Pseudomonas aeruginosa

Am J Respir Crit Care Med, 2004 Feb 15, 169(4), 459 - 67 Epub 2003 Dec 04.
Primary ciliary dyskinesia: diagnostic and phenotypic features; Noone PG et al.; Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function . We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD . Of 110 subjects evaluated, PCD was diagnosed in 78 subjects using a combination of compatible clinical features coupled with tests of ciliary ultrastructure and function . Chronic rhinitis/sinusitis (n = 78; 100%), recurrent otitis media (n = 74; 95%), neonatal respiratory symptoms (n = 57; 73%), and situs inversus (n = 43; 55%) are strong phenotypic markers of the disease . Mucoid Pseudomonas aeruginosa (n = 12; 15%) and nontuberculous mycobacteria (n = 8; 10%) were present in older (> 30 years) patients with PCD . All subjects had defects in ciliary structure, 66% in the outer dynein arm . Nasal nitric oxide production was very low in PCD (nl/minute; 19 +/- 17 vs . 376 +/- 124 in normal control subjects) . Rigorous clinical and ciliary phenotyping and measures of nasal nitric oxide are useful for the diagnosis of PCD . An increased awareness of the clinical presentation and diagnostic criteria for PCD will help lead to better diagnosis and care for this orphan disease.

Intern Med J, 2003 Dec, 33(12), 593 - 7
Adults with cystic fibrosis: meeting the challenge!
Dobbin CJ, Bye PT.
The number of adults with cystic fibrosis (CF) is increasing . They are striving for independence and a fulfilling life with focus on career, relationships, education and finances at a time when lung function is likely to be declining and complications of this multi-system disease are increasing . Maintaining the quality and improving the duration of life are continuing challenges for the -clinician and the patient . Increased hope and greater expectations have been provided by a number of recent clinical advances and active research into novel treatments, including gene therapy . There has been increased recognition of the necessity for early diagnosis, adequate monitoring and effective intervention for complications such as diabetes and osteoporosis . Research into multi-resistant bacteria and clonal strains of Pseudomonas aeruginosa is ongoing and attention has focused on infection control policies . Although more high-level evidence is required on many issues confronting people with CF, a considerable effort has been made over the last decade to provide a more evidence-based approach to therapy with a number of large controlled clinical trials . For the adult with CF, there are also more decisions to be made . There is focus on reproductive health, with most couples enjoying the real possibility of having children . For those with advanced disease, the option for lung transplantation is well established . Maintenance of quality care will require adequate planning, effective transition programmes from paediatric to adult care, specialized training for doctors, nurses and allied health professionals and the allocation of sufficient resources to accommodate the inevitable increase in patient numbers.

J Clin Laser Med Surg, 2003 Oct, 21(5), 283 - 90
Effects of low-level laser therapy (LLLT) of 810 nm upon in vitro growth of bacteria: relevance of irradiance and radiant exposure; Nussbaum EL et al.; OBJECTIVE: The aim of this study was to investigate the irradiance-dependency of low-level laser therapy (LLLT) effects on bacterial growth . BACKGROUND: LLLT is applied to open wounds to improve healing; however, its effect on wound bacteria is not well understood . MATERIALS AND METHODS: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were irradiated using a wavelength of 810 nm at irradiances of 0.015 W/cm2 (0-50 J/cm2) and 0.03 W/cm2 (0-80 J/cm2) . Bacteria were counted after 20 h of incubation . RESULTS: LLLT effects varied significantly with species . P.aeruginosa growth decreased overall dependent on an interaction of irradiance and radiant exposure; greatest inhibition was produced using high irradiance delivering radiant exposures in the range of 1-20 J/cm2 (p = 0.001-0.04) . In contrast, E . coli growth increased overall (p = 0.01), regardless of irradiance; greatest effects were produced using low radiant exposures (1-20 J/cm2) . There was a main effect for irradiance (p = 0.03) on S . aureus growth; however, growth was not different compared with controls . Additional analysis showed that there were differences in growth of P.aeruginosa when comparing samples that were matched by exposure times (66, 329, 658, 1316, 1974, and 2632 sec) rather than radiant exposure; this suggests that irradiance rather than exposure time was the significant factor in P . aeruginosa inhibition . CONCLUSION: These findings have immediate relevancy in the use of LLLT for infected wounds . Exposure to 810-nm irradiation (0.03 W/cm2) could potentially benefit wounds infected with P . aeruginosa . However, increased E . coli growth could further delay recovery.

Mol Microbiol, 2003 Dec, 50(5), 1477 - 91
Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication; Duan K et al.; The change in gene expression patterns in response to host environments is a prerequisite for bacterial infection . Bacterial diseases often occur as an outcome of the complex interactions between pathogens and the host . The indigenous, usually non-pathogenic microflora is a ubiquitous constituent of the host . In order to understand the interactions between pathogens and the resident microflora and how they affect the gene expression patterns of the pathogens and contribute to bacterial diseases, the interactions between pathogenic Pseudomonas aeruginosa and avirulent oropharyngeal flora (OF) strains isolated from sputum samples of cystic fibrosis (CF) patients were investigated . Animal experiments using a rat lung infection model indicate that the presence of OF bacteria enhanced lung damage caused by P . aeruginosa . Genome-wide transcriptional analysis with a lux reporter-based promoter library demonstrated that approximately 4% of genes in the genome responded to the presence of OF strains using an in vitro system . Characterization of a subset of the regulated genes indicates that they fall into seven functional classes, and large portions of the upregulated genes are genes important for P . aeruginosa pathogenesis . Autoinducer-2 (AI-2)-mediated quorum sensing, a proposed interspecies signalling system, accounted for some, but not all, of the gene regulation . A substantial amount of AI-2 was detected directly in sputum samples from CF patients and in cultures of most non-pseudomonad bacteria isolated from the sputa . Transcriptional profiling of a set of defined P . aeruginosa virulence factor promoters revealed that OF and exogenous AI-2 could upregulate overlapping subsets of these genes . These results suggest important contributions of the host microflora to P . aeruginosa infection by modulating gene expression via interspecies communications.

Pediatr Dermatol, 2003 Nov-Dec, 20(6), 529 - 30
Harlequin baby with ecthyma gangrenosum; Gunes T et al.; Pseudomonas aeruginosa bacteremia or sepsis often occurs in hospitals, affecting mainly children with underlying disease . Ecthyma gangrenosum is classically considered a pathognomonic sign of sepsis by P . aeruginosa . The harlequin baby, a severe variant of ichthyosis, occurs rarely, and these infants are at high risk of cutaneous infections and sepsis . We herein report a harlequin baby who developed ecthyma gangrenosum.

J Pediatr (Rio J), 2000 Jul-Aug, 76(4), 295 - 9
{Study of lung involvement in patients with cystic fibrosis}; Dornelas EC et al.; OBJECTIVE: To characterize the involvement of the respiratory apparatus of patients with cystic fibrosis in order to obtain a comprehensive view of their pulmonary picture.METHODS: Data were obtained retrospectively from the medical records of 16 patients with cystic fibrosis; arterial gas and spirometry data were obtained prospectively for the same patients, who were not in an acute pulmonary situation . The patients were subjects of both sexes aged 6 years or older who were followed up at the Pediatrics Outpatient Clinic of the University Hospital, Faculty of Medicine of Ribeirao Preto, USP.RESULTS: Median patient age was 114 months (9 years and 6 months) ranging between 72 - 360 months, and 68.75% were males . Productive cough was the most frequent symptom observed in 75% of the population studied . All patients had positive sputum culture obtained at least one year before, with Pseudomonas aeruginosa being detected in 81.25% of the cases . Arterial gases revealed some abnormalities in 81.25% of the patients and spirometry revealed abnormalities in 56.25%.CONCLUSION: All patients presented at least one type of pulmonary alteration . Measurement of arterial gases detected a larger number of patients with altered pulmonary function than did spirometry, but the two examinations complemented each other for a good evaluation of pulmonary function.

J Bacteriol, 2003 Dec, 185(24), 7297 - 300
Control of Pseudomonas aeruginosa algZ expression by the alternative sigma factor AlgT; Wozniak DJ et al.; AlgZ controls Pseudomonas aeruginosa alginate synthesis by activating algD, yet algZ expression is not detectable in nonmucoid strains . Mobility shift and Western blot assays revealed that algZ expression requires the sigma factor AlgT . The mapped algZ transcription start site revealed a consensus AlgT-dependent promoter that, when mutated, substantially reduced algZ transcription.

J Bacteriol, 2003 Dec, 185(24), 7129 - 39
Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa; Lamb JR et al.; The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C(4)-HSL), regulates gene expression in response to cell density . With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C(4)-HSL concentration . Studies with an E . coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator . Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C(4)-HSL binding . Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C(4)-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation . Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization . RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P . aeruginosa . We conclude that C(4)-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P . aeruginosa.

J Bacteriol, 2003 Dec, 185(24), 7068 - 76
FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa; Huang B et al.; Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili . Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P . aeruginosa genome sequence . Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility . fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired . The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism . Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain . Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin . These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

Biochimie, 2003 Oct, 85(10), 953 - 62
Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process; Durand A et al.; Rat kidney acylase I was characterised by performing site-directed mutagenesis and enzymatic analysis in the presence of various chemical inhibitors . Site-directed mutagenesis on E147 and overexpression of the protein in a bacterial system, revealed the importance of this residue in enzymatic activity, it corresponds to the putative catalytic E175 in carboxypeptidase G2 from Pseudomonas aeruginosa . The reactivity of histidine and cysteine residues of acylase I with diethylpyrocarbonate (DEPC) and mercuric chloride, respectively, showed that these two amino acids are required for the enzyme to be fully active . Interestingly, the effects of mercuric chloride on rat kidney acylase I were not as great as those on the porcine enzyme, in agreement with previously observed differences between the two enzymes . Moreover, N-{3-(2-furyl)-acryloyl-L-methionine} (FA-Met) a synthetic substrate of the porcine acylase I was found to be an inhibitor of the rat kidney enzyme . These results strongly suggest the existence of differences between the active site of rat and porcine kidney acylases I . Lastly, the rat kidney enzyme was as sensitive as its porcine counterpart to two metal chelating agents, 1,10-phenanthroline and ethylenediamine tetraacetate (EDTA).

FEBS Lett, 2003 Dec 4, 555(2), 297 - 301
Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa; Cioci G et al.; The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose and calcium was determined at 1.6 A resolution, and the native protein was solved at 2.4 A resolution . Each monomer adopts a beta-sandwich fold with ligand binding site at the apex . All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the co-ordination of the calcium ion . The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C-type lectins . The structure of the complex provides a framework for future design of anti-bacterial compounds.

Biomaterials, 2004 Mar-Apr, 25(7-8), 1195 - 204
The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines; West SL et al.; The comparison of copolymers containing sulfobetaine or phosphobetaine moieties for use as potential biocompatible coatings has been investigated . Two statistical copolymers were produced by a free radical polymerisation technique, one based on a sulfobetaine and the other on a phosphobetaine, both with a silyl group component to allow thermal crosslinking after coating . PMMA and glass discs were dip-coated with the polymers and their properties were compared to the uncoated controls . Bacterial adhesion to these coated materials was assessed using Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa . Human macrophages and granulocytes were used to assess the adhesion and activation of inflammatory cells whilst mouse 3T3 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion . In all cases the polymer coatings reduced cell adhesion with respect to the base materials . The phosphobetaine-based copolymer coatings were shown to be markedly superior to the sulfobetaine-based copolymer coatings.

Bioorg Med Chem Lett, 2003 Dec 15, 13(24), 4399 - 403
Synthesis and biological activity of novel 1beta-methylcarbapenems with oxyiminopyrrolidinylamide moiety; Lee JH et al.; The synthesis and antibacterial activity of novel 1beta-methylcarbapenems 1a-f bearing oxyiminopyrrolidinylamide moiety at C-5 position of pyrrolidine are described . Most compounds exhibited comparable antibacterial activity to meropenem against a wide range of Gram-positive and Gram-negative organisms including Pseudomonas aeruginosa isolates . Of these carbapenems, 1a showed potent and broad spectrum of antibacterial activity and similar stability to DHP-I to meropenem . Against clinical isolates of 40 Gram-negative bacterial species including MDR and ESBL-producing strains, the selected carbapenem 1a possessed excellent in vitro activity except for MDR P . aeruginosa, and was comparable in potency to meropenem.

Biochim Biophys Acta, 2003 Dec 5, 1624(1-3), 76 - 80
Growth and membrane polarization in Pseudomonas aeruginosa UG2 grown in randomized microgravity in a high aspect ratio vessel; England LS et al.; Growth and membrane polarization of Pseudomonas aeruginosa UG2 cells grown under randomized microgravity (RMG) and 1xg were measured in a high aspect ratio vessel (HARV) and also in batch cultures mixed at 12 and 150 rpm in Erlenmeyer shake flasks . Membrane polarization was measured using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) . No differences were observed in the growth curves or membrane polarization values (about 0.300) under all three culture conditions . However, the net effect of RMG at the single cell level may be still unknown . It may be possible that RMG effects are species-dependent or bacterial cells with a small mass and volume may be near the threshold where RMG exerts a minimal effect.

Biochemistry, 2003 Dec 9, 42(48), 14249 - 57
Molecular heterogeneity of a type III cytotoxin, Pseudomonas aeruginosa exoenzyme S; Maresso AW et al.; Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin . The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases . A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells . Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells . Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity . Bacterial-associated ExoS and ExoS that had been secreted by P . aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9 . The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers . Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis . ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS . Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS . This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS . ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.

Acta Vet Scand, 2003, 44(1-2), 35 - 42
Antibacterial effect of bovine lactoferrin against udder pathogens; Kutila T et al.; The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E . coli), Staphylococcus aureus (S . aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P . aeruginosa) and Klebsiella pneumoniae (K . pneumoniae), originally isolated from bovine mastitis . Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml . Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk . We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves . Growth inhibition was seen in the broth but hardly at all in whey . The isolates of E . coli and CNS did not grow sufficiently well in whey to draw any conclusions . The most effective inhibitory activity of Lf was seen against E . coli and P . aeruginosa . All 5 E . coil isolates had similar growth patterns . Inhibition of growth by Lf was concentration-dependent . The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.

