J Biomed Mater Res, 2003 Feb 15, 64B(2), 94 - 8
Antibiotic bone cement for the treatment of pseudomonas aeruginosa in joint arthroplasty: Comparison of tobramycin and gentamicin-loaded cements; Scott CP et al.; One hundred clinical isolates of Pseudomonas aeruginosa were collected from 22 medical centers throughout Europe and were challenged with two aminoglycoside-loaded bone cements, employing a modified in vitro Kirby-Bauer susceptibility model . The results of this study show that Simplex(R) P with tobramycin exhibits antibacterial activity against 98% of the strains tested, compared to 93% for Palacos with gentamicin . Additionally, for strains that were susceptible to the antibiotic bone cement formulations, the average zone of inhibition produced around the tobramycin-loaded cement disks was approximately 25% greater than that seen around the gentamicin-loaded cement disks . This difference was statistically significant (p << 0.01) . Tobramycin-loaded bone cement is therefore the preferred formulation when addressing Pseudomonas aeruginosa in septic joint arthroplasty .

Zhonghua Shao Shang Za Zhi, 2002 Oct, 18(5), 285 - 7
{The isolation of Pseudomonas aeruginosa from burn wound and the analysis of its antibiotic resistant spectrum}; Zhang R et al.; OBJECTIVE: To investigate the change in the antibiotic resistant spectrum of Pseudomonas aeruginosa (PA) isolated from burn wounds and the production of inducible beta-lactamase . METHODS: VITEK-AMS system (total automatic bacterial identification and drug sensitivity system) and E-test concentration gradient were employed to perform bacterial identification and antibiotic sensitivity tests . K-B method was applied to detect inducible enzyme . RESULTS: The resistance of PA to Cephalosporin and Imipenem was increased in the past 4 years from June of 1996 to June of 2000 . Whereas the resistance to Cefoperazone/Sulbactam was least . There was an obvious difference of the resistance of PA to antibiotics during the 4 years (P < 0.05) . The resistant rate to Imipenem ranged from 20% to 40% . PA was able to produce inducible enzymes among 120 strains of wild type of PA occupying 72.5% with Imipenam as the inducing agent . CONCLUSION: The analysis of the antibiotic resistance of PA and the detection of inducible enzymes could be monitored from time to time and helpful in the correction of the use of antibiotics . Constant monitoring of antibiotic resistance might be beneficial to the prevention of outbreak of epidemics of PA infection in a burn unit.

J Clin Laser Med Surg, 2002 Dec, 20(6), 325 - 33
Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro; Nussbaum EL et al.; OBJECTIVE: To examine the effects of low-intensity laser therapy (LILT) on bacterial growth in vitro . BACKGROUND DATA: LILT is undergoing investigation as a treatment for accelerating healing of open wounds . The potential of coincident effects on wound bacteria has received little attention . Increased bacterial proliferation could further delay recovery; conversely inhibition could be beneficial . MATERIALS AND METHODS: Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were plated on agar and then irradiated with wavelengths of 630, 660, 810, and 905 nm (0.015 W/cm(2)) and radiant exposures of 1-50 J/cm(2) . In addition, E . coli was irradiated with 810 nm at an irradiance of 0.03 W/cm(2) (1-50 J/cm(2)) . Cells were counted after 20 h of incubation post LILT . Repeated measures ANOVA and Tukey adjusted post hoc tests were used for analysis . RESULTS: There were interactions between wavelength and species (p = 0.0001) and between wavelength and radiant exposure (p = 0.007) in the overall effects on bacterial growth; therefore, individual wavelengths were analyzed . Over all types of bacteria, there were overall growth effects using 810- and 630-nm lasers, with species differences at 630 nm . Effects occurred at low radiant exposures (1-20 J/cm(2)) . Overall effects were marginal using 660 nm and negative at 905 nm . Inhibition of P . aeruginosa followed irradiation using 810 nm at 5 J/cm(2) (-23%; p = 0.02) . Irradiation using 630 nm at 1 J/cm(2) inhibited P . aeruginosa and E . coli (-27%) . Irradiation using 810 nm (0.015 W/cm(2)) increased E . coli growth, but with increased irradiance (0.03 W/cm(2)) the growth was significant (p = 0.04), reaching 30% at 20 J/cm(2) (p = 0.01) . S . aureus growth increased 27% following 905-nm irradiation at 50 J/cm(2) . CONCLUSION: LILT applied to wounds, delivering commonly used wavelengths and radiant exposures in the range of 1-20 J/cm(2), could produce changes in bacterial growth of considerable importance for wound healing . A wavelength of 630 nm appeared to be most commonly associated with bacterial inhibition . The findings of this study might be useful as a basis for selecting LILT for infected wounds.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1444 - 9 Epub 2003 Jan 02.
Extensive and specific responses of a eukaryote to bacterial quorum-sensing signals; Mathesius U et al.; Many bacteria use N-acyl homoserine lactone (AHL) signals to coordinate the behavior of individual cells in a local population . The successful infection of eukaryotic hosts by bacteria seems to depend particularly on such AHL-mediated "quorum-sensing" regulation . We have used proteome analysis to show that a eukaryotic host, the model legume Medicago truncatula, is able to detect nanomolar to micromolar concentrations of bacterial AHLs from both symbiotic (Sinorhizobium meliloti) and pathogenic (Pseudomonas aeruginosa) bacteria, and that it responds in a global manner by significant changes in the accumulation of over 150 proteins, 99 of which have been identified by peptide mass fingerprinting . The accumulation of specific proteins and isoforms depended on AHL structure, concentration, and time of exposure . AHLs were also found to induce tissue-specific activation of beta-glucuronidase (GUS) reporter fusions to an auxin-responsive and three chalcone synthase promoters, consistent with AHL-induced changes in the accumulation of auxin-responsive and flavonoid synthesis proteins . In addition, exposure to AHLs was found to induce changes in the secretion of compounds by the plants that mimic quorum-sensing signals and thus have the potential to disrupt quorum sensing in associated bacteria . Our results indicate that eukaryotes have an extensive range of functional responses to AHLs that may play important roles in the beneficial or pathogenic outcomes of eukaryote-prokaryote interactions.

Arch Pharm Res, 2002 Dec, 25(6), 860 - 4
Antimicrobial activity and chemical composition of some essential oils; Aridogan BC et al.; In this study the composition and antimicrobial properties of essential oils obtained from Origanum onites, Mentha piperita, Juniperus exalsa, Chrysanthemum indicum, Lavandula hybrida, Rosa damascena, Echinophora tenuifolia, Foeniculum vulgare were examined . To evaluate the in vitro antibacterial activities of these eight aromatic extracts; their in vitro antimicrobial activities were determined by disk diffusion testing, according to the NCCLS criteria . Escherichia coli (ATTC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATTC 27853 were used as standard test bacterial strains . Origanum onites recorded antimicrobial activity against all test bacteria, and was strongest against Staphylococcus aureus . For Rosa damascena, Mentha piperita and Lavandula hybrida antimicrobial activity was recorded only to Staphylococcus aureus . Juniperus exalsa, and Chrysanthemum indicum exhibited antibacterial activities against both Staphylococcus aureus and Escherichia coli . We also examined the in vitro antimicrobial activities of some components of the essential oils and found some components with antimicrobial activity.

J Infect Dis, 2003 Jan 1, 187(1), 70 - 6 Epub 2002 Dec 13.
Orally administered recombinant human interleukin-11 is protective in experimental neutropenic sepsis; Opal SM et al.; Recombinant human interleukin (IL)-11 is a multifunctional cytokine with hematopoietic, immunomodulatory, and epithelial cell protective activities . IL-11alpha receptors are expressed on the luminal surface of intestinal epithelial cells . It was hypothesized that orally administered IL-11 would prevent mucosal damage and protect against microbial invasion in a neutropenic rat model of gram-negative sepsis . IL-11 was administered daily by enteric, coated multiparticle pellets over the course of chemotherapy-induced neutropenia . Compared with the placebo group, IL-11-treated rats retained mucosal mass and had prolonged survival time, reduced pathologic changes, and reduced systemic levels of bacterial endotoxin and concentrations of Pseudomonas aeruginosa in target tissues . Enterocyte messenger RNA levels for tumor necrosis factor-alpha and interferon-gamma revealed that oral IL-11 reduced but did not prevent increased expression of these cytokine genes . These results indicate that orally administered IL-11 may preserve epithelial cell integrity in the presence of cytoreductive chemotherapy . This may represent a new treatment strategy for the prevention of infection in neutropenic hosts.

Int J Antimicrob Agents, 2003 Jan, 21(1), 8 - 12
In vitro activity of beta-lactams in combination with other antimicrobial agents against resistant strains of Pseudomonas aeruginosa; Song W et al.; Using the chequerboard titration method, the activity in combination of beta-lactams, fluoroquinolones and aminoglycosides was investigated against 24 Pseudomonas aeruginosa isolates resistant to these antibiotics . Synergy was detected with one or more antimicrobial combinations against 15 of 24 (63%) isolates and partial synergy was detected with one or more combinations against all 24 isolates . No antagonism was seen with any combination . Ceftazidime and cefepime with aztreonam, amikacin and isepamicin showed synergy or partial synergy against 12-20 (50-80%) isolates . Imipenem and meropenem with amikacin and isepamicin showed synergy or partial synergy against eight to 12 (33-50%) isolates . The results of this study indicate that against P . aeruginosa, synergy may occur between beta-lactams, fluoroquinolones and aminoglycosides although the strains are resistant to the individual antibiotics.

Scanning, 2002 Nov-Dec, 24(6), 274 - 83
Morphologic variations in bacteria under stress conditions: near-field optical studies; Cefali E et al.; The morphologic and structural variations suffered by cells of a population of Pseudomonas aeruginosa ATCC 27853 under stress conditions were investigated by using scanning near-field optical microscopy . The analysis of the images, supported by microbiological data, showed that the bacteria evolved from the initial distribution of rod-shaped cells of standard size to a population with structural and morphologic modifications . The detection of variations in the optical reflectivity over a subwavelength scale (< or = 100 nm), combined with the concurrently acquired topographical signal, allowed the visualisation of rod-shaped bacteria going towards a lytic process and entire "U"-shaped cells . In the latter cells, which derived from a morphology refolding of rod bacteria, cellular matter seemed to rearrange itself to attain a coccoid stress resistant form, responsible for the residual viability of the population.

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2347 - 55
Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa, with its application to cleaning lipid-stained contact lenses; Sugihara A et al.; With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity . A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme . The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa . The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5 . The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0 . The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate . Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids . When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.

Immunol Lett, 2003 Jan 2, 85(1), 29 - 33
Components with potential immunosuppressive activity in lipopolysaccharide of laboratory Pseudomonas aeruginosa strains; Vikstrom EV; The ability of culture filtrate (CF) of two laboratory Pseudomonas aeruginosa strains to inhibit delayed type hypersensitivity (DTH) to non-bacterial antigen in CBA mice has been studied . It was shown that intraperitoneal injection of native CF of the strains did not affect the level of DTH . However, redox treated CF expressed the immunosuppressive activity . Gel filtration of redox activated CF through Sephadex G-200 showed that CF contains three immunosuppressive components differing by their molecular weight and specificity . All components contained lipid group and O-polysaccharide chains that indicated their lipopolysaccharide (LPS) nature . These experiments show that laboratory P . aeruginosa strains have three LPS components but not all of them display the immunosuppressive activity.

J Nat Toxins, 2002 Dec, 11(4), 345 - 52
L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification, characterization, platelet aggregation-inducing and antibacterial effects; Lu QM et al.; An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography . The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits . The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme . The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs . Homology with elapid venom LAO is lower . TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium . The antibacterial effect associated with LAO activity was elminated with the addition of catalase . Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO . The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation . These results showed H2O2 formation is important for the biological effects of LAO.

Eur Respir J, 2002 Dec, 20(6), 1483 - 9
Nosocomial tracheobronchitis in mechanically ventilated patients: incidence, aetiology and outcome; Nseir S et al.; The aim of this study was to determine the incidence, the organisms responsible for and the impact on outcome of nosocomial tracheobronchitis (NTB) in the intensive care unit (ICU) . This prospective observational cohort study was conducted in a 30-bed medical/surgical ICU over a period of 6.5 yrs . All patients ventilated for >48 h were eligible . Patients with nosocomial pneumonia (NP) without prior NTB were excluded . Patients with first episodes of NTB were compared with those without NTB by univariate analysis . The study diagnosed 201 (10.6%) cases of NTB . Pseudomonas aeruginosa was the most common bacteria . NP rates were similar in patients with NTB compared with patients without NTB . Even in the absence of subsequent NP, NTB was associated with a significantly higher length of ICU stay and duration of mechanical ventilation in both surgical and medical populations . Mortality rates were similar in NTB patients without subsequent NP compared with patients without NTB . Antimicrobial treatment in NTB patients was associated with a trend to a better outcome . Nosocomial tracheobronchitis is common in mechanically ventilated intensive care unit patients . In this population, nosocomial tracheobronchitis was associated with longer durations of intensive care unit stay and mechanical ventilation . Further studies are needed to determine the impact of antibiotics on outcomes of patients with nosocomial tracheobronchitis.

Eur Respir J, 2002 Dec, 20(6), 1457 - 63
Sequential genotyping of Pseudomonas aeruginosa from upper and lower airways of cystic fibrosis patients; Jung A et al.; A controversy exists concerning the adequate specimen to characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa . Oropharyngeal, sputum and bronchoalveolar lavage samples were evaluated from 38 stable CF patients for the detection of P . aeruginosa, genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations . Bacterial isolates were typed by pulsed-field gel electrophoresis of deoxyribonucleic acid macrorestriction fragments . Sensitivity, negative and positive predictive values and specificity to detect P . aeruginosa were 35.7, 73.5, 83.3 and 96.2% for oropharyngeal cultures in nonexpectorating patients and 91.7, 94.1, 100 and 100% for sputum cultures from expectorating patients, respectively . Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively . In 62% longitudinal variations in the genotype occurred . One-half of these alterations were detectable by bronchoscopy only . In conclusion, sputum samples were of equal value as specimens from bronchoalveolar lavage to detect Pseudomonas aeruginosa colonisation . Cultures from the oropharynx are not suitable for characterising bacterial conditions in the cystic fibrosis lung . Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the bronchoalveolar lavage fluid exclusively.

Eur Respir J, 2002 Dec, 20(6), 1449 - 56
Vascular endothelial growth factor mRNA and protein expression in airway epithelial cell lines in vitro; Koyama S et al.; Vascular endothelial growth factor (VEGF) plays multifunctional roles in vascular permeability, repair and remodelling processes, in addition to the maintenance of vascular structure and function . In the present study, the potential of airway epithelial cell lines, BEAS-2B cells and A549 cells, to release and express VEGF in unstimulated and stimulated conditions was evaluated . The secretion and expression of VEGF were evaluated by enzyme-linked immunosorbant assay and by reverse transcriptase-polymerase chain reaction . The isoforms of released VEGF were determined by high-performance liquid chromatography . BEAS-2B cells and A549 cells released VEGF constitutively . Interleukin (IL)-1beta and tumour necrosis factor (TNF)-alpha augmented the release of VEGF in a time- and dose-dependent manner . The released VEGF was 165 amino acid residues in either condition . Pseudomonas aeruginosa lipopolysaccharide (LPS), interferon (IFN)-gamma, smoke extract (SE), neutrophil elastase (NE), and bradykinin stimulated the release of VEGF . Keracinocyte growth factor (KGF), which reduces vascular permeability, also stimulated both cells to release VEGF . VEGF messenger ribonucleic acid (mRNA) was expressed both time- and dose-dependently at 2 h, and declined after 2 h in response to IL-1beta and TNF-alpha . The expression of VEGF mRNA in airway epithelial cells was also augmented by LPS, IFN-gamma, SE, NE, and KGF stimulation . These data suggest that airway epithelial cells may regulate the maintenance of vascular structure and function, as well as vascular permeability, repair and remodelling processes, in a variety of lung conditions by expressing vascular endothelial growth factor.

J Med Chem, 2003 Jan 2, 46(1), 97 - 104
Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators; Chhabra SR et al.; Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described . Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described . Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile . From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity . These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents . However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity . The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

J Med Assoc Thai, 2002 Oct, 85(10), 1095 - 9
Risk factors for Pseudomonas aeruginosa bacteremia in Thai patients; Siripassorn K et al.; A case control study to determine the risk factors for P . aeruginosa bacteremia was conducted in patients admitted to Siriraj Hospital in 1998 . The case group consisted of 65 patients with P . aeruginosa bacteremia . There were 3 control groups . 65 patients with E . coli bacteremia, 64 patients with S . aureus bacteremia and 65 patients without bacteremia . Demographic information and potential risk factors i.e . type of infection, associated diseases/conditions, procedures/surgery, previous/current use of antibiotics and previous/current use of immunosuppressive/cytotoxic agents were extracted from the patients' medical records and compared . Univariate analysis revealed that the factors associated with P . aeruginosa bacteremia were infections acquired while hospitalized, hematologic malignancy, neutropenia, COPD, antibiotic receivers, cytotoxic agents receivers . However, multivariate analysis revealed that only hematologic malignancy, infections acquired while hospitalized and previous use of parenteral antibiotics were risk factors for P . aeruginosa bacteremia.

Genetika, 2002 Nov, 38(11), 1470 - 9
{Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa}; Burkal'tseva MV et al.; A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny) . We have recently sequenced the phage phi KZ genome (288,334 bp) {J . Mol . Biol., 2002, vol . 317, pp . 1-19} . By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL) . Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU) . Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus . The origin and evolution of the phi KZ-like phages are discussed . Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes . Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.

Epidemiology, 2003 Jan, 14(1), 55 - 9
Mortality of cystic fibrosis patients treated with tobramycin solution for inhalation; Rothman KJ et al.; BACKGROUND: Tobramycin solution for inhalation (TOBI; TSI) is indicated to treat patients with cystic fibrosis who are infected with Pseudomonas aeruginosa . Preliminary findings from a randomized trial indicate that patients who received TSI had about half the mortality rate of those assigned to placebo . METHODS: We used the Cystic Fibrosis Foundation registry data to conduct a retrospective cohort study of the risk of death among cystic fibrosis patients in 1999 according to their use of TSI during 1998 . We controlled for age, lung function, height and infection with; other factors were not important confounders . RESULTS: The crude risk of death among those who received TSI therapy for 4 or more months was 3.5 times greater than that among those who received no TSI (90% confidence interval = 3.0-4.2) . In general, increased TSI use was related to progressive increases in the risk of death . Such a relation is expected because TSI is used for those who are close to death, resulting in strong confounding by indication . After control of the previously mentioned confounders, the estimated risk ratio was reduced from 3.5 to 1.2 . Unfortunately, it is difficult to remove confounding by indication in its entirety . Using a method that estimates the magnitude of uncontrolled confounding, we show that the actual relation between TSI and the risk of death is likely to be protective, and may well be consistent with the results from the randomized trial . CONCLUSIONS: These data illustrate strong confounding by indication and the extent to which the interpretation of data can rest on assumptions about the data and its residual biases.

Antimicrob Agents Chemother, 2003 Jan, 47(1), 95 - 101
Regulation of membrane permeability by a two-component regulatory system in Pseudomonas aeruginosa; Wang Y et al.; Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa . By using laboratory strain PAK, a spontaneous P . aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated . By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics . Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes . Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested . This is the first demonstration that P . aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P . aeruginosa.

J Chromatogr A, 2002 Dec 6, 979(1-2), 277 - 84
Capillary electrophoresis study of outer membrane proteins of Pseudomonas strains upon antibiotic treatment; Kustos T et al.; Nosocomial wound infections by Pseudomonas aeruginosa strains have increasing importance in orthopaedic surgery . Outer membrane protein composition and cell-surface hydrophobicity of the bacteria have strong influence on adhesion to living tissues or artificial medical devices . Outer membrane proteins of five Pseudomonas strains (KT 2, KT 7, KT 25, KT 28, KT 39) isolated from orthopaedic patients' wounds and one standard strain NIH Hungary 170000 isolated from pus were examined . The capillary electrophoretic patterns of the outer membrane proteins were characteristic to each bacterial strains possessing different relative ratios of major and minor proteins . Antibiotic treatment of bacteria with three antibiotics, cefotaximum, amoxicillinum/clavulinic acid and amikacinum (applied often in prophylaxis and treatment of patients) induced changes in the electrophoretic profiles showing that outer membrane protein composition was altered significantly . The most pronounced alterations in the electrophoretic patterns after antibiotic treatment were obtained in the cases of the strains KT 2, KT 7 and KT 28 . The amikacinum administration strongly decreased the relative percentage of the 38800 rel . mol . mass protein in KT 2 (from 20 to 6%) . while the relative amount of the same protein increased significantly in KT 7 and KT 28 after cefotaximum treatment (from 2 to 16% and from 12 to 28%, respectively) . Decrease in cell-surface hydrophobicity was also observed by salt aggregation test . The results obtained can be used to determine the therapeutic concentrations of antibiotics to induce changes in the adhesion properties of bacteria.