J Bacteriol, 2003 Dec, 185(24), 7222 - 30
Transcription of quorum-sensing system genes in clinical and environmental isolates of Pseudomonas aeruginosa; Cabrol S et al.; Quorum sensing (QS)-based transcriptional responses in Pseudomonas aeruginosa have been defined on the basis of increases in transcript levels of QS-controlled genes such as lasB and aprA following the hierarchical transcriptional increases of central controllers such as the lasR gene . These increases occur at high bacterial concentrations such as early-stationary-phase growth in vitro . However, the extent to which the increases occur in a variety of clinical and environmental isolates has not been determined nor is there extensive information on allelic variation in lasR genes . An analysis of the sequences of the lasR gene among 66 clinical and environmental isolates showed that 81% have a sequence either identical to that of strain PAO1 or with a silent mutation, 15% have nucleotide changes resulting in amino acid changes, and 5% have an insertion sequence in the lasR gene . Using real-time PCR to quantify transcript levels of lasR, lasB, and aprA in the early log and early stationary phases among 35 isolates from bacteremia and pneumonia cases and the environment, we found most (33 of 35) strains had increases in lasR transcripts in early stationary phase but with a very wide range of final transcript levels per cell . There was a strong correlation (r(2) = 0.84) between early-log- and early-stationary-phase transcript levels in all strains, but this finding remained true only for the 50% of strains above the median level of lasR found in early log phase . There were significant (P < 0.05) but weak-to-modest correlations of lasR transcript levels with aprA (r(2) = 0.2) and lasB (r(2) = 0.5) transcript levels, but again this correlation occurred only in the 50% of P . aeruginosa strains with the highest levels of lasR transcripts in early stationary phase . There were no differences in distribution of lasR alleles among the bacteremia, pneumonia, or environmental isolates . Overall, only about 50% of P . aeruginosa strains from clinical and environmental sources show a lasR-dependent increase in the transcription of aprA and lasB genes, indicating that for about 50% of clinical isolates this regulatory system may not play a significant role in pathogenesis.

Environ Microbiol, 2003 Dec, 5(12), 1341 - 9
Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis; Ernst RK et al.; Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse . Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P . aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment . Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent . No specific pattern of genome mosaicism defined strains associated with CF . Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene . Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung . The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance . Further characterization of P . aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Environ Microbiol, 2003 Dec, 5(12), 1294 - 308
In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets; Potvin E et al.; Pseudomonas aeruginosa is a model for studying opportunistic pathogens that are highly resistant to most classes of antibiotics and cause chronic pulmonary infections . We have developed and adapted a multiplex polymerase chain reaction-based signature-tagged mutagenesis (STM) for high-throughput screening of a collection of 7968 P . aeruginosa mutants in a rat model of chronic respiratory infection . After three rounds of screening, a total of 214 mutants, representing transposition events into 148 open reading frames, were shown to be attenuated in lung infection and were retained for further analysis . As proof of concept supporting this technology, we identified 11 insertions in typical virulence genes such as those coding for pili implicated in motility, attachment and swarming, alginate synthesis and its expression, a mucus transcription regulator, extracellular enzymes such as alkaline protease, esterase and amino peptidase, a rhamnosyl surfactant transferase and a lipopolysaccharide glycosyl transferase . Detailed analysis of the 148 STM mutants, including seven auxotrophs, revealed insertions in 21 of the 26 known gene classes used to characterize sequenced bacterial genomes . We noted that at least 46% of STM mutants identified had insertions in hypothetical proteins or proteins of unknown function and that approximately 40% of all STM mutants had insertions in surface proteins including the outer membrane, the periplasm and the inner membrane . Interestingly, 11 STM mutants attenuated for lung infection were also identified in microarray and transcriptome for quorum sensing and mucoidy production . The remaining 130 mutants were systematically analysed for their capability to express fully known virulence factors . In addition, testing the ability of these mutants to infect alternative model host Drosophila melanogaster revealed 36 STM mutants defective in protease, twitching motility, swimming and swarming . Finally, we identified many genes, the activity of which in respiratory infection was not fully appreciated.

J Infect Dis, 2003 Dec 1, 188(11), 1695 - 706 Epub 2003 Nov 21.
Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia; Schulert GS et al.; Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality . Although the intrinsic virulence of P . aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease . In this study, the virulence of 35 P . aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia . The virulence of individual isolates differed significantly from one to another in each of these assays . Increased virulence was associated with the secretion of ExoU, a toxin transported by the P . aeruginosa type III secretion system . Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence . Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P . aeruginosa.

Invest Ophthalmol Vis Sci, 2003 Dec, 44(12), 5220 - 7
Role of Pseudomonas aeruginosa ExsA in penetration through corneal epithelium in a novel in vivo model; Lee EJ et al.; PURPOSE: The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium . The new model was then used to study the role of ExsA, a transcriptional activator of P . aeruginosa, in bacterial penetration through injured and healing corneal epithelia . METHODS: Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P . aeruginosa strain . Disease progression was monitored for 14 days . The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination . In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing . Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy . RESULTS: Corneas remained susceptible to infection 6 but not 12 hours after scratch injury . By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells . Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function . Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103) . Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances . CONCLUSIONS: The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model . Comparison of the 6- and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.

J Biol Chem, 2004 Feb 20, 279(8), 6934 - 42 Epub 2003 Nov 24.
Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported; Dhanasekaran S et al.; The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory . In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized . The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000 . The enzyme has an alkaline pH optimum of 8.0 to 9.0 . rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+ . The enzyme is inhibited by Zn2+ and succinylacetone . The presence of PfALAD in P . falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy . The enzyme has been localized to the apicoplast of the malaria parasite . Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium . It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization . Expression levels of PfALAD in P . falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite . It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.

Antimicrob Agents Chemother, 2003 Dec, 47(12), 3867 - 76
aph(3')-IIb, a gene encoding an aminoglycoside-modifying enzyme, is under the positive control of surrogate regulator HpaA; Zeng L et al.; Pseudomonas aeruginosa harbors a chromosomal aminoglycoside phosphotransferase gene, aph(3')-IIb, which confers P . aeruginosa resistance to several important aminoglycoside antibiotics, including kanamycin A and B, neomycin B and C, butirosin, and seldomycin F5 . The aph(3')-IIb gene has been found to be regulated by an AraC-type transcriptional regulator (HpaA) encoded by a gene located upstream of the aph(3')-IIb gene . In the presence of 4-hydroxyphenylacetic acid (4-HPA), HpaA activates the expression of aph(3')-IIb as well as that of the hpa regulon which encodes metabolic enzymes for the utilization of 4-HPA . hpaA and aph(3')-IIb form an operon, and in response to the presence of 4-HPA, the wild-type P . aeruginosa strain PAK (but not its hpaA mutant strain) displays increased resistance to neomycin . A survey of 39 clinical and 19 environmental isolates of P . aeruginosa demonstrated in all of them the presence of an hpaA-aph gene cluster, while 56 out of the 58 isolates are able to utilize the 4-HPA as a sole carbon source, suggesting a feature common to P . aeruginosa strains . Interestingly, a larger portion of clinical isolates than environmental isolates showed 4-HPA-induced resistance to neomycin . The aph(3')-IIb gene product is likely to function as a metabolic enzyme which has a cross-reactivity with aminoglycosides . These findings provide new insight into the possible mechanism of P . aeruginosa antibiotic resistance.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 181 - 6
Multidrug-resistant Pseudomonas aeruginosa strains harbouring R-plasmids and AmpC beta-lactamases isolated from hospitalised burn patients in a tertiary care hospital of North India; Shahid M et al.; The present study was designed to determine resistance rates and patterns in Pseudomonas aeruginosa isolates obtained from hospitalised burn patients in an Indian tertiary care hospital . To that end, we isolated plasmid(s) from the multidrug-resistant isolates, demonstrated the plasmid-mediated resistance by curing and transformation experiments, and screened all the isolates for the occurrence of AmpC beta-lactamases . Thirty isolates of P . aeruginosa were analysed for the presence of antibiotic resistance . Plasmid-curing experiments and AmpC beta-lactamase detection were performed on all the isolates and seven isolates showing the most common antibiotic resistance pattern were selected for plasmid isolation and transformation experiments . All 30 isolates were multidrug-resistant and the majority (83.3%) of isolates were resistant to seven or more antibiotics, out of 11 antibiotics tested including anti-pseudomonal and non-anti-pseudomonal antimicrobial drugs . The most striking feature was the presence of resistance to amikacin . A 48.5-kb plasmid was isolated from the isolates . Curing and transformation experiments showed that resistance to amikacin was plasmid-mediated . Phenotypic screening for the occurrence of AmpC beta-lactamases showed that 20% of isolates were AmpC producers whereas 10% of isolates were characterised as 'indeterminate' for AmpC enzyme . In conclusion, a markedly high (56.7%) resistance to amikacin was noted in the present study . Amikacin resistance was determined to be plasmid-encoded and the presence of an AmpC beta-lactamase was inferred in 20% of isolates . This is among the first reports regarding the emergence of plasmid-mediated resistance to amikacin and the occurrence of AmpC beta-lactamases in P . aeruginosa strains from India.

J Appl Microbiol, 2003, 95(5), 990 - 1000
Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp . entomocidus HD9; Cherif A et al.; AIMS: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers . METHODS AND RESULTS: Bacillus thuringiensis ssp . entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi . Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase . Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121 degrees C . Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization . After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12.4 kDa was isolated . The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells . Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B . thuringiensis HD9 was not toxic against Vero cells . CONCLUSIONS: Entomocin 9 is a novel heat-stable, bacteriocin produced by B . thuringiensis HD9 . The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments . SIGNIFICANCE AND IMPACT OF THE STUDY: New finding on entomocin 9 would make B . thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.

Eye, 2003 Nov, 17(8), 863 - 71
Further studies on the role of IL-12 in Pseudomonas aeruginosa corneal infection; Hazlett LD et al.; PURPOSE: Previous studies have shown that in Pseudomonas aeruginosa ocular infection, IL-12 drives a Th1 T-cell response and IFN-gamma production in susceptible (cornea perforates) C57BL/6 (B6) mice, and that after similar infection of resistant (cornea heals) BALB/c mice, no IL-12 is detectable in cornea at either the mRNA or protein levels . Therefore, the purpose of this study was to test whether BALB/c mice are capable of responding to exogenous IL-12 administration, and whether disease responsiveness following P . aeruginosa challenge is modified . METHODS: Immunostaining, RT/PCR, recombinant cytokine injection, and histopathology were used . Statistical analysis was performed using an unpaired, two-tailed Student's t-test . RESULTS: Injection of BALB/c mice with recombinant (r) IL-12 converted these normally resistant animals to the susceptible phenotype as evidenced by corneal perforation within 5-7 days after infection . RT-PCR analysis of the corneas of rIL-12 vs PBS/BSA-treated mice showed a significant increase in IFN-gamma and TNF-alpha mRNA levels in the rIL-12 vs PBS/BSA (vehicle)-treated mice at 3 and 5 days p.i . In addition, similar analysis of IL-4 mRNA levels showed decreased amounts of the cytokine in rIL-12 vs vehicle-treated mice . Injection of rIL-4 into susceptible B6 mice, however, failed to rescue these animals from corneal perforation following P . aeruginosa challenge . CONCLUSIONS: These data provide evidence that BALB/c mice can respond to exogenous IL-12, that the cytokine promotes susceptibility by increasing IFN-gamma and TNF-alpha production, with a concomitant reduction in IL-4 levels; and that injected rIL-4 fails to rescue susceptible B6 mice from corneal perforation after bacterial challenge.

Rev Mal Respir, 2003 Nov, 20(5 Pt 1), 711 - 8
{Reproducibility of the shuttle walk test in children with cystic fibrosis}; Pouessel G et al.; INTRODUCTION: Exercise testing is useful in the respiratory evaluation of patients with cystic fibrosis . The shuttle walk test (SWT) is a progressive, externally paced, exercise test requiring the subject to walk/run back and forth between two fixed points . The aim is to assess the reproductibility of the SWT in paediatric patients with cystic fibrosis . METHODS: This prospective study recruited 31 children with stable disease . The patients performed two SWT one day (SWT 1 and 2) and two others (SWT 3 and 4) within 15 days . Only SWT 2 and 4 were assessed for reproducibility . RESULTS: 61% were boys, median age (range): 12.9 (7-18.9) years, median Shwachman score (range): 80 (65-100), median values for FEV1 and FVC (range): 92 (55-154) and 92 (64-140)% predicted, respectively . Median distance for SWT 2-4 (range): 910 (580-1020) and 925 (540-1020) metres . Reproducibility for SWT distance and physical activity measured by an accelerometer is very good (intra-class correlation coefficient=0.90 and 0.92, respectively) . SWT distance correlated with physical activity (p=3.10(-4)) and weight (p=0.03) . SWT distance was independent of the following parameters: height, weight-for-age Z-score, FEV1, FVC, Shwachman score, colonisation with Pseudomonas aeruginosa . CONCLUSIONS: The SWT is reproducible in paediatric patients with cystic fibrosis and provides assessment of respiratory performance that complements spirometric measures of lung function.

Salud Publica Mex, 2003 Sep-Oct, 45(5), 371 - 8
{Pseudomonas aeruginosa outbreak, in the area of surgical wound ambulatory care, in postmastectomy patients}; Vilar-Compte D et al.; OBJECTIVE: To describe an outbreak due to Pseudomonas aeruginosa in postmastectomy wounds . MATERIAL AND METHODS: Cases were patients with a surgical infection caused by P . aeruginosa resistant to ciprofloxacin and gentamycin seen between March 13, 2000 and May 18, 2000, at Instituto Nacional de Cancerologia in Mexico City . Specimens for culturing were taken from faucets, antiseptics, and tap water, as well as from healthcare workers . A case-control analysis was conducted . RESULTS: Thirteen late surgical infections were caused by a ciprofloxacin and gentamycin-resistant P . aeruginosa . The causative Pseudomonas was isolated from a nurse's nostrils and non-sterile gauzes left by her on the Mayo table at the Breast Tumor ambulatory clinic . None of the closed packages was positive to Pseudomonas . On April 14, 2000, the nurse was transferred to another ward and strict infection control practices were established . After this date, 4 additional cases were diagnosed . Radiation therapy was the only risk factor for infection (Or = 5.1, 95% cI 1.1-28.4) . CONCLUSIONS: This outbreak was probably caused by a common source initially, and later disseminated by cross-infection among patients . The poor compliance with infection control practices during wound cleaning and drainage led to implementing a series of specific preventive interventions.

Med Microbiol Immunol (Berl), 2005 Jan, 194(1-2), 39 - 45 Epub 2005 Jan.
Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor; Marquart ME et al.; The effect of calcium and magnesium on protease IV production during the growth of Pseudomonas aeruginosa was investigated . Strain PA103 was grown to stationary phase in medium containing various concentrations of either calcium or magnesium . Culture supernatants were concentrated, standardized relative to cell density, and the pyoverdine concentrations were measured . Overall extracellular protease activity and specific protease IV (lysine endoproteinase) activity were measured with or without TLCK, a serine protease inhibitor effective against protease IV activity . Protease IV activity was also observed by casein zymography . Calcium and magnesium were quantified in the corneas and aqueous humor of rabbits that were inoculated intrastromally with strain PA103 . Pyoverdine production was not significantly different in cultures grown in medium with added calcium or magnesium, but extracellular caseinase activity increased in these cultures . Susceptibility of caseinase activity to TLCK inhibition and a specific assay for protease IV indicated that protease IV activity increased in cultures grown in calcium or magnesium . Casein zymography supported the observation that protease IV activity increased in the cultures with added calcium and magnesium . Addition of calcium or magnesium to the protease IV-specific assay had no effect on the catalytic activity of pure protease IV . Infection of rabbit corneas with PA103 did not change the magnesium concentration in either corneas or aqueous humor, but significantly increased the concentration of calcium in corneas . These results indicate that calcium and magnesium enhance the production of protease IV, but not pyoverdine production . Calcium increases in the cornea following infection with P . aeruginosa could favor production of protease IV.

JAMA, 2003 Nov 19, 290(19), 2588 - 98
Comparison of 8 vs 15 days of antibiotic therapy for ventilator-associated pneumonia in adults: a randomized trial; Chastre J et al.; CONTEXT: The optimal duration of antimicrobial treatment for ventilator-associated pneumonia (VAP) is unknown . Shortening the length of treatment may help to contain the emergence of multiresistant bacteria in the intensive care unit (ICU) . OBJECTIVE: To determine whether 8 days is as effective as 15 days of antibiotic treatment of patients with microbiologically proven VAP . DESIGN, SETTING, AND PARTICIPANTS: Prospective, randomized, double-blind (until day 8) clinical trial conducted in 51 French ICUs . A total of 401 patients diagnosed as having developed VAP by quantitative culture results of bronchoscopic specimens and who had received initial appropriate empirical antimicrobial therapy were enrolled between May 1999 and June 2002 . INTERVENTION: A total of 197 patients were randomly assigned to receive 8 days and 204 to receive 15 days of therapy with an antibiotic regimen selected by the treating physician . MAIN OUTCOME MEASURES: Primary outcome measures-death from any cause, microbiologically documented pulmonary infection recurrence, and antibiotic-free days-were assessed 28 days after VAP onset and analyzed on an intent-to-treat basis . RESULTS: Compared with patients treated for 15 days, those treated for 8 days had neither excess mortality (18.8% vs 17.2%; difference, 1.6%; 90% confidence interval {CI}, -3.7% to 6.9%) nor more recurrent infections (28.9% vs 26.0%; difference, 2.9%; 90% CI, -3.2% to 9.1%), but they had more mean (SD) antibiotic-free days (13.1 {7.4} vs 8.7 {5.2} days, P<.001) . The number of mechanical ventilation-free days, the number of organ failure-free days, the length of ICU stay, and mortality rates on day 60 for the 2 groups did not differ . Although patients with VAP caused by nonfermenting gram-negative bacilli, including Pseudomonas aeruginosa, did not have more unfavorable outcomes when antimicrobial therapy lasted only 8 days, they did have a higher pulmonary infection-recurrence rate compared with those receiving 15 days of treatment (40.6% vs 25.4%; difference, 15.2%, 90% CI, 3.9%-26.6%) . Among patients who developed recurrent infections, multiresistant pathogens emerged less frequently in those who had received 8 days of antibiotics (42.1% vs 62.0% of pulmonary recurrences, P =.04) . CONCLUSIONS: Among patients who had received appropriate initial empirical therapy, with the possible exception of those developing nonfermenting gram-negative bacillus infections, comparable clinical effectiveness against VAP was obtained with the 8- and 15-day treatment regimens . The 8-day group had less antibiotic use.

Nutrition, 2003 Nov-Dec, 19(11-12), 994 - 6
Antibacterial activity of vegetables and juices; Lee YL et al.; OBJECTIVE: We evaluated the antibacterial activities of various fruit and vegetable extracts on common potential pathogens including antibiotic-resistant strains . METHODS: Standardized bacterial inocula were added to serial dilutions of sterile vegetable and fruit extracts in broth, with final bacterial concentrations of 10(4-5) cells/mL . After overnight incubation at 35 degrees C, antibacterial activity was measured by minimum inhibitory and minimum bactericidal dilutions (for raw juices) or concentrations (for tea) . RESULTS: Among the vegetable and fruit extracts tested, all green vegetables showed no antibacterial activity on Staphylococcus epidermidis and Klebsiella pneumoniae . All purple and red vegetable and fruit juices had antibacterial activities in dilutions ranging from 1:2 to 1:16 . Garlic juice had significant activity, with bactericidal action in dilutions ranging up to 1:128 of the original juice . Tea also had significant activity, with bactericidal action in concentrations ranging up to 1.6 mg/mL, against a spectrum of pathogens including resistant strains such as methicillin- and ciprofloxacin-resistant staphylococci, vancomycin-resistant enterococci, and ciprofloxacin-resistant Pseudomonas aeruginosa . CONCLUSIONS: Tea and garlic have the potential for exploration of broader applications as antibacterial agents.

J Biomed Mater Res A, 2003 Dec 15, 67(4), 1276 - 83
Antibacterial properties of nitric oxide-releasing sol-gels; Nablo BJ et al.; The antibacterial characteristics of nitric oxide (NO)-releasing sol-gel coatings are described . The NO release from these surfaces is steady over short periods (approximately 1 h) and measurable over several days . The ability of NO to prevent bacterial adhesion is evaluated by exposing controls and NO-releasing sol-gels to approximately 10(8) colony-forming units (cfu)/mL saline suspensions of Pseudomonas aeruginosa . Pseudomonas aeruginosa adhesion to sol-gel controls varies depending on the sol-gel formulation . Sol-gel surfaces capable of NO release decrease bacterial adhesion by 30% to 95% relative to controls . The contact angle measurements of control and NO-releasing surfaces are similar, supporting NO's action as an antibacterial agent against bacterial adhesion .

Bioorg Med Chem Lett, 2003 Dec 1, 13(23), 4205 - 8
MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa . Part 2: achieving activity in vivo through the use of alternative scaffolds; Nakayama K et al.; Problems of low solubility, high serum protein binding, and lack of efficacy in vivo in first generation MexAB-OprM specific efflux pump inhibitors were addressed . Through the use of pharmacophore modelling, the key structural elements for pump inhibition were defined . Use of alternative scaffolds upon which the key elements were arrayed gave second generation leads with greatly improved physical properties and activity in the potentiation of antibacterial quinolones (levofloxacin and sitafloxacin) versus Pseudomonas aeruginosa in vivo.

Bioorg Med Chem Lett, 2003 Dec 1, 13(23), 4201 - 4
MexAB-OprM-specific efflux pump inhibitors in Pseudomonas aeruginosa . Part 1: discovery and early strategies for lead optimization; Nakayama K et al.; The identification of a series of compounds that specifically inhibit efflux by the MexAB-OprM pump system in Pseudomonas aeruginosa is described . Synthesis and in vitro structure-activity relationships (SARs) are outlined . Early leads lacked activity in animal models, and efforts to improve solubility and reduce serum protein binding by the introduction of polar groups are discussed.

Eur J Biochem, 2003 Dec, 270(23), 4744 - 54
Factors involved in the assembly of a functional molybdopyranopterin center in recombinant Comamonas acidovorans xanthine dehydrogenase; Ivanov NV et al.; Previous work from this laboratory has shown that the spectral and functional properties of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans show some similarities to those of the well-characterized eukaryotic enzymes isolated from bovine milk and from chicken liver {Xiang, Q . & Edmondson, D.E . (1996) Biochemistry35, 5441-5450} . Therefore, this system was chosen to study the factors involved in the expression of functional recombinant enzyme in Escherichia coli to provide insights into the assembly of the functional Mo-pyranopterin center . Genes xdhA and xdhB (encoding the two known subunits of the native enzyme) and putative genes xprA and ssuABC were sequenced . Heterologous expression of the xdhAB genes in E . coli JM109(DE3) produced active enzyme . The Mo content was 0.11-0.16 mol per alphabeta protomer, while the Fe and FAD levels were at stoichiometries similar to that of the native enzyme . The XDH activity increased sixfold when the culture was grown under conditions of low aeration (6 L.min-1) as compared with high aeration (12 L.min-1) . Co-expression of the xdhAB genes with the Pseudomonas aeruginosa PA1522 (xdhC) gene increased the level of Mo incorporated into the expressed enzyme to a 1 : 1 stoichiometry . Under these conditions, high levels of functional protein (2.284 U.mg-1 and 8.039 mg.L-1 of culture) were obtained independently of the level of culture aeration . Therefore, the assembly of a functional Mo-pyranopterin center in XDH requires the presence of a functional xdhC gene product . The purified, recombinant XDH shows spectral and kinetic properties identical to those of the native enzyme.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14339 - 44 Epub 2003 Nov 14.
Comprehensive transposon mutant library of Pseudomonas aeruginosa; Jacobs MA et al.; We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained . Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised . The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome . The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene . About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media . Based on statistical analyses and bioinformatic comparison to known essential genes in E . coli, we estimate that the actual number of essential genes is 300-400 . Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies . Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities . The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

J Clin Invest, 2003 Nov, 112(10), 1460 - 5
Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target; Smith RS et al.; Pseudomonas aeruginosa has two complete quorum-sensing systems . Both of these systems have been shown to be important for Pseudomonas virulence in multiple models of infection . Thus, these systems provide unique targets for novel antimicrobial drugs.

Mol Microbiol, 2003 Nov, 50(3), 809 - 24
A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa; Dasgupta N et al.; The single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen . In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type-a and type-b strains of P . aeruginosa respectively . The flagellar genes are clustered in three non-contiguous regions of the chromosome . A polymorphic locus flanked by flgJ and fleQ in Region I contains a glycosylation island in PAK . The expression and ordered assembly of the complex multicomponent flagellum is intricately regulated . Dedicated flagellar genes fleQ, fleS, fleR, fliA, flgM and fleN encode proteins that participate in the regulation of the flagellar transcriptional circuit . In addition, expression of the flagellum is coordinately regulated with other P . aeruginosa virulence factors by the alternative sigma factor sigma54, encoded by rpoN . In order to gain insight into the hierarchical regulation of flagellar genes, deletion mutations were constructed in fleQ, fleR, fliA and rpoN . The transcriptional impact of these mutations was examined by transcriptional profiling using a P . aeruginosa whole genome microarray . Analysis of the transcriptomes generated for each of these mutants indicates a four-tiered (Classes I-IV) hierarchy of transcriptional regulation . Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (sigma28) . Class II genes including fleSR, encoding a two-component regulatory system require FleQ and RpoN (sigma54) for their transcriptional activation . Class III genes are positively regulated by the activated response regulator FleR in concert with RpoN . The transcription of Class IV genes is dependent on the availability of free FliA following the export of the FliA specific antisigma factor FlgM through the basal body rod-hook structure (assembled from Class II and III gene products) . Two previously uncharacterized genes, which are coordinately regulated with known flagellar genes have been identified by genome-wide analysis and their role in flagellar biogenesis was analysed.

Clin Microbiol Infect, 2003 Sep, 9(9), 980 - 3
Susceptibility of multi-drug-resistant Pseudomonas aeruginosa in intensive care units: results from the European MYSTIC study group; Goossens H; The MYSTIC program monitors worldwide in vitro susceptibilities of clinical bacterial isolates from centers that prescribe meropenem . This report focuses on 107 isolates of multi-drug resistant (MDR) Pseudomonas aeruginosa, which commonly causes infections that are difficult to treat in hospitalized patients . We chose samples from patients from 33 European intensive care units (ICUs) . There was considerable inter-country variation in the proportion of P . aeruginosa that were MDR, ranging from 50% in Turkey to < or =3% in Spain, the UK, Germany, Bulgaria and Malta . Amongst the MDR isolates, the percentage resistance (MIC50/90 in mg/L) to the antibiotics tested were amikacin 18.7% (32/>128), meropenem 29.1% (8/64), imipenem 44.9% (16/64), cefepime 49.5% (32/64) and piperacillin/tazobactam 57.9% (128/>128).