Infect Immun, 2003 Jan, 71(1), 365 - 73
Selective early production of CCL20, or macrophage inflammatory protein 3alpha, by human mast cells in response to Pseudomonas aeruginosa; Lin TJ et al.; Mast cells are important as sentinel cells in host defense against bacterial infection . Much of their effectiveness depends upon recruiting other immune cells; however, little is known about the mechanisms of this response . CCL20, also known as macrophage inflammatory protein-3alpha (MIP-3alpha), Exodus, and LARC, is a chemokine known to be a potent chemoattractant for immature dendritic cells and T cells . In this study, we examined the human mast cell production of both CCL20 and granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine for innate immune responses in the lung, in response to Pseudomonas aeruginosa . Reverse transcription-PCR and Western blot analysis demonstrated that the human mast cells (HMC-1) express CCL20 mRNA and are able to produce a significant amount (32.4 ng/ml) of CCL20 protein following stimulation by calcium ionophore and phorbol myristate acetate . Importantly, P . aeruginosa potently stimulated CCL20 production in human cord blood-derived mast cells (CBMC), with production peaking at 6 h after stimulation . This time course of expression was distinct from that of GM-CSF, which peaked after 24 to 48 h . Significant CCL20 production did not occur following immunoglobulin E-mediated activation of CBMC under conditions which induced a substantial GM-CSF response . Interestingly, the CCL20 response of mast cells to P . aeruginosa was relatively resistant to inhibition by the corticosteroid dexamethasone, interleukin-10, or cyclosporine, while GM-CSF production was potently inhibited . However, P . aeruginosa-induced CCL20 production was blocked by the protein kinase C (PKC) inhibitor Ro 31-8220 and a PKC pseudosubstrate . These results support a role for human mast cells in the initiation of immune responses to P . aeruginosa infection.

J Hosp Infect, 2003 Jan, 53(1), 18 - 24
Reappraisal of attributable mortality in critically ill patients with nosocomial bacteraemia involving Pseudomonas aeruginosa; Blot S et al.; In a retrospective study, population characteristics and outcome were investigated in intensive care unit (ICU) patients with hospital-acquired Pseudomonas aeruginosa bacteraemia admitted over a seven-year period (January 1992 through December 1998) . A matched cohort study was performed in which all ICU patients with P . aeruginosa bacteraemia were defined as cases (N=53) . Matching (1:2 ratio) of the controls (N=106) was based on the APACHE II classification: an equal APACHE II score (+/-1 point) and an equal diagnostic category . Patients with P . aeruginosa bacteraemia had a higher incidence of acute respiratory failure, haemodynamic instability, a longer ICU stay and length of ventilator dependence (P<0.05) . In-hospital mortalities for cases and controls were 62.3 vs . 47.2% respectively (P=0.073) . Thus, the attributable mortality was 15.1% (95% confidence intervals: -1.0-31.2) . In a multivariate survival analysis the APACHE II score was the only variable independently associated with mortality . In conclusion, P . aeruginosa bacteraemia is associated with a clinically relevant attributable mortality (15%) . However, we could not find statistical evidence of P . aeruginosa being an independent predictor of mortality.

Avian Dis, 2002 Oct-Dec, 46(4), 1045 - 50
Characterization of Pseudomonas aeruginosa isolates associated with mortality in broiler chicks; Walker SE et al.; In mid-2000, a broiler chicken company in Alabama experienced high early mortality rates in chicks from two different hatcheries . Five isolates of Pseudomonas aeruginosa, obtained from these contaminated hatcheries and resulting broiler chicks with omphalitis, were selected to determine virulence of the bacteria . One-day-old specific-pathogen-free white leghorn chicks were placed into positive pressure isolation units (10 chicks per unit); feed and water were provided ad libitum . The five isolates of P . aeruginosa (1 x 10(1) or 1 x 10(1) colony-forming units/bird) were used to challenge two replicates of 10 chicks via yolk sac inoculation . Two control groups were injected with 0.1 ml of phosphate-buffered saline, and two groups received no treatment . Mortality was recorded daily, and the chicks that died were necropsied and liver and yolk sacs were cultured . After 14 days, the remaining chickens were euthanatized and necropsied . Bacterial isolates retrieved from liver and yolk sacs were identified by the API 20 NE typing system to confirm that they were the same as the challenge isolate . Virulence varied greatly among the isolates, resulting in mortality rates from 0 to 90% . The challenge isolates produced different and often distinctive postmortem lesion patterns . Antibiotic sensitivity tests showed that all five isolates were resistant to sulfisoxazole, ceftiofur, penicillin, lincomycin, bacitracin, oxytetracycline, erythromycin, naladixic acid, and tetracycline . The isolates varied in sensitivity to other antibiotics, but all isolates were sensitive to gentamicin.

Avian Dis, 2002 Oct-Dec, 46(4), 826 - 30
The in vitro efficacy of a quaternary ammonia disinfectant and/or ethylenediaminetetraacetic acid-tris against commercial broiler hatchery isolates of Pseudomonas aeruginosa; Walker SE et al.; Studies have indicated variations in the degree of efficacy of certain commercial disinfectants used in poultry production facilities . We used an adequate method of in vitro testing to compare the efficacy of disinfectants while testing them in conditions similar to those of the poultry facilities . BioSentry 904, ethylenediaminetetracetic acid (EDTA)-Tris, and a combination of the two were tested by this method against five field isolates of Pseudomonas aeruginosa at 10(3), 10(6), and 10(9) colony-forming units (CFU)/ml . At the 10(9) CFU/ml concentration, most compounds failed to achieve a total kill with a contact time of 15 min . When tested at bacterial concentrations of 10(3) CFU/ml, the combination of EDTA-Tris mixed at a 1:1 ratio with BioSentry 904 killed the bacteria upon initial contact (< or = 0.05 min) . This disinfectant mixture exhibited antagonistic, indifferent, or synergetic effects when exposed to different bacterial isolates at a concentration of 10(6) CFU/ml.

Protein Sci, 2003 Jan, 12(1), 56 - 65
NMR detection of multiple transitions to low-populated states in azurin; Korzhnev DM et al.; Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone (15)N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8 degrees C) . Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes . The group of (15)N nuclei involved in the faster process exhibits at 44.8 degrees C a forward rate constant of 11.7+/-2.4 s(-1) and a population of the exited state of 0.39+/-0.07% . They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane . For the slower process, the forward rate constant and population of the exited state at 44.8 degrees C are 4.1+/-0.1 s(-1) and 0.45+/-0.02%, respectively . The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 A, indicating conformational rearrangements involving the copper coordinating loops . The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35 . The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer.

Mol Microbiol, 2003 Jan, 47(1), 195 - 207
Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis; Beare PA et al.; Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine . Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA . Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors . Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR . The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS . This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease . Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein . FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced . To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).

Mol Microbiol, 2003 Jan, 47(1), 145 - 58
Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs; Kresse AU et al.; Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence . More than half of the genotypes of P . aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs) . To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined . Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism . A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability . Speciation in bacteria is accompanied by LCIs . Therefore adaptation by LCIs that allows persistence of P . aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.

Plant J, 2002 Dec, 32(6), 879 - 89
Sulfate reduction is increased in transgenic Arabidopsis thaliana expressing 5'-adenylylsulfate reductase from Pseudomonas aeruginosa; Tsakraklides G et al.; The two-electron reduction of sulfate to sulfite in plants is mediated by 5'-adenylylsulfate (APS) reductase, an enzyme theorized to be a control point for cysteine synthesis . The hypothesis was tested by expression in Arabidopsis thaliana under transcriptional control of the CaMV 35S promoter of the APS reductase from Pseudomonas aeruginosa (PaAPR) fused with the rbcS transit peptide for localization of the protein to plastids . PaAPR was chosen for the experiment because it is a highly stable enzyme compared with the endogenous APS reductase of A . thaliana, and because PaAPR is catalytically active in combination with the plant thioredoxins m and f indicating that it would likely be catalytically active in plastids . The results indicate that sulfate reduction and O-acetylserine (OAS) production together limit cysteine synthesis . Transgenic A . thaliana lines expressing PaAPR accumulated sulfite, thiosulfate, cysteine, gamma-glutamylcysteine, and glutathione . Sulfite and thiosulfate increased more than did cysteine, gamma-glutamylcysteine and glutathione . Thiosulfate accumulation was most pronounced in flowers . Feeding of OAS to the PaAPR-expressing plants caused cysteine and glutathione to increase more rapidly than in comparably treated wild type . Both wild-type and transgenic plants accumulated sulfite and thiosulfate in response to OAS feeding . The PaAPR-expressing plants were slightly chlorotic and stunted compared with wild type . An attempt to uncover the source of thiosulfate, which is not thought to be an intermediate of sulfate reduction, revealed that purified beta-mercaptopyruvate sulfurtransferase is able to form thiosulfate from sulfite and beta-mercaptopyruvate, suggesting that this class of enzymes could form thiosulfate in vivo in the presence of excess sulfite.

Eur J Clin Invest, 2003 Jan, 33(1), 82 - 7
Differential expression of alpha- and beta-defensins in human peripheral blood; Fang XM et al.; BACKGROUND: Human defensin peptides with broad-spectrum antimicrobial activity have been implicated in the human defence response towards microbial invasion . Two families of defensins designated alpha- and beta-defensins, respectively, have been identified . Little is known about the expression of both defensin families in human peripheral blood . The purpose of this study was to examine the expression of alpha- and beta-defensin genes in human peripheral blood . MATERIAL AND METHODS: Fifty-one healthy blood donors were screened for defensin expression . Blood from defensin responders was stimulated by lipopolysaccharide or heat-inactivated Pseudomonas aeruginosa ex vivo . Levels of mRNA were assessed by semiquantitative RT-PCR . Southern blot analysis and sequencing were used to confirm the identity of defensin gene transcripts . Western blotting analysis was used to detect the expression of defensin peptides . RESULTS: beta-defensin was undetected in human peripheral blood without stimulation . Following stimulation by lipopolysaccharide or heat-inactivated bacterial cells, the majority (88.2%) of healthy individuals had a detectable expression for beta-defensin-1 gene and 39.2% for beta-defensin-2 gene, compared with none for beta-defensin-3 . beta-defensin-1 and -2 mRNAs in the stimulated human peripheral blood of responders became detectable at 3 h and showed a maximum at 6 h following induction by 100 ng mL-1 of lipopolysaccharide or bacterial cells . In contrast, human alpha-defensins 1-3 mRNA are constitutively expressed in peripheral leukocytes but not up-regulated by lipopolysaccharide or bacterial cells . CONCLUSIONS: In human peripheral blood, beta-defensin-1 and -2 genes were transiently transcribed and translated following the induction of lipopolysaccharide or heat-inactivated bacterial cells, whereas alpha-defensins 1-3 genes were constitutively transcribed, and beta-defensin-3 gene was not expressed . The inducible expression of beta-defensin-1 and -2 genes showed interindividual variability.

Proteins, 2003 Feb 1, 50(2), 222 - 9
Dynamic effects of mutations within two loops of cytochrome c551 from Pseudomonas aeruginosa; Ceruso MA et al.; In this work, we investigated the structural and dynamic consequences of two substitutions, P58A and G36P, located in two different solvent-exposed loops of cytochrome c551 . The results show that both mutations affect regions that are distant from the site of mutation . Here, the two loops appear to be dynamically coupled to each other, because the substitution at one site affects the structure and the dynamics of the other site . However, the substitutions at Gly-36 and Pro-58 presented substantial differences, which were related to the mechanical (rigidity and deformability) properties of the site surrounding the mutation . Although the P58A mutant conserved a significant dynamic similarity to the wild-type protein as the immediate surroundings of position 58 became more rigid, the G36P mutant, which had deformed its flexible surroundings, presented a dynamic behavior that was markedly different from that of the wild-type protein . These results suggest that perturbation of sterically isolated and flexible regions, such as solvent-exposed loops, can have strong dynamic consequences on the protein as a whole, raising the possibility that these effects could in turn affect the stability or the function of the protein .

J Bacteriol, 2003 Jan, 185(1), 377 - 80
The Pseudomonas aeruginosa rhlAB operon is not expressed during the logarithmic phase of growth even in the presence of its activator RhlR and the autoinducer N-butyryl-homoserine lactone; Medina G et al.; The Pseudomonas aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid; rhlAB induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer N-butyryl-homoserine lactone (C(4)-HSL) . In this work we studied rhlAB induction in a P . aeruginosa and Escherichia coli background . We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C(4)-HSL . Additionally, we found that rhlAB expression is partially sigma(s) dependent.

EMBO J, 2002 Dec 16, 21(24), 6649 - 59
Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr; Lesniak J et al.; Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species . These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes . Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins . Ohr is a tightly folded homodimer, with a novel alpha/beta fold, and contains two active sites located at the monomer interface on opposite sides of the molecule . Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites . Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity . We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction.

J Gen Appl Microbiol, 2001 Feb, 47(1), 27 - 32
Roles of MexXY- and MexAB-multidrug efflux pumps in intrinsic multidrug resistance of Pseudomonas aeruginosa PAO1; Morita Y et al.; To envisage the roles of MexXY- and MexAB-multidrug efflux pumps in the intrinsic multidrug resistance of wild-type strain Pseudomonas aeruginosa PAO1, we constructed mutants lacking either individual or both efflux pumps . A mutant lacking MexXY showed increased susceptibility to aminoglycosides, erythromycin, and tetracycline, but not to beta-lactams, chloramphenicol, or quinolones . A mutant lacking MexAB showed increased susceptibility to beta-lactams, chloramphenicol, and nalidixic acid, but not to aminoglycosides, erythromycin, tetracycline, or fluoroquinolones . A mutant lacking both MexXY and MexAB showed an increased susceptibility to all antimicrobial agents tested compared with the wild type . Very similar results were obtained with a mutant lacking MexAB-OprM and a mutant lacking both MexXY and MexAB-OprM . Thus it is clear that OprM is essential not only for the function of MexAB, but also for the function of MexXY . Furthermore, we found that each pump compensated to some extent for the lack of another pump with respect to the common substrates (tetracycline, quinolones, and cefpirome) . The introduction of a plasmid carrying the mexXY genes into P . aeruginosa PAO1 cells increased the resistance to fluoroquinolones . This suggests that the mexXY genes could be involved in acquired resistance to fluoroquinolones in P . aeruginosa PAO1.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16684 - 8 Epub 2002 Dec 13.
Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP; Ishige K et al.; An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5 . Poly P chains of 30-50 residues are optimal; those of 15-700 residues can also serve . GDP is preferred over ADP among nucleoside diphosphate acceptors . This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report . The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features . Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis . PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P . aeruginosa.

Symp Ser Soc Appl Microbiol, 2002, (31), 55S - 64S
Mechanisms of bacterial biocide and antibiotic resistance; Poole K; Resistance to antibiotics is increasingly commonplace amongst important human pathogens . Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux . While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems . Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes . Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms . Still, target site mutations leading to biocide resistance, though rare, are known . Intriguingly, many multidrug efflux systems also accommodate biocides (e.g . triclosan) such that strains expressing these are both antibiotic- and biocide-resistant . Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics . Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan . These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two . Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems . Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.

Microbiology, 2002 Dec, 148(Pt 12), 3849 - 56
Beta-ketoacyl acyl carrier protein reductase (FabG) activity of the fatty acid biosynthetic pathway is a determining factor of 3-oxo-homoserine lactone acyl chain lengths; Hoang TT et al.; The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-{3-oxododecanoyl}-L-homoserine lactone (3-oxo-C(12)-HSL) are required for quorum sensing in Pseudomonas aeruginosa . These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool . This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity . Culture supernatants of P . aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown . An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P . aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase . In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C(12)-HSL . When the beta-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates . Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo . Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.

Microbiology, 2002 Dec, 148(Pt 12), 3839 - 47
Malate:quinone oxidoreductase is essential for growth on ethanol or acetate in Pseudomonas aeruginosa; Kretzschmar U et al.; Pseudomonas aeruginosa ATCC 17933 growing aerobically on ethanol uses a pyrroloquinoline quinone-dependent ethanol oxidation system . A mutant with an interrupted putative mqo gene, in which malate:quinone oxidoreductase (MQO), an enzyme involved in the citric acid cycle/glyoxylate cycle, was defective, showed a severe growth defect on ethanol and was unable to grow on acetate . Glucose, lactate, succinate or malate supported growth of the mutant . However, an NAD-dependent malate dehydrogenase activity could not be detected . Complementation of the mutant by the wild-type allele of the mqo gene restored wild-type behaviour . The wild-type expressed the dye-dependent MQO and NAD(P)-dependent malic enzymes (MEs) . Pyruvate carboxylase (PC) was found upon growth of the wild-type and the mutant on all substrates studied . PC activity in the wild-type was induced on glucose and lactate and was always higher on all substrates in the mqo mutant . In P . aeruginosa ATCC 17933, an active MQO is required for growth on ethanol or acetate, while with glucose, lactate, succinate or malate an apparent bypass route operates, with MEs using malate for generating pyruvate, which is carboxylated to oxaloacetate by PC . To the authors' knowledge, this is the first time that a specific mutant MQO phenotype has been observed, caused by the inactivation of a gene encoding MQO activity . mqo of P . aeruginosa ATCC 17933 corresponds to mqoB (PA4640) of the P . aeruginosa PAO1 genome project.

Am J Respir Crit Care Med, 2003 Mar 15, 167(6), 841 - 9 Epub 2002 Dec 12.
Significant microbiological effect of inhaled tobramycin in young children with cystic fibrosis; Gibson RL et al.; We conducted a double-blind, placebo-controlled, multicenter, randomized trial to test the hypothesis that 300 mg of tobramycin solution for inhalation administered twice daily for 28 days would be safe and result in a profound decrease in Pseudomonas aeruginosa (Pa) density from the lower airway of young children with cystic fibrosis . Ninety-eight subjects were to be randomized; however, the trial was stopped early because of evidence of a significant microbiological treatment effect . Twenty-one children under age 6 years were randomized (8 active; 13 placebo) and underwent bronchoalveolar lavage at baseline and on Day 28 . There was a significant difference between treatment groups in the reduction in Pa density; no Pa was detected on Day 28 in 8 of 8 active group patients compared with 1 of 13 placebo group patients . We observed no differences between treatment groups for clinical indices, markers of inflammation, or incidence of adverse events . No abnormalities in serum creatinine or audiometry and no episodes of significant bronchospasm were observed in association with active treatment . We conclude that 28 days of tobramycin solution for inhalation of 300 mg twice daily is safe and effective for significant reduction of lower airway Pa density in young children with cystic fibrosis.

Infection, 2002 Dec, 30(6), 387 - 91
Home and hospital antibiotic treatment prove similarly effective in cystic fibrosis; Riethmueller J et al.; BACKGROUND: Home intravenous antibiotic treatment has added to therapeutic options against Pseudomonas aeruginosa in cystic fibrosis (CF) patients leading to increased flexibility . A prospective clinical study was carried out to compare home and hospital iv antibiotic treatment in CF patients with chronic P . aeruginosa infection . PATIENTS AND METHODS: Treatment courses were planned selectively, exacerbations were excluded . 28 consecutive hospital courses (group 1) were compared with 30 home care courses (group 2) . Chest physiotherapy and nutrition therapy were carried on in both groups . Antibiotic treatment in both groups consisted of tobramycin and ceftazidime, with equal dosage and application . Groups were compared using clinical, inflammatory and microbiological parameters . RESULTS: There was a significant difference (p </= 0.05) in peripheral leukocyte counts before and after therapy in both groups . The same was true for forced expiratory volume in 1 sec (FEV(1); p </= 0.05), weight for height (p </= 0.005) and for Pseudomonas counts (p </= 0.005) in sputa . There was no statistical difference between the two groups for any of the parameters tested . CONCLUSION: It is concluded that, when exacerbations are excluded, home iv therapy is an effective therapeutic option in CF . Long-term comparison is still needed to effectively evaluate the pros and cons of home and hospital antibiotic treatment in CF.

Inflamm Res, 2002 Oct, 51(10), 506 - 10
Pseudomonas aeruginosa elastase stimulates ERK signaling pathway and enhances IL-8 production by alveolar epithelial cells in culture; Azghani AO et al.; OBJECTIVE AND DESIGN: Bacterial products as well as the host airway inflammatory responses contribute to the pathogenesis of Pseudomonas infections . We sought to determine if Pseudomonas elastase (PE) induces mitogen-activated protein (MAP) kinase activity in association with interleukin-8 (IL-8) production by alveolar epithelial cells . METHODS: We utilized Western blot analysis to detect phosphorylation of signaling intermediates and ELISA was used to measure IL-8 production . RESULTS: We found that PE induces phosphorylation of the extracellular signal-regulated (ERK1/2) proteins of the MAPK pathway in A549 epithelial cells . Similar results were obtained using primary cultures of rabbit alveolar type II epithelial cells . PE also enhanced IL-8 production, which was abolished in the presence of the ERK activation inhibitor U0126 . CONCLUSIONS: We conclude that PE activates the ERK1/2 arm of the MAPK pathway and that activation of this pathway results in enhanced IL-8 production . The results demonstrate that PE may augment pulmonary inflammation via cellular signaling that regulates expression of IL-8.