Clin Microbiol Infect, 2003 Sep, 9(9), 938 - 43
An outbreak of carbapenem-resistant Pseudomonas aeruginosa in a urology ward; Pena C et al.; OBJECTIVE: To investigate an outbreak of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a urology ward . METHODS: Patients infected or colonized with CRPA were prospectively identified by daily laboratory surveillance . Routine infection-control measures were reinforced, disinfection protocols were revised, and a surveillance program was set up, analyzing cross-transmission in the nursing ward and environment cultures from urology wards and the operating theater . CRPA isolates from clinical and environment samples were studied by pulsed-field gel electrophoresis (PFGE), following XbaI and SpeI restriction . RESULTS: From February 1998 to September 2000, 59 adult urology patients were colonized or infected by CRPA . All patients had been operated on prior to identification of the CRPA isolate and 79% of these procedures were performed in the same cystoscopy room . No patients had received prior carbapenem therapy . No cross-transmission was detected, and environment cultures from the urology ward and theater were negative except for five samples collected in the cystoscopy room . PFGE identified a single clone in the isolates from different patients and the environment samples . CONCLUSIONS: The PFGE analysis indicated that the CRPA outbreak resulted from the contamination of the cystoscopy room via an unsealed drain . The outbreak ended when the drain was sealed.

J Theor Biol, 2003 Dec 21, 225(4), 469 - 76
Differential interactions within the Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model; Ruiz-Diez B et al.; A pathogenesis model based on the interaction between Caenorhabditis elegans and bacterial opportunistic pathogens has recently been developed . In the case of Pseudomonas aeruginosa, the model is based on three different modes of nematode killing (fast killing, slow killing and lethal paralysis) by virulent bacteria that has been incubated in different nutrient media . Using parametric statistics and Probit analysis, we test the reliability of the three different killing systems with respect to bacterial virulence . To accomplish this, we use three P . aeruginosa strains, each with a different level of virulence and one strain of non-virulent Escherichia coli . Probit function proved to be effective in quantifying the virulence of P . aeruginosa . The results of the killing curve analysis using the Probit function demonstrates that the slow-killing test is the most reliable method for quantifying virulence using the C . elegans model of bacterial pathogenesis . Although the greatest virulence differences are observed after long periods of incubation, the Probit analysis clearly shows that the death kinetics of C . elegans depend on the first hours of nematode/bacteria interaction . In contrast, fast killing seems to be non-specific, at least under our experimental conditions, since the killing rates of virulent P . aeruginosa and non-virulent E . coli strains were indistinguishable.

Clin Infect Dis, 2003 Dec 1, 37(11), e154 - 60 Epub 2003 Oct 29.
Use of parenteral colistin for the treatment of serious infection due to antimicrobial-resistant Pseudomonas aeruginosa; Linden PK et al.; Serious infection due to strains of Pseudomonas aeruginosa that exhibit resistance to all common antipseudomonal antimicrobials increasingly is a serious problem . Colistin was used as salvage therapy for 23 critically ill patients with multidrug-resistant P . aeruginosa infection . Twenty-two patients who had septic shock (n=14) and/or renal failure (n=21) received mechanical ventilatory support at baseline . The most common types of infection were pneumonia (n=18) and intra-abdominal infection (n=5) . Colistin was administered for a median of 17 days (range, 7-36 days) . Seven patients died during therapy, at a median of 17 days (range, 4-26 days) after initiation of treatment . A favorable clinical response was observed in 14 patients (61%); only 3 patients experienced relapse . Bacteremia was the only significant factor associated with treatment failure (P=.02) . One patient manifested diffuse weakness that resolved after temporary cessation of colistin therapy . Colistin provides an important salvage therapeutic option for patients with otherwise untreatable serious P . aeruginosa infection.

J Med Microbiol, 2003 Dec, 52(Pt 12), 1039 - 45
Bactericidal properties of group IIa secreted phospholipase A(2) against Pseudomonas aeruginosa clinical isolates; Dubouix A et al.; It has been shown that human group IIa secreted phospholipase A(2) (sPLA(2)), found at high levels in inflammatory fluids, displays direct bactericidal properties against Gram-positive bacteria, while activity against Gram-negative bacteria requires the complement system or additional co-factors produced by neutrophils . Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common Gram-negative rod found in cystic fibrosis lung infections, where it is associated with an inflammatory environment . Because murine intestinal group II sPLA(2) produced by Paneth cells has been shown to be directly bactericidal against Gram-negative bacteria, IIa sPLA(2) activity against P . aeruginosa clinical isolates was evaluated and provides the first evidence that the enzyme can be fully bactericidal in a concentration- and time-dependent manner against Gram-negative rods . Furthermore, it was demonstrated that these bactericidal properties were unaffected by high protein and salt concentrations, as observed in cystic fibrosis secretions, and that bacterial killing paralleled phospholipid hydrolysis . Finally, no cytotoxicity was observed when IIa sPLA(2) was incubated with human pulmonary cells, highlighting its potential use to synergize bactericidal antibiotics by promoting sublethal alterations of the bacterial cell wall.

Peptides, 2003 Aug, 24(8), 1099 - 107
Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model; Phadke SM et al.; Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties . LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) . Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations . MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1 . In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability . Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.

Bioorg Med Chem Lett, 2003 Sep 1, 13(17), 2863 - 5
Synthesis of N-alkylated derivatives of imidazole as antibacterial agents; Khabnadideh S et al.; N-Alkylation of imidazole, 2-methylimidazole and 2-methyl-4-nitroimidazole have been carried out to achieve effective antibacterial agents . The products were then investigated for antibacterial activity against Escherichia coil, Staphylococcus aureus and Pseudomonas aeruginosa . Antibacterial effects of 1-alkylimidazole derivatives increase as the number of carbons in alkyl chain increases up to nine carbons . Also substitution of 2-methyl and 2-methyl-4-nitro groups on imidazole ring increases the antibacterial activity.

DNA Cell Biol, 2003 Oct, 22(10), 649 - 55
Intercellular adhesion molecule-2 (ICAM-2) and Pseudomonas aeruginosa ocular infection; Hobden JA; In a previous study, ICAM-1-deficient knockout (KO) mice were able to recruit inflammatory cells into Pseudomonas aeruginosa-infected eyes and resolve the infection as well as wild-type (WT) mice . Based on this observation, it was hypothesized that ICAM-2 could serve as a surrogate receptor for leukocyte recruitment in lieu of ICAM-1 . To test this hypothesis, ICAM-2 expression was first examined in both uninfected and P . aeruginosa-infected eyes (6 h postinfection) by immunohistochemistry and RT-PCR . Similar to ICAM-1, ICAM-2 was constitutively expressed on the vascular endothelium of the iris, ciliary body, and conjunctiva of uninfected eyes . Unlike ICAM-1, ICAM-2 was not expressed in the cornea nor upregulated following P . aeruginosa infection . The role of ICAM-2 in P . aeruginosa ocular infection was then addressed through a monoclonal antibody (MAb) blockade of ICAM-2 in infected ICAM-1 KO and WT mice . MAb blockade of ICAM-2 resulted in fewer infiltrating inflammatory cells (as ascertained by histopathology) in the anterior chamber of eyes of ICAM-1-KO and WT mice 24 h postinfection . However, a myeloperoxidase assay of infected corneas showed no statistical difference (P > 0.11) between the two groups in infiltrating PMN . Collectively, these data suggest that constitutively expressed ICAM-2 does play a role in recruiting inflammatory cells into the anterior chamber of the eye during P . aeruginosa infection . Furthermore, inflammatory cell recruitment into the P . aeruginosa-infected cornea appears to be mediated by an ICAM-independent pathway.

J Burn Care Rehabil, 2003 Nov-Dec, 24(6), 365 - 70
Comparison of surface swab cultures and quantitative tissue biopsy cultures to predict sepsis in burn patients: a prospective study; Sjoberg T et al.; This study aimed at evaluating the possibility of predicting septicemia in burn patients by using wound surface and tissue culture techniques as well as blood cultures . Fifty patients with full-thickness burn wounds covering at least 10% of the total body surface area were included . Signs of septicemia were noted in 21 patients (42%) and 29 patients died (58%) . The bacterial colonization of the burn wounds consisted mainly of Staphylococcus aureus and Pseudomonas aeruginosa . Sepsis was better correlated to quantitative burn tissue biopsy cultures than surface swab cultures but the time needed for processing limits its predictive and therapeutic value.

Exp Eye Res, 2003 Dec, 77(6), 699 - 710
Isolation of conjunctival mucin and differential interaction with Pseudomonas aeruginosa strains of varied pathogenic potential; Aristoteli LP et al.; The purpose of the study was to investigate the adhesion of Pseudomonas aeruginosa strains with varying pathogenic potential to purified ocular mucin . Bovine conjunctival mucin was purified by three sequential density gradient centrifugation steps . Immobilised mucin was probed with biotin-labelled bacteria isolated from different contact lens events and quantified by densitometry . Bacterial pili were identified by electron microscopy . The results indicate that purified ocular mucin consisted of a polydisperse high molecular weight population containing at least one species of goblet cell origin and was associated with a 97 kDa mucin-associated protein . Three pathogenic P . aeruginosa strains, Paer1 (57.5 +/- 10.8x10(6) CFU ml(-1); contact lens induced acute red eye (CLARE)), 6294 (127.0 +/- 4.7x10(6) CFU ml(-1); microbial keratitis) and Paer25 (60.5 +/- 11.3x10(6) CFU ml(-1); CLARE) exhibited a significantly higher level of adhesion to mucin than the negative control, E . coli (14.3 +/- 9.6x10(6) CFU ml(-1)) (p<0.005) . The remaining P . aeruginosa isolates, Paer3 (asymptomatic patient), Paer12 (microbial keratitis) and ATCC 15442 (standard environmental strain) did not significantly differ in their mucin adhesion from the negative control . The majority of bacterial strains tested contained pili; thus differences in mucin adhesion observed could not be solely explained by pili status . In conclusion, P . aeruginosa isolates exhibit differential adhesion patterns to purified ocular mucin . It is proposed that more avid mucin-adhering strains are given the opportunity to adhere and subsequently penetrate the mucous layer of the tear film to initiate pathogenesis.

Am J Respir Cell Mol Biol, 2004 May, 30(5), 627 - 34 Epub 2003 Nov 07.
Pseudomonas aeruginosa flagella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5; Adamo R et al.; The distribution of specific toll-like receptors and components of the signaling pathways activated by Pseudomonas aeruginosa flagella were studied in airway epithelial cells . Initially flagella bound to the apical surface of polarized epithelial cells, where they prominently colocalized with asialoGM1 . By 4 h of exposure to flagella, toll-like receptor (TLR)5 expression was induced, mobilized to the apical surface of the cells, and colocalized with superficial flagella . Interleukin-8 expression in airway cells was activated by flagella through induction of Ca(2+) fluxes, Src, Ras, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and nuclear factor-kappaB activation, a pathway previously associated with asialoGM1-mediated stimuli . There was evidence for participation of asialoGM1 and TLR2 as well as TLR5 in the response to flagella, and increased asialoGM1 correlated directly with increased signaling . TLR2 DN or TLR5 DN mutations inhibited interleukin-8 induction by 78% and 35%, respectively (P < 0.001 for each) . The participation of TLR2 as well as TLR5 was confirmed in Chinese hamster ovary cells transfected with either human TLR2 or TLR5 in which flagella activated a nuclear factor-kappaB-luciferase reporter to the same extent . Flagella signaling in airway cells can be initiated by interactions with asialoGM1 and TLR2 as well as by activation of TLR5 . The availability of exposed receptors on the apical surface of polarized airway epithelial cells is a major factor in the activation of signaling pathways by flagella.

Pediatr Res, 2004 Jan, 55(1), 69 - 75 Epub 2003 Nov 06.
Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated residual chloride secretion does not protect against early chronic Pseudomonas aeruginosa infection in F508del homozygous cystic fibrosis patients; Derichs N et al.; Cystic fibrosis (CF) disease severity is characterized by a broad variability that has been attributed, in addition to the CF transmembrane conductance regulator (CFTR) genotype, to modulating factors such as CFTR-mediated residual chloride (Cl-) secretion . Moreover, CFTR has been suggested to function as a receptor for Pseudomonas aeruginosa (PA) . In this study, we investigated whether or not the presence of residual Cl- secretion protects against early chronic PA colonization of patients' airways . Excluding influences on the phenotype caused by different CFTR mutations, we evaluated a cohort of F508del homozygous individuals with respect to the correlation between residual Cl- secretion and the age of onset of PA colonization as an important marker of clinical phenotype . A group with early chronic PA colonization before the age of 7 y (n = 14) was compared with a cohort that had no initial PA detection at least until the age of 13 y (n = 10) . We determined the Cl- transport properties by using the intestinal current measurement in rectal suction biopsies . Residual Cl- secretion, most likely due to the CFTR Cl- channel, was observed in 63% of subjects, more frequently in early chronically PA colonized than among late or not colonized patients . These results demonstrate the presence of some active F508del-CFTR in the apical cell membrane and imply that factors other than the CFTR-mediated residual Cl- secretion determine the age of onset of PA colonization.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 14315 - 20 Epub 2003 Nov 06.
Human targets of Pseudomonas aeruginosa pyocyanin; Ran H et al.; Pseudomonas aeruginosa produces copious amounts of the redoxactive tricyclic compound pyocyanin that kills competing microbes and mammalian cells, especially during cystic fibrosis lung infection . Cross-phylum susceptibility to pyocyanin suggests the existence of evolutionarily conserved physiological targets . We screened a Saccharomyces cerevisiae deletion library to identify presumptive pyocyanin targets with the expectation that similar targets would be conserved in humans . Fifty S . cerevisiae targets were provisionally identified, of which 60% have orthologous human counterparts . These targets encompassed major cellular pathways involved in the cell cycle, electron transport and respiration, epidermal cell growth, protein sorting, vesicle transport, and the vacuolar ATPase . Using cultured human lung epithelial cells, we showed that pyocyanin-mediated reactive oxygen intermediates inactivate human vacuolar ATPase, supporting the validity of the yeast screen . We discuss how the inactivation of V-ATPase may negatively impact the lung function of cystic fibrosis patients.

J Clin Microbiol, 2003 Nov, 41(11), 4991 - 7
Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing; Onteniente L et al.; We report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA) . We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains . Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes . Seventy-one different MLVA genotypes could be distinguished . With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data . The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types . Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset . Our data show that MLVA typing of P . aeruginosa based on the first set of loci has a high discriminatory power . Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P . aeruginosa . A free, online strain identification service based on the genotyping data produced herein has been developed.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2003 Nov, 15(11), 662 - 5
{Application of the acute physiology and chronic health evaluation II score system to patients with infection of Pseudomonas aeruginosa in lower respiratory tract in intensive care unit}; Shao CZ et al.; OBJECTIVE: To predict the infection and evaluate the severity of illness and prognosis for patients with infection of Pseudomonas aeruginosa (PA) in lower respiratory tract in intensive care unit (ICU) with the acute physiology and chronic health evaluation II (APACHE II) . METHODS: The clinical data of 122 cases with infection of PA in lower respiratory tract were compared and studied . These data were evaluated with APACHE II score system according to Knaus method . RESULTS: APACHE II scores of the 29 nonsurvivors were significantly higher than that of 93 survivors (18.78+/-7.13 vs . 11.70+/-5.79, t=5.43, P<0.01) . The patients with coinfections other than PA had a higher APACHE II score (14.76+/-6.89 vs . 10.08+/-6.14, P<0.01), a higher mortality (27.91 percent vs . 13.89 percent, P<0.01), and more days of stay in ICU {(28.47+/-23.59) days vs . (16.64+/-21.19) days} than those without . Patients with severe pneumonia had higher APACHE II scores (15.57+/-6.97 vs . 11.81+/-6.03) and poorer prognosis (39.22 percent vs . 12.68 percent) than those without . For all patients, when the APACHE II scores became higher and higher, the outcome became poorer and poorer, and the mortality higher and higher, and the percentage of severe pneumonia higher and higher . There was a significant correlation between APACHE II score and actual mortality (r=0.75, P<0.01) and predicted mortality (r=0.81, P<0.01) . Actual and predicted mortality increased along with the increase in APACHE II scores by 5 scores . The sensitivity and positive rate of predicted mortality was 100.00 percent and 86.72 percent respectively . CONCLUSION: APACHE II score system is highly valuable in predicting the infection and evaluating the severity of illness and prognosis in patients with infection of PA in lower respiratory tract in ICU.

Perit Dial Int, 2003 Sep-Oct, 23(5), 456 - 9
Staphylococcus aureus prophylaxis and trends in gram-negative infections in peritoneal dialysis patients; Piraino B et al.; OBJECTIVE: To examine gram-negative exit-site infection and peritonitis rates before and after the implementation of Staphylococcus aureus prophylaxis in peritoneal dialysis (PD) patients . DESIGN: Prospective data collection with periodic implementation of protocols to decrease infection rates in two PD programs . PATIENTS: 663 incident patients on PD . INTERVENTIONS: Implementation of S . aureus prophylaxis, beginning in 1990 . MAIN OUTCOME MEASURES: Rates of S . aureus, gram-negative, and Pseudomonas aeruginosa exit-site infections and peritonitis . RESULTS: Staphylococcus aureus exit-site infection and peritonitis rates fluctuated without significant trends during the first decade (without prophylaxis), then began to decline during the 1990s subsequent to implementation of prophylaxis, reaching levels of 0.02/year at risk and zero in the year 2000 . Gram-negative infections fell toward the end of the 1980s, due probably to the implementation of better connectology . However, there have been no significant changes for the past 6 years . There was little change in P . aeruginosa infections over the entire time period . Pseudomonas aeruginosa is now the most common cause of catheter infection and catheter-related peritonitis . CONCLUSIONS: Prophylaxis against S . aureus is highly effective in reducing the rate of S . aureus infections but has no effect on gram-negative infections . Pseudomonas aeruginosa is now the most serious cause of catheter-related peritonitis.

Microbiology, 2003 Nov, 149(Pt 11), 3073 - 81
Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein; Medina G et al.; The Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response . Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl . The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail . Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region . It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor sigma(54) . It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.

Microbiology, 2003 Nov, 149(Pt 11), 3051 - 72
Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella; Peabody CR et al.; Homologues of the protein constituents of the Klebsiella pneumoniae (Klebsiella oxytoca) type II secreton (T2S), the Pseudomonas aeruginosa type IV pilus/fimbrium biogenesis machinery (T4P) and the Methanococcus voltae flagellum biogenesis machinery (Fla) have been identified . Known constituents of these systems include (1) . a major prepilin (preflagellin), (2) . several minor prepilins (preflagellins), (3) . a prepilin (preflagellin) peptidase/methylase, (4) . an ATPase, (5) . a multispanning transmembrane (TM) protein, (6) . an outer-membrane secretin (lacking in Fla) and (7) . several functionally uncharacterized envelope proteins . Sequence and phylogenetic analyses led to the conclusion that, although many of the protein constituents are probably homologous, extensive sequence divergence during evolution clouds this homology so that a common ancestry can be established for all three types of systems for only two constituents, the ATPase and the TM protein . Sequence divergence of the individual T2S constituents has occurred at characteristic rates, apparently without shuffling of constituents between systems . The same is probably also true for the T4P and Fla systems . The family of ATPases is much larger than the family of TM proteins, and many ATPase homologues function in capacities unrelated to those considered here . Many phylogenetic clusters of the ATPases probably exhibit uniform function . Some of these have a corresponding TM protein homologue although others probably function without one . It is further shown that proteins that compose the different phylogenetic clusters in both the ATPase and the TM protein families exhibit unique structural characteristics that are of probable functional significance . The TM proteins are shown to have arisen by at least two dissimilar intragenic duplication events, one in the bacterial kingdom and one in the archaeal kingdom . The archaeal TM proteins are twice as large as the bacterial TM proteins, suggesting an oligomeric structure for the latter.

Int J Biol Macromol, 2003 Nov, 33(1-3), 81 - 8
13C-NMR study of the interaction of bacterial alginate with bivalent cations; Lattner D et al.; The effect of bivalent cations on solutions of extracellular polymeric substances (EPS) isolated from Pseudomonas aeruginosa was monitored by means of solid-state nuclear magnetic resonance . In particular, the binding of Ca2+ and Mg2+ to the alginate in aqueous solution was studied by determining the spin-lattice relaxation rates, line widths and line shapes of 13C nuclei under variation of the ion concentration . Both cations differ strongly in their affinity towards bacterial alginate . Spectral data indicate that the strong binding capacity of calcium is connected to the formation of a chelate complex, in which binding occurs particularly with the monomer units in alternating mannuronate-guluronate blocks . In contrast to this, binding of magnesium ions was found to be much weaker and non-specific.

Environ Technol, 2003 Sep, 24(9), 1117 - 27
Microbial and copper adsorption by smectitic clay--an experimental study; Hassen A et al.; The objective of this study was to quantify copper-, bacteria- and bacteriophage-binding capacities of natural clay with the aim of predicting the adsorption of heavy metals, human pathogenic bacteria and viruses by a clayey landfill liner . X-ray diffraction analysis of six natural clays showed that the dominant phase in all deposits consists of smectites together with illite, kaolin and, sometimes, palygorskite and sepiolite . The specific surface areas of different clay substrates were very high ranging from 293 to 351 m2 g(-1), and indicating a high proportion of phyllosilicates, consisting especially of smectites . The physico-chemical identification of separated smectites showed a high potential adsorbent character indicative of a large industrial use . The Kb12 smectite substrate chosen arbitrarily among six separated substrates, appeared as an excellent copper adsorbent . Copper was adsorbed to clay in a proportion ranging from 94.6 to 96.0% with an average of 95.1% and its adsorption occurred rapidly in less than 30 min . Organic contents of the clay substrate, evaluated as 17% of dry mass, may contribute and enhance copper adsorption . Different elution protocols using distilled water, 2 and/or 5% nitric acid revealed that while nitric acid resulted in the removal of more than 59% of the metal at the lower concentration and its complete depletion with a further elution at the higher concentration, distilled water alone was unable to remove more than 1% of adsorbed copper . This finding suggested that copper ions form high-energy bonds with layer-silicate surfaces . Interestingly, the use of a regenerated substrate as copper adsorbent subsequent to abundant washings of the used substrate consecutively, with 0.1 N HNO3 and distilled water, reduced copper adsorption by approximately 14%, suggesting a slight disturbance of clay initial structure . Batch adsorption experiments with phage T7 and raw clay Kb12 showed that the tested clay substrate appeared as a relatively moderate phage adsorbent since the quantity of adsorbed phage averaged 98.2 +/- 0.88% (2 log10 retention) as measured by infectivity for Escherichia coli ATCC 11303 . As shown by two types of separating procedures, natural sedimentation and a low speed centrifugation, bacteriophage particles were bound essentially to fine and not to relatively coarse particles of the clay suspension . The retention capacity of purified clay Kb12 appeared low, with average values lower than 60 and 50%, for Pseudomonas aeruginosa ATCC 15442 and Bacillus cereus ATCC 1135, respectively . A significant increase of retention, in the order of 30%, was found for both bacteria when the mixture clay-bacteria was incubated at laboratory temperature for 6 hours.

J Artif Organs, 2003, 6(3), 205 - 10
Selection of hemoperfusion therapy for patients with septic shock on the basis of the primary disease; Kanno Y et al.; The objective of this study was to analyze retrospectively the efficacy of polymyxin-B immobilized fiber (PMX-F) alone and in combination with continuous venovenous hemofiltration (CHF) on the prognosis of critically ill patients with sepsis using a retrospective chart review in a university hospital in Japan . A cohort of 246 patients meeting the criteria of sepsis, septic shock, or both, according to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/ACCM) Consensus Conference, were examined in this study . From these patients, 48 were selected who were found to have definitive causative bacteria and whose primary diseases were clearly identified . According to the charts, two major primary diseases were identified: one related to cardiovascular disease and the other to gastrointestinal disease . Other diseases were excluded from this study because of the small numbers of patients in categories such as malignant, hematological, genitourinary, and other diseases . Furthermore, patients who had levels of serum creatinine above 2.0 mg/dl were excluded . The prevalence of diabetes mellitus (up to 63%) was very high in both groups . There were no significant differences between the two groups in age or the Apache II scores at the start of hemoperfusion treatment; however, the gender ratio varied: 72% of the cardiovascular group were male, compared to 46% of the gastrointestinal group . The causative bacteria were markedly different between the two groups . For half of the gastrointestinal group the causative bacterium was Escherichia coli, while for half of the cardiovascular group the causative bacterium was Pseudomonas aeruginosa.The survival rate differed significantly between the two groups . The patients in the cardiovascular group survived longer than those in the gastrointestinal group . Moreover, for the patients with cardiovascular disease, there was no significant difference in the survival rate between treatment with PMX-F alone and with PMX-F and CHF in combination . In contrast, for the patients with gastrointestinal disease, there was a significant difference between treatment with PMX-F alone and with PMX-F and CHF in combination . When a patient with sepsis or septic shock is treated with hemoperfusion, the decision as to whether PMX-F should be given alone or in combination with CHF might be determined on the basis of the primary disease of the patient.

Anal Biochem, 2003 Nov 15, 322(2), 208 - 14
Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy; Pacheco R et al.; A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C . 3.5.1.4), overproduced in an Escherichia coli strain . The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time . A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency . The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM . The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively . These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer . This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.

New Microbiol, 2003 Oct, 26(4), 353 - 61
Incidence and molecular epidemiology of Pseudomonas aeruginosa bacteremias in patients with acute leukemia: analysis by pulsed-field gel electrophoresis; Fanci R et al.; The incidence and molecular epidemiology of P . aeruginosa bacteremias, were monitored in patients with acute leukemia to define mechanisms of possible nosocomial transmission . From September 1997 to March 2001 febrile episodes were examined and blood isolates of P . aeruginosa were studied employing Pulsed-Field gel Electrophoresis (PFGE) . Evaluation of DNA correlation was performed according to Tenover criteria . A total of 309 febrile episodes occurred in 187 patients . Of 139 organisms isolated in 116 bacteremias, 48% were gram negative bacilli (GNB); P . aeruginosa bacteremias were recorded in 34 (51%) of GNB sepsis . Evaluation of DNA correlation showed 2 related in 1997, 7 related in 1998, 10 related in 1999, 6 related in 2000-2001 (mainly closely and possibly related); therefore isolates closely related among themselves were also possibly related with other strains . About 60% of patients with related strains were hospitalized in the same room or in different rooms but became infected in the same period . Our data suggest a horizontal spread among the patients even if other sources were possible . The study assessed the usefulness of PFGE in bacteriological epidemiology.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 219 - 27
Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase; Kurnasov O et al.; While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria . Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria . Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway . This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans . Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R . metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU) . Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.

Infect Control Hosp Epidemiol, 2003 Oct, 24(10), 749 - 52
Pseudomonas surgical-site infections linked to a healthcare worker with onychomycosis; Mermel LA et al.; OBJECTIVE: To determine the etiology of Pseudomonas aeruginosa surgical-site infections following cardiac surgery . SETTING: University teaching hospital . PATIENTS: Those with wound cultures that grew P . aeruginosa after cardiac surgery performed from 1999 to 2001 . METHODS: Medical records and operating room (OR) records of patients with P . aeruginosa cardiac surgical-site infections from 1999 to 2001 were reviewed . Healthcare workers involved with two or more cases were interviewed and examined . Specimens for environmental cultures were obtained from the ORs and cardiac surgical equipment . Cardiac surgery cases were observed and postoperative care and the cleaning of surgical instruments were investigated . OR air handling system records during the epidemic period were reviewed . Molecular fingerprinting of available P . aeruginosa isolates from infected patients and a healthcare worker was done . RESULTS: There were five P . aeruginosa cardiac surgical-site infections from January to August 2001, compared with no such infections from 1999 to 2000 . All were adult patients . One cardiac surgeon with onychomycosis operated on all five cases . He did not routinely double glove . The involved fingernail grew P . aeruginosa . Three P . aeruginosa patient isolates were available for pulsed-field gel electrophoresis; two were identical to the isolate from the involved surgeon's onychomycotic nail . No environmental OR cultures grew P . aeruginosa . The surgeon's culture-positive nail was completely removed . There have been no P . aeruginosa surgical-site infections among cardiac surgery patients since this intervention . CONCLUSION: At least two cases of a cluster of P . aeruginosa surgical-site infections resulted from colonization of a cardiac surgeon's onychomycotic nail.