Chest, 2002 Dec, 122(6), 2127 - 36
Effects of nebulized diethylenetetraamine-NONOate in a mouse model of acute Pseudomonas aeruginosa pneumonia; Dukelow AM et al.; OBJECTIVE: Endogenous and exogenous nitric oxide (NO) may have important antibacterial effects in patients with pneumonia . NO administration has been limited to the continuous inhalation of gas-phase NO (ie, inhaled NO {iNO}) . Intermittent nebulization of NONOates, novel NO donors, may permit the continuous intrapulmonary delivery of NO . Thus, we assessed the effects of nebulized diethylenetetraamine-NONOate (DETA-NO) in a model of acute Pseudomonas aeruginosa pneumonia . DESIGN: Randomized, controlled study . SUBJECTS: Male C57Bl/6 mice . INTERVENTIONS: Pneumonia was induced by intratracheal instillation of P aeruginosa (3 x 10(7) CFU in 50 microL) . Pneumonia and sham mice were randomized to receive no treatment, nebulized DETA-NO (12.5 or 125 micromol) at 4 h and 12 h, or continuous iNO for 24 h (10 or 40 ppm) until they were killed at 24 h . MAIN RESULTS: The nebulization of DETA-NO was associated with a marked increase in mean (+/- SEM) exhaled NO levels (after nebulization, 484 +/- 34 parts per billion {ppb}; baseline, 13.4 +/- 0.4 ppb; p < 0.01) and plasma levels of nitrites/nitrates (after nebulization, 73 +/- 28 microM; at baseline, 14 +/- 3 microM; p < 0.05) . Nebulized DETA-NO decreased the pulmonary bacterial load in mice with pneumonia by 65 +/- 19% (p < 0.05 vs untreated mice) but had no effect on pulmonary leukocyte infiltration . Although the growth of P aeruginosa colonies in vitro was impaired on exposure to DETA-NO, growth was similarly impaired by exposure to DETA nucleophile/backbone alone . CONCLUSIONS: The nebulization of DETA-NO provides a method for the prolonged intrapulmonary delivery of NO . The antibacterial effect of DETA-NO in vivo and in vitro is due, in large part, to the DETA nucleophile moiety and is independent of NO, suggesting a limited therapeutic role for exogenous NO in pneumonia.

Zhonghua Yi Xue Za Zhi, 2002 Sep 10, 82(17), 1199 - 202
{Influence of high-frequency electric surgical knife on healing of abdominal incision, experimental and clinical studies}; Ji G et al.; OBJECTIVE: To study the influence of high-frequency electric surgical knife on abdominal incision healing . METHODS: Two hundred and forty Wistar rats were randomly divided into two groups of 120 rats to undergo abdominal incision by high-frequency electric knife or common lancet respectively . Each of these two groups was redivided into four subgroups that were injected hypodermically with 0.2 ml of quantitative mixture of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at the concentrations of 0.24 x 10(2) bacteria (10(2) group), 4.49 x 10(4) bacteria (10(5) group), and 4.11 x 10(7) bacteria (10(8) group) respectively, and normal saline of the same volume (10(0) group) . Eight days after the operation, the rats were killed . The infection rate of the operational wound was observed and the tissues around the wounds were examined pathologically . On the basis of the animal experiment, 220 patients undergoing abdominal operation above type II were randomly allocated into one of following three groups: high-frequency electric knife (EK) group (93 cases, high-frequency electric knife was used to cut the abdominal tissues and electro-coagulation was used for hemostasis), electro-coagulation (EC) group (55 cases, the abdominal tissues were cut with common lancet and electro-coagulation was used for hemostasis) and control group (72 cases, common lancet and silk thread suture were used) . The healing of wound was observed after operation . RESULTS: Four rats died of anesthetic accident; the other 236 rats were killed 8 days after . The wound infection rates were 23.33% and 6.60% in the experimental 10(0) group and the control 10(0) group respectively (chi(2) = 3.28, P > 0.05); 37.04% and 13.33% in the experimental 10(2) group and the control 10(2) group respectively (chi(2) = 4.31, P > 0.05), 50.00% and 24.14% in the experimental 10(5) group and the control 10(5) group respectively (chi(2) = 4.22, P > 0.05); and 63.33% and 36.67% in the experimental 10(8) group and the control 10(8) group respectively (chi(2) = 4.27, P > 0.05) . Clinical observation showed a delayed wound healing rate of 17.20% (16 cases) in EK group, 16.36% (11 cases) in EC group, and 2.86% (2 cases) in the control group . There was a statistically significant difference in delayed wound healing rate between the EK and control groups (chi(2) = 8.57, P < 0.01) and between the EC and control groups (chi(2) = 5.66, P < 0.05) . However, no significant difference in the delayed wound healing rate was seen between the EK and EC groups (chi(2) = 0.017, P > 0.05) . CONCLUSION: High-frequency electric knife remarkably delays the healing of abdominal incision . Its application should be minimized so as to reduce the possibility of postoperative complications.

J Pathol, 2003 Jan, 199(1), 122 - 9
Type 2 nitric oxide synthase and protein nitration in chronic lung infection; Hopkins N et al.; Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production . It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage . However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected . A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions . To test this hypothesis, anaesthetized, specific pathogen-free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group) . Ten to 15 days later, the lungs were isolated and fixed . Pseudomonas organisms were isolated from the lungs of the chronically infected group . These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs . Expression of iNOS was increased in the chronically infected group when compared with the control group . However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs . Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway . These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS .

Microb Pathog, 2002 Nov, 33(5), 203 - 10
Acid aspiration induces bacterial pneumonia by enhanced bacterial adherence in mice; Mitsushima H et al.; The issue of whether acid aspiration facilitates bacterial pneumonia caused by Pseudomonas aeruginosa by enhanced bacterial adherence was examined in mice . Survival or the number of bacteria in lung tissues was evaluated after an intratracheal challenge of hydrochloric acid (HCl), a sublethal dose of P . aeruginosa, or both in mice . Bacterial adherence to the tracheal epithelium after acid aspiration was also examined by scanning electron microscopy . A simultaneous intratracheal challenge of 50 microl of 10(-1) N HCl, but not 10(-2) to 10(-4) N HCl, combined with a sublethal dose of P . aeruginosa significantly increased the number of bacteria in the lung tissues and decreased survival, while all mice that received either HCl or P . aeruginosa survived . Significantly higher numbers of adherent bacteria on the tracheal epithelium were found in mice that received 10(-1)N HCl, compared with mice that received HCl (10(-2) to 10(-4) N) or saline . These data indicate that acid aspiration induced airway epithelial injury and enhanced P . aeruginosa adherence to the epithelium, and led to the subsequent development of bacterial pneumonia in mice . Enhanced bacterial adherence on the acid-injured epithelium may explain fatal bacterial pneumonias in patients with respiratory aspiration of gastric contents.

Mem Inst Oswaldo Cruz, 2002 Oct, 97(7), 1027 - 31
Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases; Holetz FB et al.; Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeasts . Of these, 10 plant extracts showed varied levels of antibacterial activity . Piper regnellii presented a good activity against Staphylococus aureus and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa, and a weak activity against Escherichia coli . Punica granatum showed good activity on S . aureus and was inactive against the other standard strains . Eugenia uniflora presented moderate activity on both S . aureus and E . coli . Psidium guajava,Tanacetum vulgare, Arctium lappa, Mikania glomerata, Sambucus canadensis, Plantago major and Erythrina speciosa presented some degree of antibacterial activity . Spilanthes acmella, Lippia alba, and Achillea millefolium were considered inactive . Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram . Of these, three plants belong to the Asteraceae family . This may mean that the same compounds are responsible for the antibacterial activity in these plants . Anticandidal activity was detected in nine plant extracts (P . guajava, E . uniflora, P . granatum, A . lappa, T . vulgare, M . glomerata, L . alba, P . regnellii, and P . major) . The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Science, 2002 Dec 6, 298(5600), 1942 - 6
Generating and exploiting polarity in bacteria; Shapiro L et al.; Bacteria are often highly polarized, exhibiting specialized structures at or near the ends of the cell . Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria . This polarization is orchestrated by complex and dynamic changes in the subcellular localization of signal transduction and cytoskeleton proteins as well as of specific regions of the chromosome . Recent work has provided information on how dynamic subcellular localization occurs and how it is exploited by the bacterial cell . The main task of a bacterial cell is to survive and duplicate itself . The bacterium must replicate its genetic material and divide at the correct site in the cell and at the correct time in the cell cycle with high precision . Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment . This involves moving toward food, adapting to environmental extremes, and, in many cases, entering and exploiting a eukaryotic host . These activities often involve processes that take place at or near the poles of the cell . Here we explore some of the schemes bacteria use to orchestrate dynamic changes at their poles and how these polar events execute cellular functions . In spite of their small size, bacteria have a remarkably complex internal organization and external architecture . Bacterial cells are inherently asymmetric, some more obviously so than others . The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria . "New" poles generated at the cell division plane differ from old poles from the previous round of cell division . Even in Escherichia coli, which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly (1), and they differ in the deposition of phospholipid domains (2) . There are many instances of differential polar functions; among these is the preferential use of old poles when attaching to host cells as in the interaction of Bradyrhizobium with plant root hairs (3) or the polar pili-mediated attachment of the Pseudomonas aeruginosa pathogen to tracheal epithelia (4) . An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus . The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell (5) . Interestingly, M . xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells.

J Immunol, 2002 Dec 15, 169(12), 6985 - 91
Cationic polypeptides are required for antibacterial activity of human airway fluid; Cole AM et al.; In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid . In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid . The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa . By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense . Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P . aeruginosa.

Exp Eye Res, 2002 Dec, 75(6), 635 - 43
Pseudomonas aeruginosa strains with lipopolysaccharide defects exhibit reduced intracellular viability after invasion of corneal epithelial cells; Evans D et al.; Pseudomonas aeruginosa is a leading cause of infectious keratitis . Many ocular isolates of this bacterium invade corneal epithelial cells in vitro and in vivo . Antibiotic survival assays have shown that a complete core lipopolysaccharide is required for full epithelial invasion by P . aeruginosa . In this study, we show that P . aeruginosa mutants with defects in their lipopolysaccharide core and O antigen exhibited reduced viability after internalization by corneal epithelial cells . Restoration of lipopolysaccharide core and O antigen expression by complementation with the plasmid pLPS1 restored intracellular survival . P . aeruginosa strains with a complete lipopolysaccharide survived and replicated within the cells . The data suggest that lipopolysaccharide is involved in the intracellular survival and/or replication of P . aeruginosa, indicating an additional mechanism by which this important virulence factor may contribute to the pathogenesis of corneal infection.

Prescrire Int, 2002 Dec, 11(62), 177 - 8
Tobramycin for nebulisation: new formulation . A high price for a small improvement in formulation; Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials; Biotechnology Research Group, School of Biological and Environmental Sciences, University of Ulster, Coleraine-BT52 1SA, Northern Ireland, UK . ksmrahman@yahoo.com

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials . Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production . Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively . The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth . Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)) . Emulsification activity of the rhamnolipid biosurfactant produced by P . aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel . P . aeruginosa GS9-119 emulsified only hexane and kerosene to that level.

J Med Microbiol, 2002 Dec, 51(12), 1063 - 70
Determination of quorum-sensing signal molecules and virulence factors of Pseudomonas aeruginosa isolates from contact lens-induced microbial keratitis; Zhu H et al.; The virulence of Pseudomonas aeruginosa in contact lens-induced microbial keratitis has been linked to various extracellular and cell-associated bacterial products, such as proteases and toxins . Recently, a group of bacterial signal molecules, N-acyl-homoserine lactones (AHLs), has been reported to play an important role in the regulation of the production of several bacterial virulence factors in P . aeruginosa . The aim of this study was to determine the signal molecules produced by P . aeruginosa keratitis strains, and to elucidate any possible correlation between the production of signal molecules and the expression of phenotypic characteristics, including protease production, bacterial invasion and acute cytotoxic activity . The presence and profiles of AHLs in ocular P . aeruginosa isolates were analysed by a combination of thin-layer chromatography and bioassay . All 17 keratitis isolates produced AHLs . There were differences both in the amounts and the types of AHL production in the various phenotypes of isolates . High levels of AHLs were found among the isolates with high protease activity and invasiveness . Acutely cytotoxic isolates displayed low AHL and protease activities . Invasive strains were more common than cytotoxic strains from keratitis patients . These results suggest that quorum-sensing systems of P . aeruginosa display a complexity even within the same species, and the production of certain AHL signal molecules may be associated with certain phenotypes in P . aeruginosa.

J Biol Chem, 2003 Feb 7, 278(6), 3615 - 27 Epub 2002 Dec 02.
Three highly conserved proteins catalyze the conversion of UDP-N-acetyl-D-glucosamine to precursors for the biosynthesis of O antigen in Pseudomonas aeruginosa O11 and capsule in Staphylococcus aureus type 5 . Implications for the UDP-N-acetyl-L-fucosamine biosynthetic pathway; Kneidinger B et al.; N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus . The three P . aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S . aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity . Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps . WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars . The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F . Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc . These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc . A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P . aeruginosa serotype O11 strain PA103 . The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen . This lipopolysaccharide deficiency could be complemented with P . aeruginosa wbjB or with the S . aureus homolog cap5E . Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S . aureus strain Newman . Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.

Burns, 2002 Dec, 28(8), 738 - 44
The evaluation of nosocomial infection during 1-year-period in the burn unit of a training hospital in Istanbul, Turkey; Oncul O et al.; An analysis of the burned patients, admitted to our eight bed burn unit and treated between 1 January and 31 December 2000, was performed . Prevalence, etiologic agents, length of hospitalization, cost of treatment and mortality rates caused by nosocomial infections (NIs) were studied . The study included 63 patients . Eighteen of these (Group-A) had 24 NI episodes . The most common NI observed was burn-wound infection (58.3%), followed by bacteraemia-sepsis (16.7%) . NIs were not detected in the rest at all (Group B) . The mean length of hospitalization was 38.5+/-19.7 days in Group A, and 20.3+/-7.6 days in Group B . The mean total burned surface area (TBSA) was 43+/-21 in Group A and 29+/-18 in Group B, while the most important independent risk factor for NI was TBSA in burned patients (OR, 1.08; CI(95), 0.93-1.24) . NI prolonged the mean hospital stay to 18 days and increased the cost of treatment by 502 US dollars . The most common bacteria isolated was Pseudomonas aeruginosa (41.7%) and the second was methicillin resistant Staphylococcus aureus (MRSA-25.0%) . All of the NI-free patients survived, while, five (28.5%) patients with NI died (P<0.01).These findings emphasized the need for careful disinfection and conscientious contact control procedures in areas that serve immunosupressed individuals, such as burned patients.

Biochemistry, 2002 Dec 10, 41(49), 14591 - 601
The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study; Folschweiller N et al.; In iron limitation conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (PaA) . PaA has an extremely high affinity for Fe(3+) but also chelates other ions such as Al(3+) and Ga(3+) with a lower affinity . The transfer of PaA-Fe(3+) across the outer membrane of the bacteria is mediated by the receptor FpvA, a TonB-dependent outer membrane transport protein . FpvA binds the iron-free and iron-loaded forms of pyoverdin with similar affinities, but only PaA-Fe(3+) is taken up by the cell, suggesting that FpvA adopts different conformations depending on its loading status . We used time-resolved fluorescence spectroscopy to characterize the different forms of FpvA-PaA in vitro . We showed that the FpvA-PaA complex adopts two different conformations depending on how it was prepared (formed in vitro or in vivo prior to purification) . The dihydroquinoline moiety of both conformers is fully protonated, or coordinated by protein charged groups, but the polarity of its environment, its solvent accessibility, and its rotational dynamics are much slower when the FpvA-PaA complex is formed in vivo than in vitro . In the presence of Ga(3+) or Al(3+) ions, the solvent accessibility and mobility of the dihydroquinoline moiety in the two FpvA-PaA complexes are intermediate between those observed for the metal-free ones . In addition, the Forster resonance energy transfer kinetics from FpvA tryptophan residues to the PaA chromophore differs from one complex to the other, revealing differences in one or more of the donor-acceptor topologies.

J Antimicrob Chemother, 2002 Dec, 50(6), 1045 - 9
Synergic activity of cephalosporins plus fluoroquinolones against Pseudomonas aeruginosa with resistance to one or both drugs; Fish DN et al.; Owing to increasing resistance in Pseudomonas aeruginosa, empirical drug regimens may include agents to which some strains may be resistant . The purpose of this study was to evaluate the in vitro activities of different combinations of cephalosporin plus fluoroquinolone against P . aeruginosa isolates with varying susceptibility to the study drugs . Broth microdilution susceptibility testing was performed with 10 clinical isolates of P . aeruginosa . The bactericidal activity of cefepime or ceftazidime alone and in combination with ciprofloxacin, levofloxacin, gatifloxacin or moxifloxacin was evaluated using time-kill methods . Colony counts were determined at 0, 4, 8 and 24 h, using antimicrobial concentrations of 0.5 x MIC . All procedures were performed in duplicate . Synergy was defined as a >2-log decrease in cfu/mL at 24 h compared with the single most active agent . The MICs for tested strains were: ceftazidime 0.75-32, cefepime 0.125-8, ciprofloxacin 0.0078-8, levofloxacin 0.023-16, gatifloxacin 0.023-16 and moxifloxacin 0.0521-32 mg/L . Four strains were susceptible to all drugs, two strains were cephalosporin susceptible and fluoroquinolone resistant, and two strains were cephalosporin resistant and fluoroquinolone susceptible . Two strains were resistant or intermediately susceptible to all drugs . Various cephalosporin and fluoroquinolone combinations were synergic against P . aeruginosa, including strains resistant to one or both agents in combination . No synergy was observed in two strains susceptible to all drugs . There were no differences noted between different cephalosporin and fluoroquinolone combinations . Concentrations used in this study are clinically achievable with recommended regimens in most cases.

J Antimicrob Chemother, 2002 Dec, 50(6), 1039 - 43
Mechanisms of beta-lactam resistance in Pseudomonas aeruginosa: prevalence of OprM-overproducing strains in a French multicentre study (1997); Cavallo JD et al.; One hundred and forty-three non-repetitive strains of Pseudomonas aeruginosa were collected in 13 French hospitals in 1997 . A decreased susceptibility or resistance to ticarcillin (MIC > 16 mg/L) was found in 61 isolates (43%) and this was attributed to three major mechanisms: (i) overexpression of OprM and hence related efflux components such as MexAB or MexXY (42.6%), (ii) production of acquired beta-lactamase (29.5%) and (iii) overexpression of chromosomally encoded AmpC cephalosporinase (21.3%) . Four of seven 'intrinsically' resistant strains (11.5%) with normal amounts of OprM were shown to produce low levels of AmpC, whereas in three isolates no resistance mechanism to beta-lactams could be identified . Overproduction of OprM thus appears as an important mechanism of ticarcillin resistance in French isolates of P . aeruginosa.

J Antimicrob Chemother, 2002 Dec, 50(6), 939 - 44
Combined bactericidal activity of perfluorooctyl bromide and aminoglycosides against Pseudomonas aeruginosa; Jung R et al.; Perfluorooctyl bromide (PFOB) may be useful as a medium for antibiotic delivery to treat pneumonia during liquid ventilation . OBJECTIVE: The purpose of the study was to determine the antibacterial activity of PFOB either alone or in combination with aminoglycosides against Pseudomonas aeruginosa . DESIGN: Modified time-kill assays were used to determine antibacterial activity: an inoculum of 1 x 10(5) cfu/mL was added to PFOB, or PFOB + an aminoglycoside (1 x MIC) . Viable counts were performed at 0, 0.25, 0.5, 0.75, 1, 2, 4 and 6 h . Electron microscopy was used to visualize the effect . Approximately 1.5 x 10(8) cfu/mL of bacteria were added to HEPES buffer (control), PFOB, gentamicin and PFOB + gentamicin . At baseline and 0.5 h, the bacteria were viewed under 20,000x magnification for both negative staining and thin-sectioning experiments . RESULTS: Exposure to PFOB alone resulted immediately in a >90% reduction in the inoculum at baseline compared with control (P = 0.001) . Following the initial reduction in colony count, bacteria grew in a similar manner to controls for PFOB-exposed strains . Aminoglycosides, alone at 1 x MIC or with PFOB, produced a bacteriostatic effect over the 6 h period . PFOB-exposed P . aeruginosa showed cell wall irregularity under electron microscopy . The gentamicin-exposed P . aeruginosa showed blebbing . PFOB + gentamicin caused extensive cell wall damage, exhibiting the additive effects of PFOB and gentamicin . CONCLUSION: PFOB appears to affect the cell wall of P . aeruginosa and enhance the bacterial cell destruction caused by aminoglycosides . The combined antibacterial effect of PFOB with the aminoglycosides is greater than that observed with these agents alone.

J Biol Chem, 2003 Jan 24, 278(4), 2085 - 8 Epub 2002 Dec 01.
An elegant means of self-protection in gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space; Eda S et al.; Infection of Pseudomonas aeruginosa in cystic fibrosis patients is a major cause of mortality . This organism shows wide ranging antibiotic resistance that is largely attributable to the expression of xenobiotic efflux pump(s) . Here, we show a novel mechanism by which the resistance-nodulation-division-type xenobiotic transporter expels potential hazards and protects the interior of the cells . The xenobiotic transporters MexB and MexY preferentially export beta-lactam and aminoglycoside antibiotics, respectively . When two large extramembrane loops of MexY were replaced by the corresponding loops of MexB, the hybrid protein exhibited beta-lactam selectivity (MexB-type), but failed to recognize aminoglycoside . As the transmembrane segment of MexB was replaced with a corresponding transmembrane segment of MexY, one-by-one for all 12 segments, all the hybrid proteins showed MexB-type antibiotic selectivity . These results clearly demonstrated that the resistance-nodulation-division-type efflux pump in P . aeruginosa selects and transports substrates via the domains that largely protrude over the cytoplasmic membrane . The transmembrane segments were unlikely to have been involved in substrate selectivity . These observations led us to propose a novel mechanism by which the xenobiotic transporters in Gram-negative bacteria select and expel substrates from the periplasmic space before potential hazards penetrate into the cytoplasmic membrane.