J Antimicrob Chemother, 2003 Dec, 52(6), 987 - 92 Epub 2003 Oct 29.
Steady-state pharmacokinetics of intravenous colistin methanesulphonate in patients with cystic fibrosis; Li J et al.; OBJECTIVES: To define the steady-state pharmacokinetics of colistin methanesulphonate and colistin in patients with cystic fibrosis (CF) following intravenous administration of the former.Materials and methods: The study was conducted in 12 patients with CF following intravenous administration of colistin methanesulphonate (1.63-3.11 mg/kg) every 8 h for at least 2 days . On the day of study, four blood samples were collected from each patient at 60, 120, 240 and 360 min after the end of the infusion . Concentrations of colistin methanesulphonate and colistin in plasma were measured separately by HPLC . RESULTS: At steady-state, colistin methanesulphonate had a mean (+/- S.D.) total body clearance, volume of distribution and half-life of 2.01 +/- 0.46 mL/min per kg, 340 +/- 95 mL/kg and 124 +/- 52 min, respectively . Colistin had a significantly longer mean half-life of 251 +/- 79 min (P<0.001) . With the regimen used, colistin methanesulphonate was well tolerated . This is the first report on the pharmacokinetics of colistin methanesulphonate in CF patients determined using concentrations of colistin methanesulphonate and colistin in plasma . CONCLUSIONS: Based on the in vitro pharmacodynamics against Pseudomonas aeruginosa previously published by our group and these pharmacokinetic findings, dose escalating trials may be warranted to maximize efficacy.

J Antimicrob Chemother, 2003 Dec, 52(6), 911 - 4 Epub 2003 Oct 29.
In vitro effects of combinations of antipseudomonal agents against seven strains of multidrug-resistant Pseudomonas aeruginosa; Oie S et al.; OBJECTIVES: The aim of this study was to evaluate the combined effects of antibiotic combinations by agar incorporation inhibitory tests and by time-kill tests on seven geographically and epidemiologically distinct isolates of multidrug-resistant Pseudomonas aeruginosa . All seven strains were resistant to piperacillin, meropenem, ceftazidime, cefoperazone-sulbactam, aztreonam, amikacin and ciprofloxacin . METHODS: Strains were distinguished by pulsed-field gel electrophoresis after DNA extraction and restriction with SpeI . MICs of the seven antibiotics listed above were determined by agar dilution . The effect of combinations of these agents was determined by agar incorporation tests and by time-kill studies . RESULTS: Among the two-drug combinations, the combination aztreonam and amikacin was the most effective, inhibiting proliferation in five of the seven strains . Among the three-drug combinations, the combinations of piperacillin, ceftazidime and amikacin, and that of ceftazidime, aztreonam and amikacin were the most effective, inhibiting proliferation in all seven strains . In the killing tests, the three-drug combination of ceftazidime, aztreonam and amikacin was the most effective . This three-drug combination had bacteriostatic effects on all seven strains 2, 4, 6 and 24 h after drug addition, synergic effects on 2-3 strains and bactericidal effects on 1-2 strains after 4, 6 and 24 h . CONCLUSIONS: The three-drug combination of ceftazidime, aztreonam and amikacin may be effective against P . aeruginosa resistant to all commonly used antipseudomonal drugs, and deserves further study.

Drugs R D, 2003, 4(6), 383 - 5
Staphylococcus aureus vaccine conjugate--Nabi: Nabi-StaphVAX, StaphVAX; Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence; Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56100 Pisa, ItalyAlthough freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice . In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold . Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS . From the enhancement factor it is estimated that, at -13 degrees C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride . The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure . Lowering the temperature from -4 degrees C to -18 degrees C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure . It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd(2+) complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent . The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.

Biomaterials, 2004 Jan, 25(1), 139 - 46
Studies on biodegradation and release of gentamicin sulphate from interpenetrating network hydrogels based on poly(acrylic acid) and gelatin: in vitro and in vivo; Changez M et al.; Interpenetrating network hydrogels (IPNs) based on poly(acrylic acid) and gelatin (Ge) were evaluated for in vitro and in vivo biodegradation and in vivo release of gentamicin sulphate . In vitro and in vivo degradation studies demonstrated that with the increase of acrylic acid content in the polymer, the rate of degradation decreases, and a reverse phenomenon was observed with increasing Ge content in the hydrogel . The rate of in vivo degradation was much lower than in vitro degradation . Incorporation of gentamicin sulphate in hydrogel further reduces their degradation . In vitro and in vivo drug release profile showed a burst effect, followed by controlled release . Drug concentration was measured in the local skin tissue, blood serum, kidney, liver and spleen . The local skin tissue concentration of 50% and 100% gentamicin sulphate, loaded full IPNs (i.e., Ax-1 and Ax-2), was found to be higher (20+/-2mug/g) than the minimum bactericidal concentration for Staphylococcus aureus (1.2mug/g) and Pseudomonas aeruginosa (10mug/g), respectively, for a study time of 60 days.

Ann Surg, 2003 Nov, 238(5), 754 - 64
Pseudomonas aeruginosa expresses a lethal virulence determinant, the PA-I lectin/adhesin, in the intestinal tract of a stressed host: the role of epithelia cell contact and molecules of the Quorum Sensing Signaling System; Wu L et al.; OBJECTIVE: We have previously demonstrated that P . aeruginosa can have profound effects on the intestinal epithelial barrier via one of its virulence factors, the PA-I lectin/adhesin . The aims of the present study were to further characterize the interaction of P . aeruginosa and the intestinal epithelium using both in vitro and in vivo approaches . METHODS: In vitro assays examining the effect of bacterial growth phase, epithelial cell contact, and butanoyl homoserine lactone (C4-HSL), a quorum sensing signaling molecule know to affect various extracellular virulence factors in P . aeruginosa, on PA-I expression in P . aeruginosa were performed . In vivo studies were carried out by modeling catabolic stress in mice using a 30% surgical hepatectomy and direct introduction of P . aeruginosa and various virulence components into the cecum . The effect of this model on PA-I expression in P . aeruginosa was determined . RESULTS: Results demonstrated that PA-I expression in P . aeruginosa is affected by its phase of growth, its contact to the intestinal epithelium, and its exposure to the quorum sensing molecule, C4-HSL . Furthermore, data from the present study suggest that the PA-I lectin/adhesin of P . aeruginosa may be increased in vivo by local factors within the cecum of mice in response to surgical stress . CONCLUSIONS: These data indicate that multiple factors present in the intestinal microenvironment of a stressed host may induce certain opportunistic pathogens to express key virulence factors leading to a state of lethal gut-derived sepsis.

Med Dosw Mikrobiol, 2003, 55(2), 165 - 71
{Fluoroquinolone susceptibility of Pseudomonas aeruginosa strains isolated from clinical materials}; Wydmuch Z et al.; The goal of our research was an analysis of sensitivity of Pseudomonas aeruginosa to fluoroquinolones (SPX, PEF, UB, LOM, CIP, ENX, OFX, NOR) . The sensitivity was tested by disk diffusion method, according to NCCLS standards . 120 strains isolated from hospitalized (76 strains) and outpatient clinic (44 strains) persons were tested . The highest sensitivity of strains was observed to norfloxacin (36.8% and 86.4% of strains, respectively) and ciprofloxacin (30.3% and 81.8%) . None of tested microorganisms was sensitive to flumequine . Resistant strains, isolated from sick persons in outpatient clinics were fewer (13.6%) as compared to hospitalized persons (51.3%).

Antimicrob Agents Chemother, 2003 Nov, 47(11), 3548 - 53
Relevance of soft-tissue penetration by levofloxacin for target site bacterial killing in patients with sepsis; Zeitlinger MA et al.; Antimicrobial therapy of soft tissue infections in patients with sepsis sometimes lacks efficiency, despite the documented susceptibility of the causative pathogen to the administered antibiotic . In this context, impaired equilibration between the antibiotic concentrations in plasma and those in tissues in critically ill patients has been discussed . To characterize the impact of tissue penetration of anti-infective agents on antimicrobial killing, we used microdialysis to measure the concentration-versus-time profiles of levofloxacin in the interstitial space fluid of skeletal muscle in patients with sepsis . Subsequently, we applied an established dynamic in vivo pharmacokinetic-in vitro pharmacodynamic approach to simulate bacterial killing at the site of infection . The population mean areas under the concentration-time curves (AUCs) for levofloxacin showed that levofloxacin excellently penetrates soft tissues, as indicated by the ratio of the AUC from time zero to 8 h (AUC(0-8)) for muscle tissue (AUC(0-8 muscle)) to the AUC(0-8) for free drug in plasma (AUC(0-8 plasma free)) (AUC(0-8 muscle)/AUC(0-8 plasma free) ratio) of 0.85 . The individual values of tissue penetration and maximum concentration (C(max)) in muscle tissue were highly variable . No difference in bacterial killing of a select Staphylococcus aureus strain for which the MIC was 0.5 microg/ml was found between individuals after exposure to dynamically changing concentrations of levofloxacin in plasma and tissue in vitro . In contrast, the decrease in the bacterial counts of Pseudomonas aeruginosa (MIC = 2 microg/ml) varied extensively when the bacteria were exposed to levofloxacin at the concentrations determined from the individual concentration-versus-time profiles obtained in skeletal muscle . The extent of bacterial killing could be predicted by calculating individual C(max)/MIC and AUC(0-8 muscle)/AUC(0-8 plasma free) ratios (R = 0.96 and 0.93, respectively) . We have therefore shown in the present study that individual differences in the tissue penetration of levofloxacin may markedly affect target site killing of bacteria for which MICs are close to 2 microg/ml.

Antimicrob Agents Chemother, 2003 Nov, 47(11), 3442 - 7
Cefepime versus imipenem-cilastatin for treatment of nosocomial pneumonia in intensive care unit patients: a multicenter, evaluator-blind, prospective, randomized study; Zanetti G et al.; In a randomized, evaluator-blind, multicenter trial, we compared cefepime (2 g three times a day) with imipenem-cilastatin (500 mg four times a day) for the treatment of nosocomial pneumonia in 281 intensive care unit patients from 13 centers in six European countries . Of 209 patients eligible for per-protocol analysis of efficacy, favorable clinical responses were achieved in 76 of 108 (70%) patients treated with cefepime and 75 of 101 (74%) patients treated with imipenem-cilastatin . The 95% confidence interval (CI) for the difference between these response rates (-16 to 8%) failed to exclude the predefined lower limit for noninferiority of -15% . In addition, therapy of pneumonia caused by an organism producing an extended-spectrum beta-lactamase (ESBL) failed in 4 of 13 patients in the cefepime group but in none of 10 patients in the imipenem group . However, the clinical efficacies of both treatments appeared to be similar in a secondary intent-to-treat analysis (95% CI for difference, -9 to 14%) and a multivariate analysis (95% CI for odds ratio, 0.47 to 1.75) . Furthermore, the all-cause 30-day mortality rates were 28 of 108 (26%) patients in the cefepime group and 19 of 101 (19%) patients in the imipenem group (P = 0.25) . Rates of documented or presumed microbiological eradication of the causative organism were similar with cefepime (61%) and imipenem-cilastatin (54%) (95% CI, -23 to 8%) . Primary or secondary resistance of Pseudomonas aeruginosa was detected in 19% of the patients treated with cefepime and 44% of the patients treated with imipenem-cilastatin (P = 0.05) . Adverse events were reported in 71 of 138 (51%) and 62 of 141 (44%) patients eligible for safety analysis in the cefepime and imipenem groups, respectively (P = 0.23) . Although the primary end point for this study does not exclude the possibility that cefepime was inferior to imipenem, some secondary analyses showed that the two regimens had comparable clinical and microbiological efficacies . Cefepime appeared to be less active against organisms producing an ESBL, but primary and secondary resistance to imipenem was more common for P . aeruginosa . Selection of a single agent for therapy of nosocomial pneumonia should be guided by local resistance patterns.

Antonie Van Leeuwenhoek, 2003, 84(3), 193 - 200
Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa; Smits TH et al.; Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P . aeruginosa isolates often grow on medium- as well as long-chain n-alkanes . Heterologous expression showed that the two alkane hydroxylase homologs of P . aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C(12)-C(16)n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P . putida GPo1 counterparts in n-octane oxidation . The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P . aeruginosa strains tested in this study.

Ophthalmologica, 2003 Nov-Dec, 217(6), 426 - 30
Antibacterial activity of intraocularly used liquids against two strains of Pseudomonas aeruginosa; Economou-Stamatelopoulou C et al.; The antibacterial effectiveness of liquids used as intraocular tamponading agents in vitrectomy was tested in this study . Two strains of Pseudomonas aeruginosa were used as challenge organisms: the ATCC 9027 strain and a randomly selected resistant strain . The liquid intraocular tamponading agents tested were perfluorocarbons, balanced salt solutions, and silicone oils . Perfluorocarbons demonstrated a significant decrease in growth of the ATCC 9027 strain and sufficient protection against the resistant strain of P . aeruginosa . The balanced salt solutions as well as the silicone oils demonstrated insufficient protection against both strains used . There is a correspondence of results in the pairs of chemically similar substances .

J Biol Chem, 2004 Jan 2, 279(1), 755 - 64 Epub 2003 Oct 22.
Probing the functional importance of the hexameric ring structure of RNase PH; Choi JM et al.; RNase PH is a phosphate-dependent exoribonuclease that catalyzes the removal of nucleotides at the 3' end of the tRNA precursor, leading to the release of nucleoside diphosphate, and generates the CCA end during the maturation process . The 1.9-A crystal structures of the apo and the phosphate-bound forms of RNase PH from Pseudomonas aeruginosa reveal a monomeric RNase PH with an alpha/beta-fold tightly associated into a hexameric ring structure in the form of a trimer of dimers . A five ion pair network, Glu-63-Arg-74-Asp-116-Arg-77-Asp-118 and an ion-pair Glu-26-Arg-69 that are positioned symmetrically in the trimerization interface play critical roles in the formation of a hexameric ring . Single or double mutations of Arg-69, Arg-74, or Arg-77 in these ion pairs leads to the dissociation of the RNase PH hexamer into dimers without perturbing the overall monomeric structure . The dissociated RNase PH dimer completely lost its binding affinity and catalytic activity against a precursor tRNA . Our structural and mutational analyses of RNase PH demonstrate that the hexameric ring formation is a critical feature for the function of members of the RNase PH family.

J Surg Res, 2003 Nov, 115(1), 139 - 47
Sepsis-induced failure of hepatic energy metabolism; Hart DW et al.; HYPOTHESIS: Recent evidence suggests that sepsis may induce an uncoupling of oxidative phosphorylation . The purpose of this study was to quantify temporal changes in hepatic oxygen consumption and cellular energy state with increasing severity of sepsis and thus assess the interrelationship of these parameters as either primary defect or compensatory response . MAIN OUTCOME MEASURES: Pseudomonas aeruginosa was infused intravenously in eight instrumented anesthetized swine inducing a progressive severity of sepsis to shock . Eight other animals served as instrumented controls . Hepatic blood flow, oxygen use, and concentrations of ATP, ADP, AMP, NAD(+), and NADH were measured at baseline and then sequentially during the study . RESULTS: Except for an increase in heart rate, there were no temporal changes in measured values for the control animals . For swine receiving P . aeruginosa, hepatic oxygen delivery and consumption increased with early sepsis whereas there were no alterations in the concentrations of adenine nucleotides or NAD(+)/NADH within liver . Septic shock was notable for a decrease in oxygen delivery yet oxygen consumption remained elevated because of an increase in percent oxygen extraction . The hepatic concentrations of ATP and NADH decreased during septic shock . CONCLUSIONS: These findings suggest that any sepsis-induced limitation in phosphorylation may be initially compensated by an increase in oxygen use . This study also suggests that decreases in NADH availability may be a principal factor in the decompensation of sepsis to shock.

Biodegradation, 2003 Oct, 14(5), 309 - 19
TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain; Oh BT et al.; Successful microbial-mediated remediation requires transformation pathways that maximize metabolism and minimize the accumulation of toxic products . Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L(-1) in culture medium within 10 h under aerobic conditions . The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) . The 2HADNT and 2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT . Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium . While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH . The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P . aeruginosa strain MX.

Curr Microbiol, 2003 Sep, 47(3), 186 - 91
Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures; Kuo TM et al.; Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) . Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor . Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition . Increases of medium volume and substrate concentration failed to enhance yield . When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable . This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture . Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel . This is the first report on a successful scale-up production of DOD.

Can J Microbiol, 2003 Jul, 49(7), 450 - 64
Regulation of toxA by PtxR in Pseudomonas aeruginosa PA103; Carty NL et al.; Exotoxin A (ETA) production in Pseudomonas aeruginosa requires the regulatory locus regAB . Pseudomonas aeruginosa PA103 produces significantly higher levels of ETA than the prototypic strain PAO1 does, partly because of differences in the regAB locus . Other factors that contribute to this variation are not known . We previously described the P . aeruginosa gene ptxR that positively regulates production of ETA through regAB . ETA production was enhanced but still iron regulated in the PAO1 strain PAO1-XR that carries two copies of ptxR on its chromosome . Here we determine whether ptxR regulation of ETA is different in PA103 . In contrast to PAO1-XR, ETA activity produced by PA103-2R, a PA103 strain carrying two copies of ptxR, is enhanced tenfold and partially deregulated in the presence of iron . Real-time PCR transcriptional analysis showed that the copy number of toxA mRNA in PA103-2R is significantly higher than in PA103 in both the presence and absence of iron, yet no similar increase in either regAB or ptxR mRNA copy number was detected . The integrated plasmid together with adjoining DNA was retrieved from the PA103-2R chromosome to determine whether integration-induced DNA changes played a role in this phenotype . Introduction of the retrieved plasmid in PA103 produced a phenotype similar to that of PA103-2R . Sequence analysis of the plasmid revealed the loss of 322 bp within the region 3' of ptxR . A plasmid construct carrying a 4-bp insertion in this same region produced in PA103 a phenotype similar to that of PA103-2R . Our results suggest that the effect of ptxR on toxA expression is different in PA103 than in PAO1 and that this variation in PA103-2R does not occur solely through regAB . Changes within the region 3' of ptxR are critical for the production of the unique PA103-2R phenotype, which occurs in trans and requires intact ptxR, but is not caused by ptxR overexpression.

Arch Otolaryngol Head Neck Surg, 2003 Oct, 129(10), 1098 - 100
Evaluation of topical povidone-iodine in chronic suppurative otitis media; Jaya C et al.; OBJECTIVES: To evaluate if povidone-iodine (PVP-I) can be used topically in the treatment of chronic suppurative otitis media-tubotympanic disease and to compare it with ciprofloxacin hydrochloride ear drops . DESIGN: Prospective double-blind randomized study . SETTING: Academic tertiary medical center . PATIENTS: Forty patients with chronic suppurative otitis media were randomized into 2 groups . INTERVENTION: One group (19 patients) received 5% PVP-I ear drops, while the other group (21 patients) received 0.3% ciprofloxacin ear drops . Both were administered topically, 3 drops 3 times daily for 10 days . These patients were followed up at weekly intervals for up to 4 weeks after commencing therapy . RESULTS: Clinical improvement at the end of study was 88% in the PVP-I group and 90% in the ciprofloxacin group . The most commonly isolated organism was Pseudomonas aeruginosa . In vitro resistance to ciprofloxacin was seen in 17% of organisms, while no resistance was seen for PVP-I . CONCLUSIONS: To our knowledge, this is the first study to evaluate the efficacy of PVP-I as a topical agent in the treatment of chronic suppurative otitis media . The results show that clinically, topical PVP-I is as effective as topical ciprofloxacin, with a superior advantage of having no in vitro drug resistance . Also, there is an added benefit of reduced cost of therapy.

Pathol Biol (Paris), 2003 Oct, 51(8-9), 460 - 3
{Which betalactam antibiotic use as a marker of multiresistance in Pseudomonas aeruginosa?}; Cavallo JD et al.; The determination of an indicating antibiotic for multiresistance, as methicillin in staphylococci, can be useful for Pseudomonas aeruginosa . Until now, the majority of the hygienists used ticarcillin, ceftazidim or imipenem in their investigations as markers of multiresistance for this species . Piperacillin has never been proposed for this purpose . To evaluate this choice, 2098 non-repetitive P . aeruginosa strains collected from 15 teaching hospitals in 1997-1999 were analysed, for eight antibiotics (ticarcillin, piperacillin, ceftazidim, imipenem, tobramycin, amikacin, ciprofloxacin, fosfomycin) according (i) to the results of the minimal inhibiting concentrations obtained by dilution in Mueller-Hinton agar, (ii) to their susceptibility following the criteria of Comite de l'antibiogramme de la Societe Francaise de Microbiologie and (iii) to the determination of the mechanisms of resistance to the beta-lactam antibiotics . The low rates of sensitivity to the beta-lactam antibiotics, aminoglycosides, ciprofloxacin and fosfomycin were more frequent for piperacillin-resistant strains than for ceftazidim-resistant ones . Resistance to the other beta-lactam antibiotics are poor markers of multiresistance . In the light of the presented data, piperacillin seems to be, among the beta-lactam antibiotics, the best candidate as a marker of multiresistance for P . aeruginosa, followed by ceftazidim . This multiresistance is mainly found in strains overproducing AmpC cephalosporinase or transferable beta-lactamases . These mechanisms are well detected by resistance to piperacillin.

Pathol Biol (Paris), 2003 Oct, 51(8-9), 443 - 8
{Mechanism of adaptive resistance to aminoglycosides of Pseudomonas aeruginosa}; Hocquet D et al.; Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, << adaptive >> resistance to these antibiotics . In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P . aeruginosa . Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1 . Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration) . In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance . Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.

Am J Respir Cell Mol Biol, 2004 Apr, 30(4), 576 - 84 Epub 2003 Oct 17.
Deficiency in neutrophil elastase does not impair neutrophil recruitment to inflamed sites; Hirche TO et al.; To reach the sites of inflammation, neutrophils traverse the endothelium, its underlying basement membrane, and other barriers depending on the localization of the insulting agent . Whether neutrophil elastase (NE) plays a role in neutrophil recruitment to inflamed sites is still debatable . By exploiting mice deficient in NE (NE(-/-)), we sought to address this dilemma . We recruited neutrophils to the lungs or the peritoneum of wild-type (WT) or NE(-/-) mice by intranasal or intraperitoneal challenge with Pseudomonas aeruginosa or its lipopolysaccharide . At designated times post-inoculation (0, 4, 24, and 48 h), groups of mice were killed to assess changes in leukocyte counts and inflammatory responses . NE(-/-) and WT mice had normal circulating leukocyte numbers including neutrophils and changes in the hemograms in the setting of acute inflammation were indistinguishable . Analyses of lung tissues or fluids from the lungs and peritoneum found that regardless of the inflammatory model, the leukocyte counts including neutrophils and the inflammatory response were similar in NE(-/-) and WT mice at all time points . In vitro, neutrophils isolated from the lungs or the peritoneum of NE(-/-) and WT mice had comparable chemotactic and respiratory-burst functions and migrated normally through Matrigel in response to various stimuli . Interestingly, preincubation of human peripheral blood neutrophils with NE physiologic inhibitors did not alter the migration of the cells through Matrigel . In sum, our findings present the first in vivo description that the absence of NE does not impair neutrophil recruitment to inflamed sites and that NE is not required for basement membrane transmigration of neutrophils.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Sep-Oct, (5), 72 - 6
{Mode of the rational use of Pseudomonas aeruginosa bacteriophages in therapeutic and epidemic control practice}; Aslanov BI et al.; Ecological aspects of the circulation of P . aeruginosa and P . aeruginosa bacteriophages under hospital conditions were under study . The statement concerning the formation of triple parasitic systems was put forward . The influence of these systems on the formation of phage and antibiotic resistance in P . aeruginosa hospital strains was studied . Spontaneous circulation of faintly virulent phages taking part in the formation of triple parasitic systems was shown not to ensure the elimination of P . aeruginosa hospital strains in clinics . Construction of highly virulent phages adapted to local P . aeruginosa strains was the only way of ensuring the protection of patients . Theoretical and practical approaches to the use of highly active bacteriophages for controlling P . aeruginosa infection were substantiated . The realization of these approaches resulted in achieving not only a clinical, but also essential epidemic control effect in cases of purulent septic infections caused by P . aeruginosa (a decreased frequentcy of hospital infections from 40.8% to 8.93%).

Med Pregl, 2003 May-Jun, 56(5-6), 237 - 42
{Current aspects in pharmacologic use of bile acids}; Mikov M et al.; EFFECTS OF BILE ACIDS AND THEIR SALTS ON ABSORPTION OF OTHER SUBSTANCES: Bile acids and their salts increase intestinal absorption of lipids and transmembrane and paracellular transfer of small and endogenous and exogenous polar molecules . It has been established that they are good promotors of insulin absorption through skin and nasal mucose, and of blood-brain barrier transfer of salycilates and quinine . EFFECTS OF BILE ACIDS AND THEIR SALTS ON ABSORPTION OF OTHER SUBSTANCES AND THEIR POTENTIAL ACTION: It has been established that combination of bile acids with amphotericin B has potential Leishmanicideal effect and combination with ciprofloxacine has improved its antibacterial activity against Pseudomonas aeruginosa in vitro . BILE ACIDS: PHARMACADYNAMIC EFFECTS: Bile acids have analgesic and hypoglycemic effect . They also have anti-HIV effect probably suppressing virus transmission from cell to cell . CONCLUSION: New studies of natural bile acids and new synthetic bile acids have revealed that they are not only adjuvants to existing active principles in pharmaceutical forms, but they can act as new therapeutic agents . However, it is necessary to study their possible mechanisms, but they are not crucial for their therapeutic application . Toxicological and pharmacological studies will determine the role of newly synthesized bile acids and their salts in current therapy.

Infection, 2003 Aug, 31(4), 208 - 15
Design of a surveillance system of antibiotic use and bacterial resistance in German intensive care units (SARI); Meyer E et al.; BACKGROUND: Data on antibiotic consumption and bacterial resistance are important for benchmarking, ensuring quality of antibiotic treatment and helping to understand the relationship between the use of antibiotics and the emergence of resistance . METHODS: The SARI project is an ecological study that has established laboratory-based surveillance in German intensive care units (ICU) . Resistance rates of 13 sentinel pathogens are reported and certain alert organisms are sent for genotyping and retesting of antimicrobial resistance . RESULTS: The project, initiated in February 2000, now includes 35 ICUs generating a total of 266,013 patient days, 354,356 defined daily doses (DDD) and providing susceptibility data on 21,354 isolates . Pooled antibiotic usage density (AD = DDD/1,000 patient days) was highest for penicillins with lactamase inhibitor (AD 338.3) followed by quinolones (AD 155.5) and second-generation cephalosporins (AD 124.6) . Total AD was calculated as 1,337 DDD/1,000 patient days . Resistance rates (RR) for laboratories testing according to the German Industrial Standard (DIN) were 19.3% for methicillin-resistant Staphylococcus aureus (MRSA), 9.5% for ciprofloxacin-resistant Escherichia coli and 25.4% for imipenem-resistant Pseudomonas aeruginosa . 40% of the laboratories did not identify the extended spectrum beta lactamase production of a Klebsiella pneumoniae strain . CONCLUSION: Focusing on German ICUs, the SARI surveillance system provides a concept that produces a benchmark for the link between antibiotic resistance and consumption.