Protein Expr Purif, 2002 Dec, 26(3), 432 - 7
Expression and purification of two recombinant forms of the type-III cytotoxin, Pseudomonas aeruginosa ExoS; Maresso AW et al.; Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin . The N-terminus (residues 1-232) possesses Rho GTPase-activating (GAP) activity, while the C-terminus (residues 233-453) comprises an ADP-ribosyltransferase domain . Amino acid residues 51-72 of ExoS are involved in membrane binding and aggregation, which has complicated purification schemes . Here, it is reported on the expression, purification, and characterization of two recombinant forms of ExoS that lack this membrane-binding domain, designated rExoS78-453 and rExoSdelta51-72 . Purification of these forms was achieved using sequential NTA/Ni(2+)-affinity, gel filtration, and anion-exchange chromatography . Both forms of ExoS possessed Rho GAP activity and ADP-ribosyltransferase activity comparable to wild-type ExoS . Mass spectrometry showed that rExoS78-453 and rExoSdelta51-72 had molecular masses similar to their predicted molecular masses.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 176 - 84 Epub 2002 Sep 28.
Investigations on the metal switch region of human porphobilinogen synthase; Jaffe EK; Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis . The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium . In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium . The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS . The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc . The specific activities of the chimeras are significantly lower than human PBGS . Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage . The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence . Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position . The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.

J Biol Inorg Chem, 2003 Jan, 8(1-2), 156 - 66 Epub 2002 Sep 19.
Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c(551); Russell BS et al.; A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and {(1)H}-(15)N heteronuclear nuclear Overhauser effect data is reported . The protein backbone is highly rigid (< S(2)>=0.924+/-0.005) and displays little variation in picosecond-nanosecond time scale dynamics over the structure . The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content . Also reported are protection factors calculated from hydrogen-exchange rates . These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions . These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (< S(2)>=0.80+/-0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3 . This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties.

Can J Microbiol, 2002 Sep, 48(9), 810 - 20
Bactericidal effect of gentamicin-induced membrane vesicles derived from Pseudomonas aeruginosa PAO1 on gram-positive bacteria; MacDonald KL et al.; Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens . This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria . Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected . g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic . Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B . subtilis (hydrophilic) than to predominantly hydrophobic E . hirae, L . monocytogenes, and S . aureus . Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E . hirae were sensitive to the 26-kDa autolysin to varying degrees . Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis . Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2150 - 2 Epub 2002 Nov 23.
Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of Pseudomonas aeruginosa L-arginine deiminase; Oudjama Y et al.; Pseudomonas aeruginosa L-arginine deiminase, an enzyme catalyzing the irreversible catabolism of arginine to citrulline, has been produced in selenomethionyl form . The protein was purified and crystallized by the sitting-drop vapour-diffusion method using a precipitant solution consisting of 55% MPD, 100 mM cacodylate pH 6.5, 20 mM MgCl(2) . Crystals display tetragonal symmetry (P4(1)2(1)2 or P4(3)2(1)2), with unit-cell parameters a = b = 106.0, c = 300.2 A, and diffract to 2.7 A resolution . A complete MAD data set was collected to 3.2 A resolution on beamline BM30 at ESRF.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2145 - 6 Epub 2002 Nov 23.
Crystallization and preliminary X-ray crystallographic analysis of acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa; Eom SJ et al.; Acetohydroxy acid isomeroreductase (AHIR) is involved in the biosynthetic pathway of branched-chain amino acids in microorganisms and plants . AHIR from Pseudomonas aeruginosa has been overexpressed in Escherichia coli and crystallized at 297 K using potassium/sodium tartrate as a precipitant . X-ray diffraction data have been collected to 2.0 A resolution at 100 K using synchrotron radiation . The crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 184.38 A, alpha = beta = gamma = 90 degrees . Six monomers are present in the asymmetric unit, giving a V(M) of 2.34 A(3) Da(-1) and a solvent content of 47.4%.

Thorax, 2002 Dec, 57(12), 1015 - 20
Oropharyngeal carriage and lower airway colonisation/infection in 45 tracheotomised children; Morar P et al.; BACKGROUND: A study was undertaken to determine the oropharyngeal carrier state of potentially pathogenic microorganisms (PPM) and the magnitude of colonisation and infection rates of the lower airways with these PPM in children requiring long term ventilation first transtracheally and afterwards via a tracheotomy . METHODS: A 5 year, prospective, observational cohort study was undertaken in 45 children (33 boys) of median age 6.4 months (range 0-180) over a 5 year period at the Royal Liverpool Children's NHS Trust of Alder Hey, a university affiliated tertiary referral centre . The children were first admitted to the 20-bed paediatric intensive care unit (PICU) and, following placement of a tracheotomy, they were transferred to a four bedded respiratory ward . The two main indications were neurological disorders and airway obstruction . All children were ventilated transtracheally for a median period of 12 days (range 0-103) and, after placement of the tracheotomy, for a similar period of 12 days (range 1-281) . Surveillance cultures of the oropharynx were taken on admission to the PICU and on the day of placement of the tracheotomy . Throat swabs were taken twice weekly during ventilation, both transtracheal and via the tracheotomy . Tracheal aspirates were taken once weekly and when clinically indicated (in cases where the lower airway secretions were turbid) . RESULTS: Twenty five patients (55%) had abnormal flora, mainly aerobic Gram negative bacilli (AGNB), particularly Pseudomonas aeruginosa, while the community PPM Staphylococcus aureus was present in the oropharynx of 37% (17/45) of the study population . The lower airways were sterile in six children; the other 39 patients (87%) had a total of 82 episodes of colonisation . "Community" PPM significantly increased once the patients received a tracheotomy, independent of the number of patients enrolled, episodes of colonisation/infection, and the number of colonised/infected patients . "Hospital" PPM significantly decreased after tracheotomy only when episodes were compared . CONCLUSIONS: While P aeruginosa present in the admission flora caused primary endogenous colonisation/infection during mechanical ventilation on the PICU, S aureus not carried in the throat was responsible for the exogenous colonisation/infection once the patients had a tracheotomy . This is in sharp contrast to adult studies where exogenous infections are invariably caused by AGNB . This discrepancy may be explained by chronic underlying conditions such as diabetes, alcoholism, and chronic obstructive pulmonary disease which promote AGNB, whereas the children were recovering following tracheotomy.

J Clin Microbiol, 2002 Dec, 40(12), 4607 - 11
Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa; Parsons YN et al.; A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom . Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1 . Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined . Several sequences were found only in the Liverpool epidemic strain . Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production . The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island . Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened . None of the sequences identified was present in the Manchester strain . However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain . All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene . A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.

Invest Ophthalmol Vis Sci, 2002 Dec, 43(12), 3654 - 9
Pseudomonas aeruginosa binds to extracellular matrix deposited by human corneal epithelial cells; Esco MA et al.; PURPOSE: To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding . METHODS: Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2) . P . aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2) . The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2) . Binding in the presence of antibodies directed against laminin-5 was also measured . RESULTS: Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660 . In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660 . Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660 . Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control . CONCLUSIONS: The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P . aeruginosa . Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix . The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

Invest Ophthalmol Vis Sci, 2002 Dec, 43(12), 3646 - 53
Influence of wear and overwear on surface properties of etafilcon A contact lenses and adhesion of Pseudomonas aeruginosa; Bruinsma GM et al.; PURPOSE: To determine changes in physicochemical surface properties of contact lenses (CLs) during daily wear and effects of lens wear on adhesion of a Pseudomonas aeruginosa strain from a patient with CL-related keratitis . METHODS: Ten new CL wearers used ionic, etafilcon A lenses with 58% water on both eyes for approximately 10 hours each day during 10 and 50 days . All lenses were treated daily with an appropriate lens care solution . After the CLs were worn for 10 days (first pair of lenses) and 50 days (second pair, representing overwear), hydrophobicity by water contact angles, surface roughness by atomic force microscope, elemental surface composition by x-ray photoelectron spectroscopy (XPS), and adsorbed proteins by SDS-PAGE were determined on one lens . The lens from the contralateral eye was placed in a parallel plate flow chamber for bacterial adhesion after each time interval . RESULTS: Water contact angles on lenses changed from 45 degrees on unused lenses to 61 degrees +/- 25 degrees after 10 days of wear and changed significantly (P < 0.05) to 27 degrees +/- 14 degrees after 50 days of wear . Surface roughness increased significantly (P < 0.05) from 4 +/- 2 nm (unused) to 10 +/- 7 nm after 50 days of wear . These changes were accompanied by adsorption of proteinaceous material, as evidenced by XPS and SDS-PAGE, demonstrating adsorption of lysozyme, tear lipocalin, and a 30-kDa protein . Initial bacterial adhesion to worn CLs was lower than to unworn CLs . Furthermore, detachment of adhering bacteria from worn lenses was easier than from unworn lenses . The changes observed in the physicochemical surface properties of the lenses after the CLs were worn for 50 days were accompanied by reports of discomfort by 6 of the 10 new CL wearers . Multiple regression analysis revealed that the most predictive variables for an effect on initial deposition after 10 days of wear were hydrophobicity, roughness, the presence of nitrogen-rich material, including the presence of a 30-kDa protein, and the presence of oxygen-rich material-that is, the type of oxygen adsorbed (O equal or parallel C or Ocjs0807;C) . After 50 days of wear, roughness and the presence of tear lipocalin were most predictive . CONCLUSIONS: This study demonstrates that the physicochemical surface properties changed after wear and overwear, whereas overwear of the lenses decreased initial adhesion of P . aeruginosa #3 under the present experimental conditions.

Phytochemistry, 2002 Dec, 61(8), 907 - 12
Meroterpenes from Penicillium sp found in association with Melia azedarach; Geris dos Santos RM et al.; A Penicillium sp was isolated from the root bark of Melia azedarach and cultivated over sterilized rice . After chromatographic procedures, two meroterpenes, named preaustinoid A and B, were obtained in addition to the known alkaloid verruculogen . Their structures were identified by extensive spectroscopic studies, and they exhibited moderate bacteriostatic effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus sp.

Mol Microbiol, 2002 Dec, 46(5), 1381 - 90
Intracellular localization modulates targeting of ExoS, a type III cytotoxin, to eukaryotic signalling proteins; Pederson KJ et al.; ExoS is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa . Residues 96-232 comprise the Rho GTPase activating protein (Rho GAP) domain, whereas residues 233-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain . Earlier studies showed that the N-terminus targeted ExoS to intracellular membranes within eukaryotic cells . This N-terminal targeting region is now characterized for cellular and biological contributions to intoxications by ExoS . An ExoS(1-107)-green fluorescent protein (GFP) fusion protein co-localized with alpha-mannosidase, which indicated that the fusion protein localized near the Golgi . Residues 51-72 of ExoS (termed the membrane localization domain, MLD) were necessary and sufficient for membrane localization within eukaryotic cells . Deletion of the MLD did not inhibit type III secretion of ExoS from P . aeruginosa or type III delivery of ExoS into eukaryotic cells . Type III-delivered ExoS(DeltaMLD) localized within the cytosol of eukaryotic cells, whereas type III-delivered ExoS was membrane associated . Although type III-delivered ExoS(DeltaMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP-ribosylate Ras . Type III-delivered ExoS(DeltaMLD) and ExoS showed similar capacities for eliciting a cytotoxic response in CHO cells, which uncoupled the ADP-ribosylation of Ras from the cytotoxicity elicited by ExoS.

Semin Perinatol, 2002 Oct, 26(5), 332 - 9
Pseudomonas aeruginosa infections in the neonatal intensive care unit; Foca MD; Pseudomonas aeruginosa is a highly adaptable gram-negative bacillus with the ability to cause serious disease in vulnerable populations . This article reviews the relevant epidemiology of this pathogen in the hospital setting with particular attention to the neonatal unit . Issues related to reservoirs of the organism with special consideration of the hands of staff are also addressed . Virulence factors and pathogenic mechanisms are highlighted as well as the important role of antimicrobial resistance patterns . Finally, there is a discussion of the clinical syndromes found in neonates and the appropriate antibiotic usage strategies for effective treatment of this pathogen of continuing importance.

Appl Environ Microbiol, 2002 Dec, 68(12), 6343 - 52
A novel fluorescent protein-based biosensor for gram-negative bacteria; Goh YY et al.; Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria . EGFP mutants (EGFP(i)) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host . The EGFP(i) proteins were purified and tested for their efficacy as a novel fluorescent biosensor . After efficient removal of lipopolysaccharide from the E . coli lysates, the binding affinities of the EGFP(i) G10 and G12 to lipid A were established . The K(D) values of 7.16 x 10(-7) M for G10 and 8.15 x 10(-8) M for G12 were achieved . With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP(i) biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants . The EGFP(i) specifically tagged gram-negative bacteria like E . coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment . This dual function of the EGFP(i) in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Jul-Aug, (4), 40 - 3
{Pseudomonas aeruginosa vaccine on the basis of antigens isolated from the supernatant of culture media K-4}; Nuriddinova NR et al.; The possibility of using experimental culture medium K-4 prepared on the basis of casein hydrolysate peptides with the isoelectric point 4.1 for obtaining antigens from P . aeruginosa strains was evaluated . Two antigenic fractions were isolated from the culture fluid containing extracellular slime . The study of the toxicity of the antigenic preparations revealed that one of these fractions had low toxicity for mice (the second antigenic fraction was highly toxic) . The former P . aeruginosa antigenic fraction was used for obtaining pyocyanic vaccine . One vaccination dose of this vaccine contained 0.2 mg of the antigen adsorbed on aluminum hydroxide . Pyocyanic vaccine ensured the active protection of mice challenged with P . aeruginosa homologous and heterologous strains.

Spec Care Dentist, 2002 Jul-Aug, 22(4), 137 - 41
The effect of a disinfectant/coolant irrigant on microbes isolated from dental unit water lines; Epstein JB et al.; The purpose of this study was to assess water samples from a hospital dental clinic to determine whether a disinfectant/coolant irrigant containing chlorhexidine (Lines, Micrylium Laboratories) affects the presence of microbial organisms in dental unit waterlines . Water samples from three hospital dental operatories were collected at baseline and after overnight treatment with a disinfectant-containing irrigant followed by sterile water irrigation . Saliva of treated patients and sterile water rinse specimens were collected from the waterlines of these operatories for three consecutive days, then weekly for eight weeks after treatment . Specimens were cultured to identify total heterotrophic plate counts as well as presence of Pseudomonas aeruginosa and Candida species . Baseline organism counts varied from 10(3) to 10(5) colony-forming units per milliliter . After treatment, no organisms were detected in waterline discharge . Decontamination of dental unit waterlines is possible using a disinfectant/irrigant followed by sterile water irrigation . The potential for contamination of the lines from patients' saliva may have been reduced due to use of anti-retraction valves and the disinfectant/sterile water irrigation, as conducted in this study.

Eur Respir J, 2002 Nov, 20(5), 1284 - 91
Surfactant in children with malignancies, immunosuppression, fever and pulmonary infiltrates; Griese M et al.; In children with malignancies and immunosuppression, significant morbidity and mortality result from respiratory complications . The aim of the present study was to investigate whether or not this is associated with altered surfactant components or functions . Bronchoalveolar lavage fluid from 24 children with malignancies, immunosuppression, pulmonary infiltrates and fever unresponsive to empirical antibiotic treatment were compared to that from 24 healthy children . Levels of surfactant protein (SP) A and D and their binding capacity for Pseudomonas aeruginosa, as well as levels of SP-B and SP-C, were assessed by enzyme-linked immunosorbent assay . The large and small surfactant aggregate forms were separated and the biophysical activity of large surfactant aggregates was determined using a pulsating bubble surfactometer . Compared to healthy controls, SP-A levels were increased four-fold, the increase being most pronounced in those children with pathogens recovered from their bronchoalveolar lavage fluid . In children with malignancies, levels of SP-C were increased two-fold and of small surfactant aggregates five-fold . No differences were observed in levels of SP-B or SP-D, binding capacity of SP-A or SP-D or the surface activity of large surfactant aggregates . The increased levels of surfactant protein A, particularly in children with recovered microorganisms, and unchanged binding capacity of surfactant protein A are consistent with upregulated local host defence mechanisms . Increased surfactant protein A and C may also be responsible for the conserved biophysical activity of surfactant in children with malignancies, immunosuppression, pulmonary infiltrates and fever.

Eur Respir J, 2002 Nov, 20(5), 1263 - 70
Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor; Kohri K et al.; Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production . Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype . This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production . Human airway epithelial (NCI-H292) cells were stimulated with PA culture supernatant (Sup) . MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting . PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression . Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor . Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation . The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.

Ugeskr Laeger, 2002 Oct 28, 164(44), 5145 - 7
{Ear piercing and auricular chondritis caused by Pseudomonas aeruginosa}; Andersen HT; This paper describes five patients who had undergone transcartilaginous ear piercing and were infected with Pseudomonas aeruginosa, which led to perichondrial abscess . Early treatment of chondritis with aggressive surgical therapy combined with relevant antibiotics results in good healing and a minimal loss of cartilage . The literature review and cases presented show that piercing is not harmless and that regulation of the piercing business would seem to be desirable.

Ugeskr Laeger, 2002 Oct 28, 164(44), 5144 - 5
{Perichondritis caused by high ear piercing . Therapeutic and legal aspects}; Eckhardt LR et al.; Three cases of auricular perichondritis caused by infection with Pseudomonas aeruginosa are described to draw attention to the high incidence of piercing-related infections and the risk of structural deformities to the outer ear . Various therapies and outcomes are described and current Danish legislation is highlighted.

J Cutan Med Surg, 2003 Jan-Feb, 7(1), 13 - 9 Epub 2002 Nov 27.
In-vitro analysis for microbial barrier properties of 2-octyl cyanoacrylate-derived wound treatment films; Narang U et al.; BACKGROUND: In recent years, 2-octyl cyanoacrylate monomer has been formulated for various wound care products that have been approved by the Food and Drug Administration (FDA) . PURPOSE: To evaluate the in vitro effectiveness of 2-octyl cyanoacrylate formulation-based films as barriers to various pathogens, including bacteria, fungi, and yeast . METHODS: The barrier properties of the cyanoacrylate films prepared by the following two methods were tested: (1) prepolymerized film and (2) in situ polymerized film . The upper surface of films was inoculated such that the microorganisms would have to penetrate the film to colonize the media beneath . Nine different organisms were used . Plates were observed for growth at two, four, and seven days after inoculation . RESULTS: No growth was observed in any test, with the exception of prepolymerized film challenged with Pseudomonas aeruginosa . The pattern of growth observed suggests that the bacteria colonized the medium by traveling around and not through the film . Conclusions: 2-Octyl cyanoacrylate-based films are excellent microbial barriers.

Free Radic Biol Med, 2002 Dec 1, 33(11), 1527 - 33
Pyocyanin induces oxidative stress in human endothelial cells and modulates the glutathione redox cycle; Muller M; Pyocyanin is a redox active virulence factor produced by the human pathogen Pseudomonas aeruginosa . Treatment of endothelial cells with pyocyanin (1-50 microM) resulted in the dose-dependent formation of hydrogen peroxide that was detected in the extracellular medium . Total intracellular glutathione levels decreased in response to pyocyanin in a dose-dependent manner from a control value of 19.9 +/- 2.7 nmol/mg protein to 10.0 +/- 2.4 nmol/mg protein . Prior treatment of cells with catalase afforded complete protection against loss of glutathione . Total intracellular soluble thiols decreased from 95.0 +/- 6.2 nmol/mg protein to 78.6 +/- 2.3 nmol/mg protein at the highest test dose . Intracellular levels of NADPH increased up to 2.4-fold in response to pyocyanin exposure . It is concluded that pyocyanin exposes endothelial cells to oxidative stress by the generation of hydrogen peroxide, which subsequently depletes intracellular glutathione and increases intracellular levels of mixed disulfides.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 57 - 63
Characterization of outer membrane efflux proteins OpmE, OpmD and OpmB of Pseudomonas aeruginosa: molecular cloning and development of specific antisera; Murata T et al.; The third genes, opmE, opmD and opmB, of multidrug efflux operons deduced from the Pseudomonas aeruginosa PAO1 genome data were cloned by polymerase chain reaction . The opmB gene product showed functional cooperation with inner membrane-associated components, MexAB, MexCD and MexXY, of the previously characterized multidrug efflux systems responsible for resistance to antimicrobial agents and extrusion of ethidium . The opmE and opmD gene products did not show functional cooperation . Immunoblots using a specific rabbit antiserum demonstrated, through exponential to stationary phases, constant expression of opmB and growth phase-dependent expression of opmD.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 31 - 5
Characterization of the GO system of Pseudomonas aeruginosa; Oliver A et al.; The mutT, mutM, and mutY genes of the GO system of the Pseudomonas aeruginosa PAO1 strain have been characterized by cloning, sequencing, and complementation analysis . The three genes, when cloned in a plasmid, were able to complement the high mutation frequency of the corresponding Escherichia coli deficient strains . Our results demonstrate that the putative mutT, mutM, and mutY gene products from P . aeruginosa are able to perform the expected activity . In addition, the sequence of the P . aeruginosa mutT gene strongly suggested that the product of this gene has a bifunctional activity in P . aeruginosa, being the C-terminal part 40% identical to a consensus sequence of thiamine monophosphate synthases . Our results also demonstrated that the N-terminal part of the protein is necessary and sufficient for the 8-oxodGTP hydrolase activity.

Virology, 2002 Oct 25, 302(2), 333 - 41
Chimeric ecotropic MLV envelope proteins that carry EGF receptor-specific ligands and the Pseudomonas exotoxin A translocation domain to target gene transfer to human cancer cells; Erlwein O et al.; Redirecting retroviral vector transduction simply by insertion of a ligand into the envelope (Env) protein has met with several obstacles . For example, virions targeted to epidermal growth factor receptor (EGFR), after receptor binding, rapidly traffic to the lysosomes, where they are degraded . Exotoxin A of Pseudomonas aeruginosa has the ability to translocate from endosomes to the cytoplasm by means of a translocation domain (TLD) . We generated a series of chimeric Env proteins of Moloney murine leukemia virus containing EGFR ligands, where TLD was inserted into different regions . These chimeric proteins were successfully produced, if the translocation domain was not located at the immediate N-terminus of Env . The ability to transduce murine cells via the ecotropic receptor varied but correlated with the amount of Env proteins incorporated into the virions . Chimeric vector particles could bind to EGFR, demonstrating the functional exposure of the peptide ligand . However, transduction of human cells expressing EGFR but not the ecotropic receptor by virions carrying the chimeric protein was not observed.

Z Naturforsch {C}, 2002 Sep-Oct, 57(9-10), 954 - 6
Diastereomeric pyoverdin-chromium(III) complexes; Budzikiewicz H et al.; Coordination isomeric diastereomeric Cr3+ complexes of the pyoverdin of Pseudomonas aeruginosa ATCC 15692 could be separated by chromatography and characterized by spectroscopic methods.

Infect Immun, 2002 Dec, 70(12), 7136 - 9
The furin inhibitor hexa-D-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo; Sarac MS et al.; The Pseudomonas aeruginosa exotoxin A (PEA) protein requires furin-mediated cleavage for manifestation of toxicity . We show here that the small stable furin inhibitor hexa-D-arginine amide effectively blocks PEA-induced cell lysis and is itself noncytotoxic . Administration of hexa-D-arginine to PEA-treated mice significantly improves their survival rate and also decreases circulating levels of tumor necrosis factor alpha.

Infect Immun, 2002 Dec, 70(12), 7054 - 62
The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53; Yamada T et al.; Two redox proteins, azurin and cytochrome c(551) elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages . Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis . Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin . Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level . A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity . Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 247 - 51
The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component; Murata T et al.; The tripartite efflux systems MexAB-OprM and MexCD-OprJ of Pseudomonas aeruginosa each display characteristic substrate specificity against a variety of antimicrobial agents . The chimeric efflux system MexC-MexB-OprJ/DeltaMexD constructed by exchange of MexD with MexB endowed the recombinant host the same resistance profile as MexAB-OprM rather than MexCD-OprJ . The change of substrate specificity was shown to be due to extrusion from the chimeric efflux system by cellular accumulation experiments using tetracycline, erythromycin, and ethidium bromide . Thus, we conclude that MexB and MexD are primary components of the efflux system responsible for sorting extrusion substrates.

J Laryngol Otol, 2002 Sep, 116(9), 686 - 9
Changes in bacteriology of discharging ears; Hwang JH et al.; A bacteriological study on 161 consecutive out-patients presenting with otorrhoea was performed prospectively at a local teaching hospital in Taiwan between August 2000 and June 2001 . A total of 177 isolates were recovered . Staphylococcus aureus was found in 77 (43.5 per cent) isolates, and non-Staphylococcus aureus in 100 (56.5 per cent) isolates . Pseudomas sp was found to be the most common pathogen (28.8 per cent) in the non-Staphylococcus aureus group . Staphylococcus aureus had become more common than Pseudomonas aeruginosa in acute otitis externa, granular myringitis, and chronic otitis media in Taiwan . Methicillin-resistant Staphylococcus aureus (MRSA) was also an increasing problem in all three disease entities . The prevalence of community-acquired MRSA infections in discharging ears was found to be 13.7 per cent (22/161) . MRSAs were highly susceptible to vancomycin, teicoplanin, fusidic acid, and minocycline . More studies should be done to determine the susceptibility of MRSA to ofloxacin in the future.

Ann Clin Microbiol Antimicrob . 2002 Oct 9;1(1):2 {Epub ahead of print}
Susceptibility patterns and cross resistances of antibiotics against Pseudomonas aeruginosa in a teaching hospital of Turkey; Gencer S et al.; BACKGROUND: Pseudomonas aeruginosa is the third most common pathogen responsible for nosocomial infections and the prevalence of multiple resistant isolates has been increasing . Ninety-nine clinical isolates were studied in order to assess the current levels of susceptibility and cross-resistances of widely used antipseudomonal antibiotics against P . aeruginosa and to determine some resistance mechanisms by phenotypic methods . METHODS: MICs of isolates for nine antipseudomonal antibiotics were determined by the E test method . RESULTS: Thirty-six percent of isolates were resistant to more than one group of antibiotics . The rates of susceptible isolates were ciprofloxacin 75%, amikacin 73%, ceftazidime 65%, meropenem 63%, imipenem 63%, piperacillin/tazobactam 60%, cefoperazone/sulbactam 59%, cefepime 54% and tobramycin 44% . The majority of carbapenem resistant isolates were susceptible to ciprofloxacin and amikacin . CONCLUSION: Ciprofloxacin seems to be the most active agent against P . aeruginosa followed by amikacin in our unit . The usefulness of combinations of these antibiotics and beta-lactams should be tested in treating multi-drug resistant P . aeruginosa.

J Biol Chem, 2003 Feb 14, 278(7), 4524 - 30 Epub 2002 Nov 14.
Mutation or overexpression of a terminal oxidase leads to a cell division defect and multiple antibiotic sensitivity in Pseudomonas aeruginosa; Tavankar GR et al.; Mutation of the cyanide-insensitive terminal oxidase of Pseudomonas aeruginosa leads to pleiotropic effects . A cio mutant and strains, including the wild-type, carrying the cioAB genes on a multicopy plasmid were temperature-sensitive and had a cell division defect, leading to the formation of non-septate, multinucleated filaments . Such strains of this intrinsically antibiotic-resistant bacterium were more sensitive to a range of antibiotics including chloramphenicol, beta-lactams, quinolones, aminoglycosides, and macrolides . The effect of cio mutation on Deltap-dependent accumulation of chloramphenicol suggested that antibiotic sensitivity resulted from loss of or damage to a multidrug efflux pump . The ability of reducing agents and catalase to suppress the temperature-sensitive phenotype and of catalase to partially suppress antibiotic sensitivity suggested that increased levels of reactive oxygen species might be the cause of the observed phenotypes . Consistent with this was the increased sensitivity of strains to H(2)O(2) and their increased protein carbonyl content, an indicator of oxidative protein modification . The temperature-dependent synthesis of a specific catalase was absent in the cio mutant and in strains carrying multiple plasmid-borne copies of cioAB . We propose that reduced catalase levels result in oxidative modification and consequent loss of function of proteins involved in a range of cellular functions . How mutation or overexpression of the cyanide-insensitive terminal oxidase leads to a loss of catalase activity is unknown at present.

Antimicrob Agents Chemother, 2002 Dec, 46(12), 4026 - 8
Novel variant (bla(VIM-4)) of the metallo-beta-lactamase gene bla(VIM-1) in a clinical strain of Pseudomonas aeruginosa; Pournaras S et al.; A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece . The isolate carried a class 1 integron that contained as a sole cassette the gene bla(VIM-4), a novel variant of bla(VIM-1), with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme . This is the first detection of a VIM-1 variant after its appearance in Italy.

Biopolymers, 2002 Dec 15, 65(6), 395 - 407
Molecular structure of the outer bacterial membrane of Pseudomonas aeruginosa via classical simulation; Shroll RM et al.; A detailed structural analysis has been performed of the outer bacterial membrane of Pseudomonas aeruginosa using a parameterized classical simulation model (R . D . Lins and T . P . Straatsma, Biophysical Journal, 2001, Vol . 81, pp . 1037-1046) with modest modifications . The structural analysis of the membrane is presented and newly discovered characteristics of the membrane are discussed . Simulations indicate that the relative contribution of different ligands to calcium ion coordination varies across the membrane, while maintaining a constant average coordination number of 6.1 . Water penetrates the surface of the membrane to a depth of about 30 A . The hydration of ions and phosphate groups is shown to depend on location within the membrane . A measure of saccharide residue orientation is defined and average orientations are presented . Saccharide residues possess varying degrees of motion with a trend of greater mobility at the membrane surface . However, their motion is limited and even in the membrane outer core region the average structure appears fairly rigid over a period of 1 ns .

Am J Respir Crit Care Med, 2003 Mar 15, 167(6), 828 - 34 Epub 2002 Nov 14.
Nontuberculous mycobacteria . I: multicenter prevalence study in cystic fibrosis; Olivier KN et al.; Nontuberculous mycobacteria (NTM) are potential respiratory pathogens in cystic fibrosis (CF) . To assess the species-specific prevalence and risk factors for acquisition, we conducted a prospective, cross-sectional study of the prevalence of NTM and clinical features of patients at 21 U.S . centers . Almost 10% of patients with CF who were 10 years or older were included (n = 986) . The overall prevalence of NTM in sputum was 13.0% (range by center, 7-24%) . Mycobacterium avium complex (72%) and Mycobacterium abscessus (16%) were the most common species . When compared with patients with CF without NTM, culture-positive subjects were older (26 vs . 22 years, p < 0.001), had a higher FEV1 (60 vs . 54%, p < 0.01), higher frequency of Staphylococcus aureus (43 vs . 31%, p < 0.01), and lower frequency of Pseudomonas aeruginosa (71 vs . 82%, p < 0.01) . Molecular typing revealed that almost all patients within each center had unique NTM strains . In summary, NTM are common in patients with CF, but neither person-to-person nor nosocomial acquisition explained the high prevalence . Older age was the most significant predictor for isolation of NTM . The clinical significance of NTM in CF is incompletely defined, but patients with these organisms should be monitored with repeat cultures.

Med Clin (Barc), 2002 Nov 9, 119(16), 605 - 9
{Cystic fibrosis in adults: study of 111 patients}; de Gracia J et al.; BACKGROUND: Our goal was to establish the clinical and genetic characteristics of patients diagnosed with adult-onset cystic fibrosis (CF) . PATIENTS AND METHOD: This was a retrospective observational descriptive comparative study of CF patients according to their age at the time of diagnosis . All adult patients (> 16 years old) attended in our CF Unit until November 2001 were included in the study . Those patients diagnosed of CF at their childhood (< 16 years old) were categorized as Group A patients, and those diagnosed in adulthood (>= 16 years old) were categorized as group B patients . Anthropometric parameters, respiratory and digestive clinical abnormalities, chest and abdominal radiological exams, sputum bacteriology, respiratory function and genetic tests were evaluated . Statistical analysis between groups was performed by comparing chi square test for qualitative values and the Student t test for quantitative values . RESULTS: One hundred and eleven patients (60 women, mean age 28, range 16-69 years) out of a total sample of 245 (45.3%) patients attended at the CF unit were enrolled in the study . Group A included 61 patients (32 women; mean age 23) and group B included 50 patients (28 women; mean age 32) . The comparative study between both groups showed that patients in group B were older, had a higher weight and less incidence of initial digestive abnormalities, pancreatic insufficiency, malnutrition, hepatic disease, chronic bronchial colonization by Pseudomonas aeruginosa, admissions, lung transplantation and deaths due to CF . On the contrary, these patients had a higher incidence of pancreatitis, allergic bronchopulmonary aspergillosis at diagnosis and better respiratory function test parameters . The sweat test was negative in 4 patients of group B and 1 of group A . The genetic study showed 31 different CF mutations, from which only 10 were observed in group B . CONCLUSIONS: CF can also be diagnosed in adult age . Patients diagnosed in adulthood have less digestive abnormalities, better lung function and different genetic mutations . The sweat test can be negative or undetermined . These patients also display a better prognosis.

J Burn Care Rehabil, 2002 Nov-Dec, 23(6), 366 - 70
The efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected burns; Cooper RA et al.; Because there is no ideal therapy for burns infected with Pseudomonas aeruginosa, there is sufficient need to investigate the efficacy of alternative antipseudomonal interventions . Honey is an ancient wound remedy for which there is modern evidence of efficacy in the treatment of burn wounds, but limited evidence for the effectiveness of its antibacterial activity against Pseudomonas . We tested the sensitivity of 17 strains of P . aeruginosa isolated from infected burns to two honeys with different types of antibacterial activity, a pasture honey and a manuka honey, both with median levels of activity . All strains showed similar sensitivity to honey with minimum inhibitory concentrations below 10% (vol/vol); both honeys maintained bactericidal activity when diluted more than 10-fold . Honey with proven antibacterial activity has the potential to be an effective treatment option for burns infected or at risk of infection with P . aeruginosa.

Int J Antimicrob Agents, 2002 Nov, 20(5), 384 - 6
The comparison of in the vitro effect of imipenem or meropenem combined with ciprofloxacin or levofloxacin against multidrug-resistant Pseudomonas aeruginosa strains; Erdem I et al.; This study evaluated the in vitro effects of the combination of a carbapenem (imipenem or meropenem) with a quinolone (ciprofloxacin or levofloxacin) using a microbroth dilution chequerboard technique and multidrug-resistant Pseudomonas aeruginosa strains . The ciprofloxacin and meropenem combination was only synergistic against 2 strains (6.2%) and ciprofloxacin and imipenem against 1 strain (3.1%) . Levofloxacin and imipenem or meropenem were not synergistic for against any strain . None of the combinations showed an antagonistic effect.

Int J Antimicrob Agents, 2002 Nov, 20(5), 380 - 3
Epidemiological typing of imipenem-resistant Pseudomonas aeruginosa; Muller-Premru M et al.; The level of genetic heterogeneity of nine isolates of Pseudomonas aeruginosa resistant or intermediately susceptible to imipenem was determined by epidemiological typing using macrorestriction analysis of chromosomal DNA . The strains were isolated between December 2000 and February 2001 from a variety of specimens from nine patients hospitalized in five different departments of the University Medical Center in Ljubljana and in the nursing home . They belonged to seven genotypes or clones (A-G) . Six isolates were heterogeneous, with different B-G genotypes . Three isolates had an identical A genotype but it is more likely that this genotype was more prone to develop imipenem resistance than to spread among the patients.

Ophthalmologe, 2002 Nov, 99(11), 849 - 53
{Diffuse lamellar keratitis . Postoperative prophylactic treatment with corticosteroids in an experimental animal study}; Holzer MP et al.; PURPOSE: The aim of this experimental ani-mal study was to induce diffuse lamellar keratitis (DLK), and investigate a prophylactic treatment with corticosteroids . MATERIALS AND METHODS: A corneal flap was cut in 40 eyes from 20 Dutch-belted rabbits and the interface inoculated with either Pseudomonas aeruginosa lipopolysaccharide (LPS) endotoxin ( n=21) or Palmolive Ultra soap ( n=19) . Half of the eyes were treated with topical corticosteroids and the other half remained untreated . Slitlamp examinations were performed 1, 3, 5 and 7 days postoperatively and DLK was graded from I-IV . RESULTS: At the end of the study 33 eyes were available for evaluation and 94% of the non-treated eyes developed DLK . Out of those eyes treated with steroids 19% developed DLK during the 1 week follow-up period . This was statistically significantly lower ( P=0.018) when compared to the untreated group . CONCLUSION: Pseudomonas aeruginosa LPS endotoxin as well as Palmolive((R)) Ultra caused a very high rate of DLK in rabbit eyes . The postoperative prophylactic treatment with corticosteroids showed a statistically significant lower DLK rate in this rabbit eye model.

Microbiology, 2002 Nov, 148(Pt 11), 3423 - 30
Static growth of mucoid Pseudomonas aeruginosa selects for non-mucoid variants that have acquired flagellum-dependent motility; Wyckoff TJ et al.; When mucoid (alginate-producing) Pseudomonas aeruginosa FRD1 is grown under low oxygen conditions in liquid culture (static), non-mucoid variants appear and eventually predominate . This conversion is not readily observed in aerobic, shaken cultures or static cultures containing the alternative electron acceptor nitrate . In this study, it is shown that the non-mucoid variants that arise under static growth conditions are almost exclusively algT mutants . It has been shown that AlgT not only positively regulates alginate biosynthesis, but also directly or indirectly negatively regulates flagellum synthesis . Indeed, during static growth, conversion to the non-mucoid phenotype is accompanied by the acquisition of flagellum-mediated motility . Surprisingly, by using a reporter gene fusion with the fliC promoter (pfliC::xylE), it was found that fliC expression begins within hours of static growth and is reversible after returning the culture to shaking conditions . The ability of the strain to produce alginate seems to be irrelevant to this phenomenon, as an AlgT(+) deltaalgD strain showed identical results . Thus, it is suggested that the first effect of static growth is to induce motility as an adaptive measure in the presence of wild-type algT . This may afford P . aeruginosa the ability to swim towards areas of higher oxygen concentrations . Subsequent to this, algT mutations are likely to secure the motile phenotype.

Proc Natl Acad Sci U S A, 2002 Nov 26, 99(24), 15699 - 704 Epub 2002 Nov 08.
A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis; Silo-Suh L et al.; A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease . Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection . However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection . A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection . Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50% . A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth in planta . FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection . Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis . In contrast, in a non-CF strain, PAO1, an algT mutation did not affect its virulence on alfalfa . Conversely, PAO1 virulence was reduced in a mucA mutant that overproduced alginate . These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection . These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of P . aeruginosa in CF.

J Bacteriol, 2002 Dec, 184(23), 6499 - 507
Chimeric analysis of the multicomponent multidrug efflux transporters from gram-negative bacteria; Tikhonova EB et al.; Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters . RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane . They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria . The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown . We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from Escherichia coli and Pseudomonas aeruginosa, respectively . The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from E . coli, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from P . aeruginosa . We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters . Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux.

J Bacteriol, 2002 Dec, 184(23), 6472 - 80
Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa; Gallagher LA et al.; A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1 . The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects . The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes . Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented . Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions . The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation . Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.

Biochimie, 2002 May-Jun, 84(5-6), 499 - 510
The pyocins of Pseudomonas aeruginosa; Michel-Briand Y et al.; Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins . The pyocin genes are located on the P . aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C . Three types of pyocins are described . (i) . R-type pyocins resemble non-flexible and contractile tails of bacteriophages . They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation . (ii) . F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure . (iii) . S-type pyocins are colicin-like, protease-sensitive proteins . They are constituted of two components . The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5) . It interacts with the small component (immunity protein) . The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN . R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively . The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.

Immunology, 2002 Nov, 107(3), 297 - 305
Influence of gender and interleukin-10 deficiency on the inflammatory response during lung infection with Pseudomonas aeruginosa in mice; Guilbault C et al.; Cystic fibrosis females have a worse prognosis compared to male patients . Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and lower levels of IL-10 are found in the bronchoalveolar lavage fluid compared to healthy controls . The present study was aimed at investigating the importance of gender and IL-10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa . We found that wildtype females were more susceptible than males to infection, as we observed greater weight loss, higher bacterial load, and inflammatory mediators in their lungs . IL-10 knockout mice, both females and males, had higher levels of TNF-alpha in the lungs compared to wildtype mice and maintained higher levels of polymorphonuclear cells and lower levels of macrophages for a longer period of time . Our results demonstrate that the number of bacteria recovered from the lungs of IL-10 knockout male mice was significantly higher than that observed in their wildtype male counterparts and we show that neutralization of IL-10 in infected female mice for a prolonged period of time leads to increased susceptibility to infection . Results reported in this study clearly demonstrate that females, both wildtype and IL-10 knockout mice are more susceptible to Pseudomonas aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs.

Am J Rhinol, 2002 Sep-Oct, 16(5), 255 - 60
A comparison of endoscopic culture techniques for chronic rhinosinusitis; Tantilipikorn P et al.; BACKGROUND: Recent evidence suggests that endoscopically obtained cultures from the middle meatus give comparable results to antral puncture for acute sinusitis . The best method for obtaining middle meatal cultures remains somewhat controversial because it has been theorized that specimens obtained with a swab are contaminated easily . This study compares endoscopic culture results from two different methods: swab and aspiration . Specifically, this study sought to determine whether or not the culture contamination rate is higher using the swab versus an aspiration technique . METHODS: One hundred consecutive culture specimensfrom 81 chronic rhinosinusitis patients were compared . Fifty cultures were obtained using a swab technique (group I) and another 50 cultures were obtained by aspirating pathological material into a sterile suction trap (group II) . The patient populations in each group were similar; there were no differences in terms of age, gender, comorbid medical conditions, or prior medical therapy . Cultures were considered contaminated if they yielded normal nasal flora or if rare or few Staphylococcus coagulase-negative colonies grew after no bacteria was identified in gram stain . Staphylococcus aureus, Staphylococcus coagulase-negative, and Pseudomonas aeruginosa were the three most common organisms in both groups . RESULTS: Gram-negative bacteria were noted in 21/60 (35%) positive cultures . Although the contamination rate of the suction aspiration group (14%) was less than the endoscopic swab group (10%), this did not approach statistical significance (p = 0.75) . CONCLUSIONS: Data from this study suggest that endoscopically guided aspiration of pathological material is no better than properly obtained swabs in directing antimicrobial therapy for chronic rhinosinusitis.

Folia Microbiol (Praha), 2002, 47(4), 445 - 9
Toxinogenicity and markers of pathogenicity of Pseudomonas aeruginosa strains isolated from patients with tumor diseases; Majtan V et al.; Potential virulence factors (elastase, proteinase, lipase, phospholipase C, alginate) as well as surface properties (hydrophobicity, motility) were determined in 103 Pseudomonas aeruginosa strains isolated from patients with cancer . Nontypable strains were the dominant group (60%), followed by serotypes O11 (17%), O12 (7%) and O4 (5%) . Seventy-one strains (69%) produced high level of elastase (10-60 mg/L), 87% of the strains possessed high activity of proteinase (bacterial) (10-250 mg/L) and 69% of the strains demonstrated higher level of lipase (20-150 U/mL); these elevated levels of enzymes were associated mainly with nontypable strains . On the other hand, 79% of the strains did not produce or produced only a low level of phospholipase C and 60% of isolates did not manifest any or very low production of alginate . Hydrophobicity demonstrated by adherence of the bacteria to xylene was shown by 69% of strains; 94% of strains aggregated with ammonium sulfate . Motility in the range of 31-80 mm was found in 76 strains (74%) . The considerable virulence of tested P . aeruginosa strains was confirmed . The nontypable strains manifested the most frequent group with high level of elastase, proteinase, lipase, hydrophobicity and motility.

Folia Microbiol (Praha), 2002, 47(4), 379 - 84
Effect of outer-membrane permeabilizers on the activity of antibiotics and plant extracts against Pseudomonas aeruginosa; Guha A et al.; Several known outer membrane permeabilizers increased susceptibility of a highly resistant pathogenic strain Pseudomonas aeruginosa to different antibiotics and plant extracts . Of all the chemicals tested, EDTA, sodium citrate and sodium hexametaphosphate (HMP) were found to be potent permeabilizers as shown by enhanced lysis of the bacteria in the presence of lysozyme . In the presence of EDTA and sodium citrate susceptibility of the strain to gentamicin and rifampicin increased markedly . The strain was resistant to vancomycin but became susceptible when grown in the presence of increasing amounts of EDTA and sodium citrate . Similar results were obtained for erythromycin when treated with sodium citrate . EDTA was found to be most potent permeabilizer in enhancing the activity of the plant extracts . Though HMP was an effective permeabilizer it had a weak or no effect on the activity of the antibiotics and plant extracts.

Mol Microbiol, 2002 Nov, 46(4), 1123 - 33
ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon; McCaw ML et al.; Expression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations . We report that ExsD, a unique protein, is a negative regulator of the type III regulon . Localization studies indicate that ExsD is not secreted by P . aeruginosa . To determine the role of exsD, a non-polar deletion was returned to the chromosome by allelic exchange . The delta exsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells . To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome . Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the delta exsD background . Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon . Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a delta exsD, pscC:: omega double mutant is derepressed . Bacterial two-hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA . We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.

J Biomed Opt, 2002 Oct, 7(4), 576 - 86
Single live cell imaging for real-time monitoring of resistance mechanism in Pseudomonas aeruginosa; Kyriacou SV et al.; We have developed and applied single live cell imaging for real-time monitoring of resistance kinetics of Pseudomonas aeruginosa . Real-time images of live cells in the presence of a particular substrate (EtBr) provided the first direct insights of resistance mechanism with both spatial and temporal information and showed that the substrate appeared to be accumulated in cytoplasmic space, but not periplasmic space . Three mutants of P . aeruginosa, PAO4290 (a wild-type expression level of MexAB-OprM), TNP030#1 (nalB-1, MexAB-OprM over expression mutant), and TNP076 (DeltaABM, MexAB-OprM deficient mutant), were used to investigate the roles of these three membrane proteins (MexAB-OprM) in the resistance mechanism . Ethidium bromide (EtBr) was chosen as a fluorescence probe for spectroscopic measurement of bulk cell solution and single cell imaging of bulk cells . Bulk measurement indicated, among three mutants, that nalB-1 accumulated the least EtBr and showed the highest resistance to EtBr, whereas DeltaABM accumulated the most EtBr and showed the lowest resistance to EtBr . This result demonstrated the MexAB-OprM proteins played the roles in resistance mechanism by extruding EtBr out of cells . Unlike the bulk measurement, imaging and analysis of bulk cells at single cell resolution demonstrated individual cell had its distinguished resistance kinetics and offered the direct observation of the regulation of influx and efflux of EtBr with both spatial and temporal resolution . Unlike fluorescent staining assays, live cell imaging provided the real-time kinetic information of transformation of membrane permeability and efflux pump machinery of three mutants . This research constitutes the first direct imaging of resistance mechanism of live bacterial cells at single cell resolution and opens up the new possibility of advancing the understanding of bacteria resistance mechanism.

Am J Pharmacogenomics, 2002, 2(4), 235 - 43
Chromosomally-encoded resistance mechanisms of Pseudomonas aeruginosa: therapeutic implications; Lister PD; Pseudomonas aeruginosa is an important nosocomial pathogen that presents a difficult therapeutic challenge . Although P . aeruginosa has been shown to acquire resistance mechanisms encoded on plasmids, this pathogen comes armed with multiple chromosomally-encoded mechanisms of resistance that can provide impressive intrinsic resistance, as well as the potential to mutate to high-level multi-drug resistance . Recent analysis of the sequenced genome of P . aeruginosa PAO1 suggested that we have just started to unlock the resistance potential of this pathogen . One of the most serious threats to the usefulness of beta-lactams against P . aeruginosa is the chromosomal AmpC cephalosporinase . When AmpC production increases through mutational events, overproduction of this cephalosporinase provides high-level resistance to all beta-lactams except the carbapenems . Carbapenem resistance typically requires down-regulation of the outer membrane protein (OprD), which serves as the primary route of entry for carbapenems . Perhaps the most threatening of the resistance mechanisms encoded on the P . aeruginosa chromosome are the multi-drug efflux pumps . These pumps have the ability to extrude multiple classes of antibiotics from the periplasmic space, as well as the cytoplasm . Natural expression of efflux pumps in 'wild-type' cells plays an important role in the relatively decreased susceptibility of P . aeruginosa to antibiotics . However, the greatest therapeutic problems occur when these pumps are overproduced in mutants and high-level, multi-drug resistance develops . Although the development of infections with highly resistant strains of P . aeruginosa can present serious therapeutic challenges, the most troublesome threat associated with the chromosomally-encoded resistance mechanisms is the potential for high-level resistance to emerge during the course of therapy . When resistance emerges during therapy, clinical failure can occur and the therapeutic options for second-line therapy can become severely limited . Unfortunately, the emergence of resistance during therapy is not a rare event with P . aeruginosa and these three resistance mechanisms . Therefore, clinicians must be mindful of this threat when choosing an appropriate therapy, and usually appropriate therapy includes a combination of drugs . Since the standard combination of an aminoglycoside and a beta-lactam has been shown to be ineffective in preventing the emergence of some resistance problems, the search for more effective combinations must be a priority.

J Enzyme Inhib Med Chem, 2002 Apr, 17(2), 117 - 22
Antibacterial Co(II), Cu(II), Ni(II) and Zn(II) complexes of thiadiazole derived furanyl, thiophenyl and pyrrolyl Schiff bases; Chohan ZH et al.; 2-Amino-1,3,4-thiadiazole undergoes a condensation reaction with furane-, thiophene- and pyrrole-2-carboxaldehyde to form tridentate NNO, NNS and NNN donor Schiff bases . These Schiff bases were further used to obtain complexes of the type {M(L)2}X, where M = Co(II), Cu(II), Ni(II) or Zn(II), L = L1, L2 or L3 and X = Cl2 . The new compounds described here have been characterized by their physical, spectral and analytical data, and have been screened for antibacterial activity against several bacterial strains such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa . The antibacterial potency of the Schiff bases increased upon chelation/complexation in comparison to the uncomplexed Schiff bases against the tested bacterial species thus, opening new approaches to find new ways in the fight against antibiotic-resistant strains.

J Enzyme Inhib Med Chem, 2002 Apr, 17(2), 101 - 6
Antibacterial cobalt(II), nickel(II) and zinc(II) complexes of nicotinic acid-derived Schiff-bases; Chohan ZH et al.; Nicotinic acid derived Schiff bases and their transition metal {cobalt(II), nickel(II) and zinc(II)} complexes have been prepared and characterized by physical, spectral and analytical data . The Schiff bases act as deprotonated tridentate ligands for the complexation of the above mentioned metal ions . These complexes, possessing the general formula {M(L)2} {where M = Co(II), Ni(II) and Zn(II) and L = HL1-HL4} showed an octahedral geometry of the metal ions . For determining the effect of metal ions upon chelation, the Schiff bases and their complexes have been screened for antibacterial activity against several pathogenic strains of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa . The new metal derivatives reported here were more bactericidal against one or more bacterial species as compared to the uncomplexed Schiff bases.

J Biol Chem, 2003 Jan 24, 278(4), 2212 - 8 Epub 2002 Nov 04.
Posttranslational modification of serine to formylglycine in bacterial sulfatases . Recognition of the modification motif by the iron-sulfur protein AtsB; Marquordt C et al.; Calpha-formylglycine is the catalytic residue of sulfatases . Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif . The modifying enzymes are unknown . AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae . Here we show (i) that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added . Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species . AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pull-down experiments . This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide.

FEBS Lett, 2002 Nov 6, 531(2), 209 - 14
If space is provided, bulky modification on the rim of azurin's beta-barrel results in folded protein; Pozdnyakova I et al.; Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold . Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7) . The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7) . Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure . In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7) . We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.

Nat Struct Biol, 2002 Dec, 9(12), 918 - 21
Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients; Mitchell E et al.; Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients . The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure . Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate-protein interactions . Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le(a)) series may be the preferred ligands of this lectin . Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.

Am J Physiol Lung Cell Mol Physiol, 2003 Feb, 284(2), L420 - 30 Epub 2002 Nov 01.
Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells; O'Malley YQ et al.; The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation . Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied . Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined . Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively . Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential . These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin . Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion . This suggests that loss of aconitase activity is not responsible for ATP depletion . How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.

Ophthalmology, 2002 Nov, 109(11), 1957 - 69
Effects of daily and overnight wear of a novel hyper oxygen-transmissible soft contact lens on bacterial binding and corneal epithelium: a 13-month clinical trial; Cavanagh HD et al.; OBJECTIVE: To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear . DESIGN: Prospective, randomized, double-masked, single-center, 13-month clinical trial . PARTICIPANTS: One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98) . One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n = 55, 30 nights) . TESTING: Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW . MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH) . RESULTS: Daily wear with control lens increased PA binding from 5.90 +/- 2.60 to 7.81 +/- 3.04 bacteria per cell (P < 0.01); HOTL wear increased PA binding significantly less (5.31 +/- 1.87-5.98 +/- 2.26; P < 0.01) . Daily wear produced no significant changes in CET or SCA . Significant decreases in DESQ were seen with both lenses with no significant intergroup differences . Tear LDH increased significantly in DW with HOTL wear versus control (P = 0.0017), but not after 1 month of subsequent EW (P = 0.533) . One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear (P < 0.01); binding adaptively decreased thereafter, returning to baseline at 9 to 12 months . Lens EW produced significantly enlarged SCA, thinning of CET (except 6-night HOTL wear), and decreased DESQ (P < 0.01) . Some adaptive recovery was seen with CET and DESQ, but not SCA; importantly, the data indicated no significant difference between 6- or 30-night EW for all outcomes . CONCLUSIONS: Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs . 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year . These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.

Bioorg Med Chem, 2002 Dec, 10(12), 3997 - 4004
Synthesis and evaluation of anticancer benzoxazoles and benzimidazoles related to UK-1; Kumar D et al.; UK-1 is a structurally unique bis(benzoxazole) natural product isolated from a strain of Streptomyces . UK-1 has been reported to possess anticancer activity but no activity against bacteria, yeast, or fungi . Previous work has also demonstrated the ability of UK-1 to bind a variety of di- and tri-valent metal ions, particularly Mg(2+) ions, and to form complexes with double-stranded DNA in the presence of Mg(2+) ions . Here we report the activity of UK-1 against a wide range of human cancer cell lines . UK-1 displays a wide spectrum of potent anticancer activity against leukemia, lymphoma, and certain solid tumor-derived cell lines, with IC(50) values as low as 20 nM, but is inactive against Staphylococcus aureus, a methicillin-resistant strain of S . aureus, or Pseudomonas aeruginosa . A series of analogues of the bis(benzoxazole) natural product UK-1 in which the carbomethoxy-substituted benzoxazole ring of the natural product was modified were prepared and evaluated for their anticancer and antibacterial properties . An analogue of UK-1 in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy-substituted benzimidazole ring was inactive against human cancer cell lines and the two strains of S . aureus . In contrast, a simplified analogue in which the carbomethoxy-substituted benzoxazole ring was replaced with a carbomethoxy group was almost as active as UK-1 against the four cancer cell lines examined but lacked activity against S . aureus . Metal ion binding studies of these analogues demonstrate that they both bind Zn(2+) and Ca(2+) ions about as well as UK-1 . The non-cytotoxic benzimidazole UK-1 analogue binds Mg(2+) ions 50-fold weaker than UK-1, whereas the simple benzoxazole analogue binds Mg(2+) ions nearly as well as UK-1 . These results support a role of Mg(2+) ion binding in the selective cytotoxicity of UK-1 and provide a minimal pharmacophore for the selective cytotoxic activity of the natural product.

Bull Acad Natl Med, 2002, 186(3), 635 - 45; discussion 645-8
{Epidemiology of nosocomial infections after cataract surgery and role of the Infection Control Committee in prevention}; Marty N et al.; Despite of a low incidence (3 for 1,000), post-cataract surgery endophthalmitis remains a serious complication with a poor prognosis . The nosocomial definition is almost always present despite the endogenous origin; the latter is associated with the risk prone operative procedure, and the presence of numerous normal flora on skin and conjunctiva . Within this context, the incriminated bacteria are coagulase-negative Staphylococcus . Micro-outbreaks of postoperative Pseudomonas aeruginosa endophthalmitis have an exogenous origin . In order to prevent these nosocomial infections, the role of the "infection control committee" and the operational hygiene team is very important: 1) survey of new infectious cases and of the hospital environment; 2) infection control measures about pre and intra operative preparation of the patient; 3) operating room maintenance.

Mol Microbiol, 2002 Nov, 46(3), 889 - 901
On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition; Mao W et al.; Tripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds . To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD-OprJ pump from Pseudomonas aeruginosa . These mutations enabled the pump to extrude the normally non-transported beta-lactam antibiotic carbenicillin . All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD . Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8 . These mutations also had a substantial impact on the MexCD-OprJ-mediated transport of numerous other substrates . Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD-OprJ susceptible to inhibition by a thiol-reactive agent, MIANS . Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu-Nap) substrates of the pump . Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates . These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.

Mol Microbiol, 2002 Nov, 46(3), 677 - 86
A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa; Maseda H et al.; The nfxC-type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF-OprN xenobiotic transporter . The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy-dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively . The nfxC-type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS-polyacrylamide gel electrophoretogram . The mutant cells lacking MexF produced undetectable OprN and only a full-size of MexE even though the cells had unimpaired oprN and mexE . Expression of the plasmid-borne MexF in this mutant fully restored OprN and three MexE bands . Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN . To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl-terminal-truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE . The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C-terminal truncated MexE is essential for stabilization and the proper assembly of OprN . Nucleotide sequencing of mutant mexFs, which produce a wild-type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1-2 and 7-8 . Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.

Arch Soc Esp Oftalmol, 2002 Nov, 77(11), 589 - 95
{Diffuse lamellar keratitis: prophylactic treatment with ketorolac tromethamine 0.5% in an animal model}; Sandoval HP et al.; PURPOSE: The objective of this study is to evaluate the use of a topical non-steroidal anti-inflammatory drug in the treatment of induced diffuse lamellar keratitis (DLK) in an animal model . MATERIALS AND METHODS: A corneal flap was created in 40 eyes of 20 Dutch-belted rabbits using the ASC microkeratome . The interface was inoculated with either Pseudomonas Aeruginosa Lipopolysaccharide (LPS) endotoxin or Ultra Palmolive liquid dish washer . The rabbits were divided in two groups: Group I (n=20) treated with ketorolac tromethamine ophthalmic solution 0.5% 4 times a day and the group II (n=20) used as control . The rabbits were examined at the slit lamp at day 1, 3, 5 and 7 postoperatively . DLK was graded from I-IV . RESULTS: At the end of the study 31 eyes were available for evaluation . 28 eyes (90%) developed DLK: 86% of the treated group and 94% of the control group during the follow-up . The treated group showed a lower rate of DLK as well as a lower severity . However, no statistically significant difference was found when comparing both groups (P>0.05) . CONCLUSION: Pseudomonas aeruginosa LPS endotoxin and Palmolive Ultra can induce DLK in rabbit eyes . The postoperative prophylactic treatment with a topical non-steroidal anti-inflammatory drug showed a tendency towards a lower DLK rate as well as the severity of the disease.

Am J Infect Control, 2002 Nov, 30(7), 425 - 9
Pseudomonas aeruginosa infections in a neonatal intensive care unit; Zafar AB et al.; This report describes a cluster of nosocomial infections with Pseudomonas aeruginosa in a neonatal intensive care nursery . All 5 cases of P aeruginosa infection were clustered in September 1999 . Aggressive infection control measures were instituted, including installation of a user-friendly handwashing soap and environmental cleaning . On the basis of the finding of persistent dirty equipment, a new full-time position was created that was dedicated to equipment cleaning . These measures were effective in eliminating the cluster . The nursery has remained free of P aeruginosa infection for more than 2 years, attesting to the success of our program.

Am J Infect Control, 2002 Nov, 30(7), 411 - 6
Analysis of antimicrobial resistance among gram-negative bacilli and antimicrobial use in intensive care unit patients for 5 years in a Veterans Affairs medical center; Gentry C et al.; BACKGROUND: Gram-negative bacilli antimicrobial resistance remains a significant problem for patients in the intensive care unit (ICU) . Patterns of antimicrobial use may be a contributing factor . METHODS: Gram-negative bacilli from ICU patients of a Veterans Affairs (VA) medical center were tested to determine in vitro antimicrobial susceptibility (205 isolates in 1995 and 209 in 1999) . Antimicrobial use was calculated from 1995 through 1999 . RESULTS: For Pseudomonas aeruginosa, significant declines in susceptibility to ciprofloxacin (medical ICU {MICU} individually and all units) and aztreonam (all units) were noted . For gram-negative bacilli that was non-P aeruginosa, significant increases in susceptibility to ceftazidime (MICU, surgical ICU, and all units), gentamicin (all units), and ticarcillin/clavulanate (MICU) were noted . The most notable trends in antimicrobial usage were sharp increases in fluoroquinolone use in the MICU and surgical ICU and substantial decreases in the use of third-generation cephalosporins, monobactams, and aminoglycosides . In each instance of significant change in the susceptibility of a group of organisms to an antibiotic, there was a corresponding inverse change in the use of the antibiotic and/or its antimicrobial category (except for aztreonam) . CONCLUSIONS: Significant changes in antimicrobial use may affect certain gram-negative bacilli antimicrobial susceptibilities in ICUs.

Pediatr Res, 2002 Nov, 52(5), 636 - 44
Apoptosis induced by Pseudomonas aeruginosa in antigen presenting cells is diminished by genetic modification with CD40 ligand; Worgall S et al.; Persistent colonization with Pseudomonas aeruginosa (PA) is a hallmark of the lung disease associated with cystic fibrosis (CF) . Based on the concept that PA is not cleared from the lung by the host response in individuals with CF, we analyzed the capacity of PA to induce cell death in human alveolar macrophages (AM) and murine dendritic cells (DC), antigen presenting cells that play a central role in the initiation of pulmonary host defenses against pathogens, and evaluated if genetic modification can lead to protection against PA induced cell death . AM and DC were susceptible to cell death induced by the laboratory PA isolates PAO1, PAK and PA103, as well as a mucoid derivative of PAO1 and PA isolates derived from sputum of individuals with CF . Apoptosis, analyzed by TUNEL assay, was detectable in AM and DC as early as 3 h after infection with PA . In contrast, the same strains and doses of PA had little effect on the lung epithelial cell line A549 and primary cultures of human bronchial epithelial cells in vitro . Pretreatment of DC with the caspase inhibitors VAD-fmk and YVAD-cmk reduced PA induced cell death (p < 0.05) . Finally, genetic modification of DC to express CD40L using an adenovirus vector decreased the susceptibility of DC to cell death induced by PAO1 compared with DC infected with a control Ad vector (p < 0.01) . The data demonstrate that DC and AM are susceptible to apoptosis induced by PA and that this response can be partially reversed by genetic modification with CD40L, a CD4+ T cell molecule that plays a central role in activating antigen presenting cells . These observations suggest a potential mechanism contributing to the persistence of PA in CF and suggest that genetic manipulation of antigen presenting cells with anti-apoptotic genes may be able to strengthen host defenses in CF.

Nucleic Acids Res, 2002 Nov 1, 30(21), 4700 - 8
Vanadate inhibits the ATPase activity and DNA binding capability of bacterial MutS . A structural model for the vanadate-MutS interaction at the Walker A motif; Pezza RJ et al.; MutS, a member of the ABC ATPases superfamily, is a mismatch DNA-binding protein constituent of the DNA post-replicative mismatch repair system (MMRS) . In this work, it is shown that the ATPase activity of Pseudomonas aeruginosa and Escherichia coli MutS is inhibited by ortho- and decavanadate . Structural comparison of the region involved in the ATP binding of E.coli MutS with the corresponding region of other ABC ATPases inhibited by vanadate, including the myosin- orthovanadate-Mg complex, showed that they are highly similar . From these results it is proposed that the orthovanadate inhibition of MutS ATPase can take place by a similar mechanism to that described for other ATPases . Docking of decavanadate on the ATP-binding region of MutS showed that the energetically more favorable interaction of this compound would take place with the complex MutS- ADP-Mg, suggesting that the inhibitory effect could be produced by a steric impediment of the protein ATP/ADP exchange . Besides the effect observed on the ATPase activity, vanadate also affects the DNA-binding capability of the protein, and partially inhibits the oligomerization of MutS and the temperature-induced inactivation of the protein . From the results obtained, and considering that vanadate is an intracellular trace component, this compound could be considered as a new modulator of the MMRS.

J Clin Microbiol, 2002 Nov, 40(11), 4388 - 90
Simple microdilution test for detection of metallo-beta-lactamase production in Pseudomonas aeruginosa; Migliavacca R et al.; A microdilution test measuring imipenem MICs in the presence or absence of a mixture of EDTA plus 1,10-phenanthroline was developed and tested on 190 Pseudomonas aeruginosa isolates, including 18 VIM- and 4 IMP-type metallo-beta-lactamase (MBL) producers . The chelator mixture reduced by fourfold or more the imipenem MICs for MBL producers, while a lower effect or no effect was usually observed with MBL nonproducers.

Plant Cell Physiol, 2002 Oct, 43(10), 1127 - 36
The cyanobacterial PilT protein responsible for cell motility and transformation hydrolyzes ATP; Okamoto S et al.; The unicellular cyanobacterium, Synechocystis sp . PCC 6803 is motile . A homologue of the PilT protein family, required for twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was found to be necessarily associated with cyanobacterial motility . The pilT1 (slr0161) mutant shows a pleotropic phenotype, defects in individual cell motility, and an increased number of long surface pili . Furthermore, the mutant loses its ability of natural competency . These findings demonstrate that PilT1 is essential for both cell motility and competency . Since the pilT gene contains a consensus ATP-binding motif (Walker boxes), the PilT protein is suggested for supplying energy for cell motility . The product of pilT1, overproduced in Escherichia coli and purified by Ni-affinity chromatography, hydrolyzes ATP in vitro.

Am J Respir Crit Care Med, 2003 Feb 1, 167(3), 390 - 4 Epub 2002 Aug 15.
Endothelial nitric oxide synthase variants in cystic fibrosis lung disease; Grasemann H et al.; Variants in the genes encoding for the nitric oxide synthases may act as disease modifier loci in cystic fibrosis, affecting both an individual's nitric oxide level and pulmonary function . In this study, the 894G/T variant in exon 7 of the endothelial nitric oxide synthase gene was related to exhaled nitric oxide and pulmonary function in 70 cystic fibrosis patients who were aged 14.8 +/- 6.9 years (mean +/- SD), with a FEV1 of 69.4 +/- 24.8% predicted . Although there was no association between endothelial nitric oxide synthase genotypes and exhaled nitric oxide in males, nitric oxide levels were significantly higher in female cystic fibrosis patients with an 894T mutant allele, compared with female patients homozygous for the 894G wild-type allele (7.0 +/- 4.4 versus 3.6 +/- 1.9 parts per billion, p = 0.02) . Furthermore, in female patients, colonization of airways with Pseudomonas aeruginosa was significantly (p < 0.05) less frequent when carrying an 894T mutant allele as compared with wild type . These data suggest that the 894T variant in the endothelial nitric oxide synthase gene is associated with increased airway nitric oxide formation in female cystic fibrosis patients, possibly affecting colonization of airways with P . aeruginosa.

Mol Microbiol, 2002 Oct, 46(2), 519 - 30
Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity; DiGiandomenico A et al.; The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin . Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin . Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin . Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P . aeruginosa PA103 (LPS serotype O11) in P . aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens . The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry . Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens . A role for pilO in the glycosylation of pilin in P . aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P . aeruginosa strains . Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin . These results show that the pilin glycan of P . aeruginosa 1244 is a product of the O-antigen biosynthetic pathway . In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.

Am J Respir Crit Care Med, 2002 Nov 1, 166(9), 1248 - 56
Inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis; Aldallal N et al.; The concept that inflammatory gene expression is dysregulated in airway epithelial cells from patients with cystic fibrosis (CF) is controversial . To examine this possibility systematically, responses to inflammatory stimuli were compared in CF airway epithelial cell lines without versus with wild-type CF transmembrane conductance regulator (CFTR) complementation and in tracheobronchial epithelial cells from patients with versus without CF . Epithelial cell expression of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) and release of the neutrophil chemoattractant interleukin (IL)-8 were determined under basal conditions or after exposure to stimuli important in CF airway inflammatory responses . We found that uncorrected CF airway epithelial cell lines inconsistently expressed higher ICAM-1 and IL-8 levels . Human CF tracheobronchial epithelial cells in primary culture released moderately increased IL-8 only after exposure to Pseudomonas aeruginosa . In CF cells with higher IL-8 release, transient expression of wild-type CFTR using an adenoviral vector did not specifically affect cytokine levels . The results indicate that there is considerable variability in airway epithelial cell responses to inflammatory stimuli among different individuals and cell models systems . Although increased ICAM-1 and IL-8 expression are observed in some CF airway epithelial cell models, many CF cells do not exhibit significant dysregulation of these important inflammatory genes.

J Med Assoc Thai, 2002 Aug, 85 Suppl 2, S564 - 8
Outcome of pediatric oncologic patients in the respiratory intensive care unit: Siriraj Hospital; Susiva C et al.; OBJECTIVES: To review the characteristics and outcome of patients with childhood malignancy requiring respiratory intensive care treatment and to assess the outcome of these patients . DESIGN: Retrospective review of 22 oncological patients admitted to the pediatric respiratory intensive care unit between January 1, 1996 and December 31, 1998 (total 3 years.) RESULTS: The overall survival at discharge from the intensive care unit was 10 out of 22 (45%) . The mean age of the patients was 4 years 5 months old (range 1 month to 14 years) . Male:Female ratio was 1.2:1.21 patients had fever . All patients with a systemic or respiratory infective illness were neutropenic with a positive hemoculture in 17 out of 21 (81%) and 10 out of 20 (50%), respectively . The most common organisms detected were coagulase negative Staphylococcal aureus and Escherichia Coli . Sputum culture in the respiratory failure group was positive in 3 out of 7 patients, all of them grew Pseudomonas aeruginosa . Antibiotics were given to all oncological patients presenting with fever . The most common antibiotics administered were Ceftazidime, Amikacin and Imipenem . Fourteen patients needed mechanical ventilation . 11 of these 14 patients had respiratory tract infections, 1 patient had acute respiratory distress syndrome and the remainder were in a coma as a result of brain metastasis . Only 2 of them survived . The mean duration of stay in the respiratory intensive care unit was 10.9 days . CONCLUSIONS: There has been an improvement in the survival of oncology patients admitted to the intensive care unit especially for those with either a systemic or respiratory infection . Early and full intensive care treatment should be provided for these patients in order to improve the outcome.

Am J Respir Cell Mol Biol, 2002 Nov, 27(5), 561 - 7
Bacterial stimulation of epithelial G-CSF and GM-CSF expression promotes PMN survival in CF airways; Saba S et al.; Airway epithelial cells provide an immediate response to bacterial pathogens by producing chemokines and cytokines that recruit polymorphonuclear leukocytes (PMNs) to the site of infection . This response is excessive in patients with cystic fibrosis (CF) who have bacterial contamination of their airways . We postulated that CF airway pathogens, in activating nuclear factor-kappaB-dependent gene transcription in epithelial cells, would promote expression of cytokines that inhibit constitutive apoptosis of recruited PMNs . Epithelial cell culture supernatants from CF (IB-3) and corrected (C-38) epithelial cells stimulated by Staphylococcus aureus or Pseudomonas aeruginosa, increased survival of PMNs by 2- to 5-fold . Enhanced PMN survival was attributed to effects of epithelial granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression, which inhibit PMN apoptosis, and was negated by neutralizing antibody to either cytokine . Both CF and normal cells responded to bacteria with increased cytokine production . Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression were activated by ligation of asialoGM1, a receptor for P . aeruginosa and S . aureus, and by S . aureus lipoteichoic acid . Lipopolysaccharide was not a potent stimulus of cytokine expression, and P . aeruginosa algC (lipopolysaccharide) and lasR (quorum sensing) mutants were fully capable of activating epithelial cells . Induced expression of cytokines by airway cells repeatedly exposed to bacteria, as occurs in CF, serves not only to recruit and activate PMNs, but also to enhance their survival.

Crit Care Med, 2002 Oct, 30(10), 2222 - 8
Analysis of transmission pathways of Pseudomonas aeruginosa between patients and tap water outlets; Reuter S et al.; OBJECTIVE: To study the association between infection and faucet contamination in a surgical intensive care unit (SICU) . DESIGN: Prospective cohort study . SETTING: One SICU and 12 peripheral wards . PATIENTS: From 45 patients colonized or infected with P . aeruginosa, 87 positive isolates were collected . INTERVENTIONS: P . aeruginosa also was found in 150 of 259 (58%) tap water samples taken from patient rooms . MEASUREMENTS AND MAIN RESULTS: Clonal relationships between patient and tap water isolates were established by random amplification of polymorphic DNA-polymerase chain reaction . A long-time contamination (144 wks) with a single specific genotype for each of the faucets in our SICU was observed . Additional genotypes found in tap water from these faucets were only isolated over short periods of time . P . aeruginosa was shown to reside in single faucets and did not originate from the supplying mains . In 15 of 45 patients (33%), P . aeruginosa genotypes were identical to those from the faucets in the patient rooms . In six other patients, the same genotype was found in faucets from neighboring rooms . Faucets served as the source of infection for patients in 35% of cases, and on the other hand a retrograde contamination of faucets by patients was observed in 15% of cases . CONCLUSIONS: Tap water from faucets contaminated with P . aeruginosa plays an important role in the propagation of this pathogen among patients . A high number of transmissions were shown to occur both from faucet to patient and from patient to faucet . Our SICU served as an epicenter for the spread of P . aeruginosa to peripheral wards . It appears prudent to follow strict hygienic precautions such as wearing gloves and performing thorough alcoholic rub disinfection of hands after patient care and after hand washing at locations known to harbor.

Blood, 2002 Dec 15, 100(13), 4660 - 7 Epub 2002 Aug 08.
Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation; BitMansour A et al.; Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections . In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa . Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP . Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen . A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival . Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase . Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection . These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

Int J Pediatr Otorhinolaryngol, 2002 Nov 11, 66(2), 167 - 73
Quantity of aerobic bacteria in the bony portion of the external auditory canal of children; Stenfors LE et al.; OBJECTIVE: the human external auditory canal (EAC) hosts a commensal bacterial flora of mainly non-pathogens, but bacterial pathogens may also be present . The latter are important in the aetiopathogenesis of external otitis and middle ear cholesteatoma with discharge . The purpose of this study was to quantify the bacteria normally harboured in the healthy EAC and on a well-defined region of the cavum conchae (CC) of children . METHODS: bacterial samples were collected from the CC and bony portion of the EAC of 32 children (18 boys, 14 girls; median age 5 years) . Prior to sampling, the EACs were either left untreated, anaesthetized with Bonain's solution, or washed with 70% alcoholic solution . The samples were incubated at 37 degrees C and evaluated regarding bacterial species and number . RESULTS: the predominant bacterial non-pathogens were coagulase-negative Staphylococci and coryneforms (diphtheroids) and pathogens Staphylococcus aureus and Pseudomonas aeruginosa . Total bacterial counts of the CC ranged between 2 x 10(3) and 4.6 x 10(4) CFU/cm(2) (median value 7 x 10(3); n = 14) . Total bacterial counts from the bony portion of the EAC ranged between 0 and 5.7 x 10(7) CFU/EAC (median value 8 x 10(3); n = 32) . Pre-treatment of the EACs with Bonain's solution containing the highly bactericidal substance phenol or with 70% alcoholic solution did not sterilize the EAC . CONCLUSIONS: small numbers of bacterial non-pathogens (and sometimes pathogens) are found in the EAC of children . Neither phenol nor 70% alcoholic solution can inhibit or eradicate all these microorganisms .

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 41 - 6
A bacterial cell to cell signal in the lungs of cystic fibrosis patients; Collier DN et al.; Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients . This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems . One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone . This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P . aeruginosa infection . Previous studies have implied that the intercellular signals of P . aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections . In this report, three types of samples from CF patients infected with P . aeruginosa were analyzed for the presence of PQS . Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P . aeruginosa infecting the lungs of CF patients.

Biosens Bioelectron, 2002 Dec, 17(11-12), 1051 - 7
Copper proteins immobilised on gold electrodes for (bio)analytical studies; Lisdat F et al.; Copper electrochemistry at modified gold electrodes was investigated with two different states of the metal ion: first bound in azurin from Pseudomonas aeruginosa and second introduced via metal ion uptake in metallothionein (MT) from rabbit liver . Azurin was immobilised on a mercaptosuccinic acid (MSA) layer self-assembled on gold . The redox behaviour in the adsorbed as well as in the covalently immobilised state was found to be quasi-reversible with a formal potential of +198 mV versus Ag/AgCl . The pH variation suggests an optimal pH range for efficient electrode communication in the neutral range . MT was fixed at electrochemically cleaned gold using the accessible cysteins of the protein . Copper was found to bind to the MT-modified gold electrode . The electrochemical behaviour of the bound copper was characterised in copper-free solution with a formal potential of +245 mV versus Ag/AgCl . Stability and potential use is discussed.

J Hosp Infect, 2002 Oct, 52(2), 136 - 40
Impact of antibiotic changes in empirical therapy on antimicrobial resistance in intensive care unit-acquired infections; Allegranzi B et al.; We conducted a one-year prospective study on intensive care unit (ICU)-acquired infections and antimicrobial resistance patterns in an 18-bed medical-surgical ICU of a tertiary-care university hospital . We divided the study into two six-month periods in order to evaluate the impact of antibiotic changes in empirical therapy on antimicrobial resistance profiles of the principal isolated micro-organisms . In the first period no changes were made to the previously applied empirical antibiotic protocol; at the end of this period we found high rates of methicillin resistance (MR) among staphylococci, 93% for Staphylococcus aureus (69 isolates) and 79% for coagulase-negative staphylococci (CNS) (48 isolates), and of multiple drug resistance for Pseudomonas aeruginosa (57 isolates), in particular 67% resistance to piperacillin/tazobactam (PIP/TZ) . We therefore decided to substitute PIP/TZ with imipenem in nosocomial pneumonia and with cefepime plus metronidazole in peritonitis . We also considered the previous use of amoxicillin/clavulanate (AM/CL) at admission in critically ill patients inadequate; we therefore advised that no antibiotics should be given unless fever developed and eventually to replace AM/CL with trimethoprim/sulfamethoxazole (TMP/SMX) . At the end of this intervention period, we observed a significant decrease of S . aureus MR (93 vs . 73%, P = 0.003) and of P . aeruginosa resistance to PIP/TZ (67 vs . 29%, P < 0.001) . A reduction in MR was also seen in CNS (79 vs . 64%, P = 0.09) . Other resistance patterns also improved among staphylococci; in contrast P . aeruginosa resistance to imipenem increased in the second period (24 vs . 41%, P = 0.06) . A non-premeditated change of antibiotics in empirical therapy, on the basis of detected resistance patterns, provided promising results in reducing some antimicrobial resistance rates . We believe, however, that antibiotic changes must be tailored to local microbiological situation monitoring, and that a repeated rotation is crucial to limit the emergence of new resistance profiles . Furthermore the adoption of this policy should be accompanied by other infection control practices aimed at reducing antimicrobial resistance and nosocomial infection rates .

J Hosp Infect, 2002 Oct, 52(2), 93 - 8
Pseudomonas aeruginosa outbreak in a haematology-oncology unit associated with contaminated surface cleaning equipment; Engelhart S et al.; An outbreak of six cases of hospital-acquired Pseudomonas aeruginosa infections (two pneumonia two septicaemia, two skin/wound infection) occurred between August and September 2000 in an adult haematology-oncology unit at a tertiary-care centre . During the outbreak, hospital-acquired infection (HAI) incidence density rates rose from 29.4 to 62.3 (P < 0.05) infections per 1000 days at risk (i.e., neutropenic days) . A systematic outbreak management system was actioned in accordance with a German draft guideline . Multiple samples from the patients' environment were tested for the presence of P . aeruginosa . A total of 4.5% of samples from sanitary equipment and 20.0% of samples from surface cleaning equipment were found to be contaminated with P . aeruginosa . Genotypic analysis by pulsed-field gel electrophoresis showed different patterns for all (N = 6) of the patient isolates, however, two of the patient isolates were identical in comparison with environmental isolates from cleaning equipment (four samples) and sanitary equipment (one sample) . Our investigation revealed that the cleaning staff had used cleaning solution instead of disinfectants for decontamination of the patients' environment . The outbreak was terminated after re-adoption of surface disinfection, application of sterile filters on taps and shower heads, chemical disinfection of the washbasin drains, and appointment of a hospital hygiene nurse to a previously unfilled position . After institution of the control measures, HAI incidence densities decreased to pre-outbreak level . This investigation emphasizes the need to carefully evaluate cleaning and disinfection practices for patient care, particularly in neutropenic patients .

Biochem Pharmacol, 2002 Nov 1, 64(9), 1407 - 13
Enhanced activity of liposomal polymyxin B against Pseudomonas aeruginosa in a rat model of lung infection; Omri A et al.; The bactericidal effectiveness of liposomal polymyxin B against Pseudomonas aeruginosa was investigated in an animal model of pulmonary infection . Polymyxin B was incorporated into liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) (2:1) . Lung infection was induced in rats following intratracheal instillation of 10(7) colony-forming units (CFU) of P . aeruginosa (ATCC 27853) embedded in agar beads . Starting on day 3 post-infection, animals were treated daily, for 3 consecutive days, with saline, empty liposomes, free polymyxin B, or liposomal polymyxin B (2mg polymyxin B/kg body weight) by intratracheal instillation; animals were killed 24hr after the third drug instillation . Treatment of infected animals with liposomal polymyxin B significantly reduced the pulmonary bacterial counts (3.7+/-0.4log CFU/paired lungs) as compared with that of free polymyxin B (5.1+/-0.2log CFU/paired lungs) . Treatment of infected animals with empty liposomes gave pulmonary bacterial counts similar to those obtained from the saline-treated group . Pulmonary infection with P . aeruginosa also resulted in lung injury as evidenced by increases in wet lung weight and decreases in angiotensin converting enzyme activity as well as increases in myeloperoxidase activity, an index of the inflammatory response . Treatment with free polymyxin B ameliorated the lung injuries induced by the microorganism, a protective effect that was more pronounced in the liposomal polymyxin B-treated group . The levels of polymyxin B in the lungs of the infected animals treated with the liposomal suspension were significantly higher (42.8+/-6.2 microg/paired lungs) compared with those treated with the free drug (8.2+/-0.4 microg/paired lungs) . These data suggest that direct delivery of liposomal polymyxin B to the lung can be effective in the treatment of pulmonary infection with P . aeruginosa by enhancing retention of the antibiotic in the lung.

Am J Physiol Lung Cell Mol Physiol, 2003 Feb, 284(2), L307 - 15 Epub 2002 Sep 27.
Signaling intermediates required for NF-kappa B activation and IL-8 expression in CF bronchial epithelial cells; Li J et al.; Ligation of the asialoGM1 Pseudomonas aeruginosa pilin receptor has been demonstrated to induce IL-8 expression in airway epithelial cells via an NF-kappaB-dependent pathway . We examined the signaling pathways required for asialoGM1-mediated NF-kappaB activation in IB3 cells, a human bronchial epithelial cell line derived from a cystic fibrosis (CF) patient, and C-38 cells, the rescued cell line that expresses a functional CF transmembrane regulator . Ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells . AsialoGM1-mediated activation of NF-kappaB, IkappaB kinase (IKK), and ERK was also greater in IB3 cells . With the use of genetic inhibitors, we found that IKK-beta and NF-kappaB-inducing kinase are required for maximal NF-kappaB transactivation and transcription from the IL-8 promoter . Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no effect on IKK or NF-kappaB activation, suggesting that ERK regulates IL-8 expression in an NF-kappaB-independent manner.

Glycoconj J, 2001 Sep, 18(9), 709 - 13
Recognition of mucin components by Pseudomonas aeruginosa; Ramphal R et al.; Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis . This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs . The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms . While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g . increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF . The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins . Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens . There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known . However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.

Glycoconj J, 2001 Sep, 18(9), 699 - 708
FAB-MS characterization of sialyl Lewis x determinants on polylactosamine chains of human airway mucins secreted by patients suffering from cystic fibrosis or chronic bronchitis; Morelle W et al.; Although a large body of structural data exists for bronchial mucins from cystic fibrosis (CF) and chronic bronchitis (CB) patients, little is known about terminal structures carried on poly-N-acetyllactosamine antennae . Such structures are of interest because they are potential ligands for bacterial adhesins and other lectins . In this study, we have used fast atom bombardment mass spectrometry (FAB-MS) to examine terminal sequences released by endo-beta-galactosidase from O-glycans obtained by reductive elimination of bronchial mucins purified from the sputum of 8 CF and 8 CB patients . Our data show that, although the polylactosamine antennae of CF and CB mucins have several terminal sequences in common, they differ significantly in their sialyl Lewis(x) (NeuAcalpha2-3Galbeta1-4{Fucalpha1-3}GlcNAcbeta1-) content . Thus all examined mucins from CF patients carry sialyl Lewis(x) on their polylactosamine antennae, whereas this type of epitope is present on only three out of the eight CB mucins examined, notably in the airways of one CB patient which were heavily infected by Pseudomonas aeruginosa as are the airways of all the CF patients . This suggests that, in airway mucins, the expression of sialyl Lewis(x) on polylactosamine antennae is probably more related to inflammation and infection than to a direct effect of the CF defect.

Glycoconj J, 2001 Sep, 18(9), 661 - 84
Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis; Lamblin G et al.; Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity . Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7) . The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines . Human airway mucins are highly glycosylated (70-80% per weight) . They contain from one single to several hundred carbohydrate chains . The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules . Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars) . The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases . The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin . There is growing evidence that the apomucin sequences influence the first glycosylation reactions . The elongation of the chains leads to various linear or branched extensions . Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants . The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases . Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa . Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease . However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected . Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection . These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNFalpha . In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.

Urology . 2002 Oct;60(4):698.
Pseudomonas aeruginosa sacroiliitis and osteomyelitis of pelvic bones after radical prostatectomy; Andonian S et al.; A 66-year-old diabetic man presented with acute incapacitating pelvic pain 6 weeks after radical prostatectomy . Symphysis pubis biopsy showed chronic osteomyelitis, and culture grew Pseudomonas aeruginosa . Despite a 7-week course of intravenous piperacillin and ceftazidime, he returned 6 months later with the same symptoms . Imaging studies and biopsy indicated right sacroiliitis and persistent pseudomonas osteomyelitis of the pelvic bones . He refused surgical debridement and was successfully treated with the same antibiotics for 8 more weeks . We emphasize the importance of bone biopsy and culture to expedite effective intravenous antibiotic therapy.

Fitoterapia, 2002 Oct, 73(6), 536 - 9
Antimicrobial activity of licorice flavonoids against methicillin-resistant Staphylococcus aureus; Fukai T et al.; Nineteen flavonoids isolated from licorice (Glycyrrhiza glabra, G . inflata and G . uralensis) were tested for their antimicrobial activities against methicillin sensitive Staphylococcus aureus, methicillin resistant S . aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa .

Fitoterapia, 2002 Oct, 73(6), 529 - 31
Antibacterial activity of Borreria verticillata roots; de Sa Peixoto Neto PA et al.; Borreria verticillata roots methanolic extract exhibited a broad antibacterial activity against multiresistant strains of Pseudomonas aeruginosa .

Microb Pathog, 2002 Oct, 33(4), 153 - 66
Type III secretion-mediated killing of endothelial cells by Pseudomonas aeruginosa; Saliba AM et al.; Pseudomonas aeruginosa, a common agent of septicemia, enters into human endothelial cellsin vitro but the effects of bacterial infection have not been addressed properly . In this study, human umbilical vein endothelial cells (HUVEC) were infected by the noninvasive PA103 and the invasive PAO1 P . aeruginosa strains and the viability of infected cells was assessed by the methyltiazole tetrazolium (MTT) assay . Both strains were cytotoxic within 3h of infection . To ascertain the role of proteins secreted by the type III secretion system (TTSS) in HUVEC killing, defective mutants of PAO1 and PA103 were constructed by plasmid insertion in exsA or pscC genes . ExsA is a transcriptional regulator that controls the expression of most TTSS related genes whereas pscC encodes a protein from the secretion machinery . Parental bacteria were significantly more cytotoxic to HUVEC than the mutants . Inactivation ofexsA reverted the inability of PA103 to enter into HUVEC but did not modify the invasiveness of PAO1 . Cytofluorometric analysis of infected HUVEC labeled by DiOC(6)(3) showed that cell killing was associated with mitochondrial depolarization, an early event reported in apoptosis . However, infected cells did not show ultrastructural or DNA fragmentation features of apoptosis . Our results suggest that TTSS effectors mediate P . aeruginosa killing of HUVEC by a mechanism distinct from apoptosis.

Int J Antimicrob Agents, 2002 Oct, 20(4), 289 - 92
Re-evaluations of disk diffusion quality control ranges for 11 drugs: report from the QC working group of the national committee for clinical laboratory standards; Jones RN et al.; A multi-laboratory re-evaluation protocol was initiated in 2001 to address broad-based concerns about the appropriateness of contemporary disk diffusion quality control (QC) guidelines of several antimicrobials in the tables of the M2-A7 standard of the National Committee for Clinical Laboratory Standards (NCCLS) . This study that conformed to the NCCLS M23-A2 guideline demonstrates the dynamic processes that result in modification or development of these 11 QC zone diameter ranges . These changes included long-term QC problems for cefazolin (new range, 21-27 mm) and trimethoprim/sulphamethoxazole (new range, 23-29 mm) when tested against Escherichia coli ATCC 25922, and ticarcillin (new range, 21-27 mm) versus Pseudomonas aeruginosa ATCC 27853 . All proposed ranges have been approved by the NCCLS for publication in M100-S12 tables in 2002.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3624 - 6
Clinical strain of Pseudomonas aeruginosa carrying a bla(TEM-21) gene located on a chromosomal interrupted TnA type transposon; Dubois V et al.; A clinical isolate of Pseudomonas aeruginosa was found to produce a clavulanic acid-inhibited extended-spectrum beta-lactamase with a pI of 6.4 . PCR, cloning, and sequencing experiments showed that the corresponding bla(TEM-21) gene was part of a chromosomally located Tn801 transposon disrupted by an IS6100 element and adjacent to an aac(3)-II gene.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3574 - 9
Determination of antibiotic effect in an in vitro pharmacodynamic model: comparison with an established animal model of infection; Bonapace CR et al.; Animal infection models have historically been used to study pharmacodynamic relationships . Similar results could theoretically be produced by using an in vitro pharmacodynamic model as an alternative to animal models . We compared the antibiotic effects of ticarcillin administered in various doses and dosing regimens against Pseudomonas aeruginosa ATCC 27853 under conditions analogous to those previously employed in a neutropenic-mouse thigh infection model (B . Vogelman et al., J . Infect . Dis . 158:831-847, 1988) . Ticarcillin dosages of either 96, 192, or 384 mg/day were administered at 1-, 2-, 3-, 4-, 8-, 12-, or 24-h intervals into a two-compartment model in order to duplicate the concentration-time profiles of the animal model . Colony counts were enumerated at 0 and 24 h . Linear regression and sigmoidal maximum-effect (Emax) model fitting were used to assess the relationship between the percentage of time that the concentration remained above the MIC (%T>MIC) or above four times the MIC (%T>4xMIC) and the change in the log(10) CFU per milliliter (Deltalog(10) CFU/ml) in the central and peripheral compartments . Statistical analysis of the Deltalog(10) CFU/ml values was performed for matched regimens of the in vitro and animal models based on the %T>MICs . The slopes of the regression equations of %T>MICs relative to Deltalog(10) CFU/ml values were similar for the in vitro and animal models, but the y intercept was greater with the in vitro model . The Deltalog(10) CFU/ml values of the 0- to 24-h colony counts at equivalent %T>MICs in the two models were not statistically different (P = 0.087) . Overall, the peripheral compartment of the in vitro model was a better predictor of effect than the central compartment . This study, which compares pharmacodynamic principles between an in vitro and an animal model, demonstrated similar relationships between %T>MICs and effects.

Eur J Biochem, 2002 Oct, 269(20), 4921 - 9
A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo; Henriksson ML et al.; 14-3-3 proteins play an important role in a multitude of signalling pathways . The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner . Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa . In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3 . Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3 . In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay . We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested . Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g . Ras, and shows a reduced capacity to change the morphology of infected cells.

J Biol Chem, 2002 Dec 27, 277(52), 50293 - 302 Epub 2002 Oct 14.
Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa; Finking R et al.; Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism) . For these proteins to become functionally active, they need to be primed with the 4'-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase) . Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase . Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism . We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS . Gene deletion experiments clearly show that PcpS is essential for growth of P . aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism . At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme . However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp . Thus, the situation in P . aeruginosa is not simply the result of the loss of an AcpS-type PPTase . PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form . This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases.

J Microbiol Immunol Infect, 2002 Sep, 35(3), 168 - 72
Nosocomial bloodstream infection in a neonatal intensive care unit of a medical center: a three-year review; Tseng YC et al.; Bloodstream infections are the most frequent nosocomial infections in neonatal intensive care units . This retrospective study surveyed the epidemiologic characteristics of nosocomial bloodstream infections which occurred in the neonatal intensive care unit from January 1, 1997 to December 31, 1999 . The overall infection patient rate was 5.5% in the 3-year period, and the overall infection patient-day rate was 4.4 per 1000 patient-days . Low birth weight was a risk factor for bloodstream infections . The rate of infection for neonates with birth weight below 1000 g ranged from 36.6% to 45.8% (1997: 36.6%; 1998: 45.8% and 1999: 38.9%) . The most common pathogens causing nosocomial bloodstream infection were: Staphylococcus aureus (18.5%) (with 92% oxacillin-resistant), Acinectobacter baumannii (16.3%), Klebsiella pneumoniae (11.9%), Escherichia coli (9.6%), and Pseudomonas aeruginosa (8.1%) . The mortality due to nosocomial bloodstream infection was highest among gram-negative bacteria, especially with P . aeruginosa (45.5%) . Therefore, surveillance of nosocomial bloodstream infection and successful strategies to decrease nosocomial bloodstream infection, such as infection control and optimal antibiotic use, are warranted.

Singapore Med J, 2002 Jun, 43(6), 296 - 9
Microbiology of chronic suppurative otitis media in Singapore; Loy AH et al.; The objective of the study was to study the microflora and the antibiograms of patients with chronic suppurative otitis media (CSOM) in Singapore . Ninety patients with CSOM were prospectively studied . They had chronic ear discharge and had not received antibiotics for the previous five days . Swabs were taken, and cultured for bacteria . Antibiotic testing was done using modified Kirby Bauer disk diffusion method . In addition to the usual antibiotics, the three most common topically available antibiotics (chloramphenicol, gentamicin and neomycin) were tested . There were 135 positive cultures for organisms from the 90 patients.The most common causal organisms isolated were Pseudomonas aeruginosa (33.3%) and Staphylococcus aureus (33.3%) followed by coagulase negative Staphylococcus (21.1%) . Fungi accounted for 8.8% of isolates while 6.6% were anaerobes . Of the three antibiotics commonly available as topical eardrops, gentamicin has the highest susceptibility rate (82.6%), followed by neomycin (67.8%) and chloramphenicol (62.8%).

Infect Immun, 2002 Nov, 70(11), 6399 - 408
Modulation of cytosolic Ca(2+) concentration in airway epithelial cells by Pseudomonas aeruginosa; Jacob T et al.; Modulation of cytosolic (intracellular) Ca(2+) concentration (Ca(i)) may be an important host response when airway epithelial cells are exposed to Pseudomonas aeruginosa . We measured Ca(i) in Calu-3 cells exposed from the apical or basolateral surface to cytotoxic and noncytotoxic strains of P . aeruginosa . Apical addition of either noncytotoxic strains or cytotoxic strains failed to affect Ca(i) over a 3-h time period, nor were changes observed after basolateral addition of noncytotoxic strains . In contrast, basolateral addition of cytotoxic strains caused a slow increase in Ca(i) from 100 nM to 200 to 400 nM . This increase began after 20 to 50 min and persisted for an additional 30 to 75 min, at which time the cells became nonviable . P . aeruginosa-induced increases in Ca(i) were blocked by the addition of the Ca channel blocker La(3+) to the basolateral but not to the apical chamber . Likewise, replacing the basolateral but not the apical medium with Ca-free solution prevented P . aeruginosa-mediated changes in Ca(i) . With isogenic mutants of PA103, we demonstrated that the type III secretion apparatus, the type III-secreted effector ExoU, and type IV pili were necessary for increased Ca(i) . We propose that translocation of ExoU through the basolateral surface of polarized airway epithelial cells via the type III secretion apparatus leads to release of Ca stored in the endoplasmic reticulum and activation of Ca channels in the basolateral membranes of epithelial cells.

Infect Immun, 2002 Nov, 70(11), 6172 - 9
Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin, Pseudomonas aeruginosa exotoxin A, and ricin; Teter K et al.; Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function . One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER) . These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER . Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway . We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation . Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin . Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism . Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of alpha1-antitrypsin (alpha1AT-Z) . Compared to the parental CHO cells, the rate of alpha1AT-Z degradation was decreased in two independent mutant cell lines . Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide . These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.

Infect Immun, 2002 Nov, 70(11), 6083 - 93
The transcriptional regulator AlgR is essential for Pseudomonas aeruginosa pathogenesis; Lizewski SE et al.; Chronic Pseudomonas aeruginosa lung infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients . One P . aeruginosa virulence factor unique to CF isolates is overproduction of alginate, phenotypically termed mucoidy . Mucoidy is the result of increased transcription from the algD gene and is activated by the transcriptional regulator AlgR . Mutations in algR result in a nonmucoid phenotype and loss of twitching motility . Additionally, AlgR controls transcription of algC, encoding a dual-function enzyme necessary for both lipopolysaccharide (LPS) and alginate production . Therefore, to determine the effect of algR on P . aeruginosa virulence, an algR mutant was examined for sensitivity to reactive oxygen intermediates, killing by phagocytes, systemic virulence, and the ability to maintain a murine lung infection . We found that P . aeruginosa PAO700 (algR::Gm(r)) was less lethal than PAO1, as tested in an acute septicemia infection mouse model, and was cleared more efficiently in a mouse pneumonia model . Additionally, the algR mutant (PAO700) was more sensitive to hypochlorite . However, PAO700 was more resistant to hydrogen peroxide and killed less readily in an acellular myeloperoxidase assay than PAO1 . There was little difference in killing between PAO1 and PAO700 with macrophage-like J774 cells and human polymorhonuclear leukocytes . Two-dimensional gel analysis of P . aeruginosa algR mutant and wild-type protein extracts revealed 47 differentially regulated proteins, suggesting that AlgR plays both a positive role and a negative role in gene expression . Together, these results imply that AlgR is necessary for virulence and regulates genes in addition to the genes associated with alginate and LPS production and pilus function.

Am J Respir Crit Care Med, 2002 Oct 15, 166(8), 1038 - 43
Impact of invasive strategy on management of antimicrobial treatment failure in institutionalized older people with severe pneumonia; El-Solh AA et al.; The aim of the study was to investigate the etiology and the impact of invasive quantitative sampling on the management of severe pneumonia in institutionalized older people with antimicrobial treatment failure . Fifty-two institutionalized patients aged 70 years and older hospitalized with a presumptive diagnosis of severe pneumonia and failure to respond to treatment after 72 hours of initiation of outpatient antimicrobial therapy were enrolled . Microbial investigation included blood culture, serology, pleural fluid, and bronchoalveolar samples . A definite etiology could be established in 24 of 52 (46%) patients . Methicillin-resistant Staphylococcus aureus (33%), enteric Gram-negative bacilli (24%), and Pseudomonas aeruginosa (14%) accounted for most isolates . Atypical infections (2%) were uncommon . Invasive bronchial sampling directed a change of microbial therapy in 8 (40%) and discontinuation of antibiotics in 2 of 20 cases (10%) of definite pneumonia . Overall hospital mortality was 42% . There was no difference in mortality among definite or unverified cases or those who had invasive bronchial sampling-guided change in therapy . We conclude that antimicrobial therapy should be targeted toward "nosocomial" pathogens in those institutionalized patients who received prior antibiotic treatment . When combined with microbial investigation, direct visualization of the tracheobronchial tree might be useful in determining the presence of bacterial pneumonia.

Jpn J Antibiot, 2002 Aug, 55(4), 440 - 5
{In vitro combination effect of pazufloxacin with various antibiotics against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus}; Maekawa M et al.; The in vitro combination effects of pazufloxacin (PZFX) with various antibiotics were investigated by the checkerboard dilution method using piperacillin (PIPC), tazobactam/piperacillin (TAZ/PIPC), ceftazidime (CAZ), cefozoprane (CZOP), imipenem/cilastatin (IPM/CS), meropenem (MEPM), panipenem/betamipron (PAPM/BP), amikacin (AMK) and isepamicin (ISP) for clinical isolates of 27 Pseudomonas aeruginosa strains, vancomycin (VCM), teicoplanin (TEIC) and arbekacin (ABK) for clinical isolates of methicillin-resistant 26 Staphylococcus aureus (MRSA) strains, respectively . The following results were obtained . 1 . For 27 P . aeruginosa strains, the synergistic effects were observed with the combination of PZFX and CAZ or MEPM (11.1%: 3 strains), and PZFX and CZOP or PAPM/BP (3.7%: 1 strain), respectively . The additive and synergistic effects of PZFX were observed with the combination in all beta-lactams tested in the strains more than 50% . No antagonistic effect was observed . The additive effects were also observed with the combination of PZFX and AMK or ISP in the strains more than 50% of the test strains and no antagonistic effect was observed . 2 . For 26 MRSA strains, no antagonistic effect was observed with the combination of all antibiotics tested . The indifference was observed with the combination of PZFX and VCM or ABK in the strains more than 60%, and the additive effects were observed with the combination of TEIC in the strains more than 80% . In conclusion, no antagonistic effect was observed in PZFX with the combination of beta-lactams and anti-MRSA agents, suggesting that the combination therapy of PZFX with these antibiotics would be possible to use for the infections caused by P . aeruginosa and MRSA.

Diagn Microbiol Infect Dis, 2002 Sep, 44(1), 35 - 41
Cefepime, piperacillin/tazobactam, gentamicin, ciprofloxacin, and levofloxacin alone and in combination against Pseudomonas aeruginosa; Burgess DS et al.; A beta-lactam plus an aminoglycoside is the standard for treating severe Pseudomonas aeruginosa infections . However, the fluoroquinolones are safer and have been widely used as an alternative to the aminoglycosides in this setting . In this study we compared the synergistic activities of piperacillin/tazobactam and cefepime when either drug was combined with gentamicin, ciprofloxacin, or levofloxacin against P . aeruginosa . Susceptibility testing and time-kill curves were performed against 12 clinical isolates of P . aeruginosa . All combinations were bactericidal and retained this activity over the 24 hr period except for piperacillin/tazobactam in combination with levofloxacin or ciprofloxacin against 2 isolates and cefepime in combination with levofloxacin against 1 isolate . None of the combinations were antagonistic . No statistical difference in the frequency of synergy exists between the beta-lactam plus gentamicin (79%) and the beta-lactams plus either ciprofloxacin or levofloxacin combinations (58%, 67%) . Furthermore, no differences in synergistic activity were noted between ciprofloxacin combinations (58%) and levofloxacin combinations (67%) . In conclusion, the degree of synergy between a beta-lactam plus aminoglycoside and a beta-lactam plus fluoroquinolone seem to be comparable . Furthermore, there is a similar rate of synergy among different fluoroquinolone-based combinations . However, faster killing, less regrowth, and decrease in the development of resistance were seen with the beta-lactam plus aminoglycoside combination.

Pneumologie, 2002 Oct, 56(10), 599 - 604
{Ciprofloxacin in the treatment of hospital-acquired pneumonia: a surveillance study in 676 patients}; Kljucar S et al.; BACKGROUND: Controlled clinical trials have shown efficacy of high-dose ciprofloxacin for hospital-acquired (HAP) or nosocomial pneumonia . But it has yet to be demonstrated whether this good efficacy also holds true for routine intensive-care patients outside of controlled trials . PATIENTS: In a post-marketing surveillance study at 87 intensive-care units in Germany we analyzed 676 cases of nosocomial pneumonia treated with intravenous ciprofloxacin in a daily dosage of at least 400 mg . RESULTS: 538 (80 %) patients were evaluable for efficacy . Cure or improvement was reported in 76 % of the cases . Clinical success rate was higher in previously untreated patients receiving ciprofloxacin as monotherapy (85.3 %) or in combination with other antibiotics (78.4 %) than in those who received ciprofloxacin as monotherapy (73.1 %) or as combination therapy (69.2 %) after an antibiotic pretreatment . In the 66 patients with Pseudomonas aeruginosa as causal pathogen, clinical success rate was 86.4 % . 32 adverse events classified as possibly or probably related to ciprofloxacin occurred in 3.1 % of patients; all of those were reversible . CONCLUSIONS: Due to the high success rate, even in cases with failed antimicrobial pretreatment, and the favourable risk-benefit ratio of high-dose ciprofloxacin, ciprofloxacin appears to be an attractive choice in the empiric treatment of hospital-acquired pneumoniaPublication Types:

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