Curr Eye Res, 2003 Nov, 27(5), 289 - 99
A comparison of invasive and cytotoxic Pseudomonas aeruginosa strain-induced corneal disease responses to therapeutics; Lee EJ et al.; PURPOSE: During corneal infection, cytotoxic Pseudomonas aeruginosa strains remain mostly extracellular, while invasive strains can enter corneal cells and replicate within them . We tested the hypothesis that ofloxacin, which easily penetrates host cell membranes, would be more effective than the less cell-permeable antibiotic tobramycin, for treatment of corneal infection by an invasive P . aeruginosa strain . METHODS: A murine model of P . aeruginosa keratitis was used to compare the response to ofloxacin, tobramycin, prednisolone acetate, and non-preserved saline treatment, as well as combination antibiotic-corticosteroid therapy for infection caused by a cytotoxic strain (6206) and an invasive strain (PAO1) . Treatment involved hourly eye drop administration for 12 hours . RESULTS: As expected, tobramycin was less effective at eradicating viable bacteria from corneas infected with the invasive strain . Despite rapid sterilization of corneas in other antibiotic treated groups, disease progression occurred during the 12 hour treatment period . Both antibiotics hastened disease resolution over the next 7 days for infections caused by either strain . Corticosteroid use during the 12 hour treatment period was of little added benefit . CONCLUSIONS: Differences between invasive and cytotoxic strain infections in their early response to the different therapeutic regimens did not translate to notable differences after 7 days, but the effects of antibiotics in halting disease progression were delayed for both strain types . These results suggest that successful management might be improved by addressing factors contributing to disease progression during sterilization of the cornea by antibiotics.

Shock, 2003 Nov, 20(5), 437 - 43
Bcl-2 inhibits gut epithelial apoptosis induced by acute lung injury in mice but has no effect on survival; Husain KD et al.; Gut epithelial apoptosis is increased in human studies and animal models of noninfectious inflammation and sepsis . Elevated intestinal cell death appears to be physiologically significant in sepsis . Previous studies demonstrate that overexpression of the antiapoptotic protein Bcl-2 in the gut epithelium of transgenic mice is associated with improved survival from Pseudomonas aeruginosa pneumonia and cecal ligation and puncture . The functional significance of elevated gut apoptosis in noninfectious inflammation has not been examined . We hypothesized that intestinal apoptosis would be detrimental to survival in noninfectious critical illness . To address this issue, acute lung injury (ALI) was induced with intratracheal injection of lipopolysaccharide (LPS, 800 microg) in wild-type (WT) FVB/N mice and transgenic mice that overexpress Bcl-2 in their intestinal epithelium . Guts were harvested at 12, 24, 48, and 72 h and assessed for apoptosis by both hematoxylin and eosin and active caspase-3 staining in 100 contiguous crypts . ALI increased gut epithelial apoptosis 12 h after LPS instillation compared with shams (P < 0.01), whereas overexpression of Bcl-2 decreased intestinal apoptosis compared with WT animals with ALI when assayed by active caspase-3 (P < 0.05) . Plasma levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-10 were similar between WT and transgenic animals with ALI, both of which had elevated IL-10 levels at 12 h and elevated IL-6 levels at 24 h compared with sham animals . In a separate experiment, transgenic and WT animals with ALI were followed for mortality to determine whether gut overexpression of Bcl-2 conferred a survival advantage . Survival at 10 days was 73% in WT animals (n = 33) and 65% in Bcl-2 animals (n = 23, P = ns) . These results indicate that while gut epithelial apoptosis is elevated in multiple models of critical illness, prevention of intestinal cell death by overexpression of Bcl-2 is associated with a disparate survival effect between sepsis and noninfectious inflammation.

Nutrition, 2003 Oct, 19(10), 880 - 5
Effects of dietary glutamine on antioxidant enzyme activity and immune response in burned mice; Yeh SL et al.; OBJECTIVES: We investigated the effect of dietary glutamine (Gln) 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 (PEIF) . We also evaluated the survival rate of vaccinated and non-vaccinated burned mice infected with Pseudomonas aeruginosa . METHODS: There were three consecutive experiments . In experiment 1, 30 BALB/c mice were assigned to one of two groups . The control group was fed casein as the protein source; the Gln group received 4% Gln (w/w) to replace part of the casein . Mice were immunized twice with PEIF, and the production of specific antibodies against PEIF was measured every week . Eight weeks after immunization, all mice received a 30% body surface area burn injury . Mice were killed 24 h after the burn . The antioxidant enzyme activities and lipid peroxides in the tissues and specific antibody production were analyzed . In experiment 2, 12 mice were assigned to a control or a Gln group and fed with one the experimental diets for 4 wk . Then burn injury was induced, and mice were killed 24 h later . In vitro, splenocytes were cultured, and interleukin (IL)-4 and IL-10 were measured after mitogen stimulation . In experiment 3, survival rates of vaccinated and non-vaccinated burned mice complicated with P . aeruginosa infection were evaluated . The survival rate was observed for 8 d after the burn . RESULTS: Antioxidant enzyme activities and lipid peroxides in tissues tended to be lower in the Gln group than in the control group after the burn . Specific antibody production against P . aeruginosa increased significantly in the Gln group at 4 and 7 wk after immunization and at 24 h after the burn . IL-4 concentrations in mitogen-stimulated splenocytes were significantly higher in the Gln group than in the control group . Survival rates of non-vaccinated burned mice in the Gln group were significantly higher than those in the control group, whereas there was no difference in the survival of vaccinated burned mice after bacterial infection . CONCLUSIONS: These results suggested that vaccinated mice receiving a Gln-enriched diet may have enhanced humoral immunity and attenuated oxidative stress induced by burn injury . Also, Gln supplementation improved the survival of burned mice complicated with P . aeruginosa infection.

FEMS Immunol Med Microbiol, 2003 Oct 24, 39(1), 37 - 44
Pseudomonas aeruginosa-specific IgG1 and IgG2 subclasses in enhancement of pulmonary clearance following passive immunisation in the rat; Dunkley ML et al.; Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which causes serious debilitating infections in patients with compromised lung function . The mechanism by which P . aeruginosa is cleared from the lung is not fully defined, although our previous studies have established a role for cellular immunity in protection against P . aeruginosa infections . This study aimed to evaluate the role of P . aeruginosa-specific IgG in protection against P . aeruginosa in a rat model of acute pulmonary infection . Immunoaffinity chromatography was used to purify total rat IgG from rat immune serum (rats immunised with P . aeruginosa) and non-immune serum . Untreated recipient rats were injected intravenously with different concentrations of pure IgG prepared from serum of unimmunised rats (non-immune IgG) or from rats immunised intestinally with killed P . aeruginosa (immune IgG) and infected intratracheally with P . aeruginosa 18 h later . The protective capability of the purified IgG against P . aeruginosa was assessed by measurement of reduction in P . aeruginosa infection in the lung 4 h after instillation of bacteria . Enhanced bacterial clearance induced by IgG was determined to be dose-dependent with a 1 mg dose failing to enhance clearance, whereas 5 mg of immune IgG enhanced clearance from the airways and the lung tissue . Measurement of the IgG1, IgG2a and IgG2b isotypes in serum and the lung lavage following transfer of P . aeruginosa-specific IgG found that all three were present . These results demonstrate that anti-P . aeruginosa IgG can enhance bacterial clearance from the airways in an acute infection and identify an important role for IgG in acute respiratory infections caused by P . aeruginosa.

Eye Contact Lens, 2003 Oct, 29(4), 255 - 7
Pseudomonas keratitis associated with continuous wear silicone-hydrogel soft contact lens: a case report; Lee KY et al.; PURPOSE: To report a case of Pseudomonas aeruginosa culture-positive microbial keratitis in a patient wearing continuous-wear silicone hydrogel soft contact lenses . METHODS: A 23-year-old white woman in good health had been wearing silicone hydrogel (lotrafilcon A) soft contact lenses continuously for 26 days when she was examined for a corneal ulcer in her left eye . She had given a history of water jet skiing and diving while wearing her contact lenses . Scrapings of the corneal ulcer were positive for P . aeruginosa, and the patient was treated with fortified topical cefazolin and gentamicin for 1 week and subsequently with topical ciprofloxacin for 2 weeks . RESULTS: The microbial keratitis resolved with successful treatment . However, the patient had a residual visual deficit secondary to stromal scarring . CONCLUSIONS: The recently introduced continuous-wear silicone hydrogel soft contact lenses, with their hyper oxygen permeability (Dk), have been shown to overcome hypoxia-associated complications and to have less P . aeruginosa binding to the corneal epithelium . Our case shows that sight-threatening microbial keratitis can still occur even with silicone hydrogel soft contact lenses . Contact lens practitioners should educate patients on the risk of sight-threatening microbial keratitis, the need for patient compliance, and prompt assessment of contact lens-related complaints.

Eye Contact Lens, 2003 Oct, 29(4), 207 - 9
Ulcerative keratitis in contact lens wearers; Mela EK et al.; PURPOSE: To determine the clinical microbiological characteristics of corneal ulcers in contact lens wearers . METHODS: A retrospective study of 23 patients admitted to our department with contact lens-related corneal ulcers during a 43-month period . Detailed demographic data, the type of contact lens, duration of lens wear, and wearing schedule were derived from a self-administered questionnaire . The severity of the ulcer; cultures of corneal scrapings, storage solutions, and contact lenses; treatment; and final outcome were evaluated . RESULTS: Of the 86 cases of ulcerative keratitis admitted during the study period, 23 (26.74%) were attributed to contact lens use . Most patients were young women from urban areas . All of them were using soft contact lenses for 3 days to 20 years . Five patients used daily-wear lenses as extended-wear lenses . Most ulcers (47.82%) were mild; 30.43% were moderate; and 21.47% were severe . Corneal scrapings for cultures were obtained in 15 of the cases and were positive in 10 (43.47%) of them, whereas in 33.33% of the culture-positive storage solutions and in 66.67% of the culture-positive contact lenses, corneal scrapings were negative . Pseudomonas aeruginosa was the most frequent isolated pathogen (60%) . The final visual acuity was 20/40 or better in 60.87% of the cases . CONCLUSIONS: Contact lens use is an important risk factor for the development of ulcerative keratitis, with P . aeruginosa remaining the predominant pathogen . It seems important to culture contact lenses and contact lens storage solutions, in addition to the corneal scrapings, and the role of initial therapy for the corneal ulcers remains important.

Chest, 2003 Oct, 124(4), 1460 - 8
The effect of pregnancy on survival in women with cystic fibrosis; Goss CH et al.; STUDY OBJECTIVES: Patients with cystic fibrosis (CF) are currently living to their fourth decade and are making reproductive decisions . Information concerning the reproductive health of women with CF has been limited to small or single-center studies . DESIGN: We conducted a matched parallel-cohort study to assess the impact of pregnancy on the survival of women with CF . PARTICIPANTS: A parallel-cohort study included all women > 12 years of age who were enrolled in the US Cystic Fibrosis Foundation National Patient Registry from 1985 to 1997 . MEASUREMENTS AND RESULTS: Six hundred eighty of the 8,136 women in the cohort became pregnant . These 680 women were matched on an index year to 3,327 control women with CF . At the inception of entry into the cohort, women who reported pregnancy were more likely to have had a higher percentage of predicted FEV(1) (67.5% predicted vs 61.7% predicted, respectively; p < 0.001) and a higher weight (52.9 vs 46.4 kg, respectively; p < 0.001) . Using Kaplan-Meier survival curves, the 10-year survival rate in pregnant women (77%; 95% confidence interval {CI}, 71 to 82%) was higher than in those women who did not become pregnant (58%; 95% CI, 55 to 62%) . A separate analysis, matching pregnant patients on FEV(1) percent predicted, age, Pseudomonas aeruginosa colonization, and pancreatic function, obtained similar results . Using Cox proportional hazard modeling to adjust for baseline age, FEV(1) percent predicted, weight, height, and pulmonary exacerbation rate per year, pregnancy was not associated with an increase risk of death . Pregnancy was not harmful in any subgroup including patients with FEV(1) < 40% of predicted or diabetes mellitus . CONCLUSIONS: Women with CF who became pregnant were initially healthier and had better 10-year survival rates than women with CF who did not become pregnant . After adjustment for the initial severity of illness, women who became pregnant did not have a significantly shortened survival.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2003 Oct, 15(10), 625 - 7
{Change of the erythrocyte chemokine receptor binding activity in scalded rats with Pseudomonas aeruginosa infection}; Chen XD et al.; OBJECTIVE: To explore the influence of infection on the erythrocyte chemokine receptor (ECKR) binding activity in severely scalded rats . METHODS: The Sprague-Dawley (SD) rats were randomly divided into three group: sham scald group (A), burn and infection group (B) and infection group (C) . The B group rats were scalded with 30% total body surface area (TBSA) of III degree, and the rats ECKR binding activity with interleukin-8 (IL-8) as ligand were detected by enzyme-linked immunosorbent assay (ELISA) at 1, 2, 4, 6 and 8 hours after infection . RESULTS: Compared to that in A group, ECKR binding activities declined significantly (both P<0.01) after infection in both of B and C groups, but they increased at 8 hours (P<0.01) . ECKR binding activity in B group was significantly less at 2, 8 hours than that in C group after infection (both P<0.05) . The declining range of ECKR binding activity was more in B group, in which the decline of ECKR binding activity appeared earlier (2 hours) than that in C (6 hours) group . CONCLUSION: The infection lead to the decline of ECKR binding activity in burned rats, and the erythrocytes might participate in the chemokine regulation and play a novel role in the infection.

Environ Toxicol Chem, 2003 Oct, 22(10), 2306 - 11
Effect of sorption on benzene biodegradation in sandy soil; Kim SB et al.; The effect of sorption on benzene biodegradation in sandy soil was studied by conducting kinetic microcosm batch tests in soil-free solution and in the presence or absence of bacteria in soil materials with varying degrees of powdered activated carbon (PAC) . In the soil-free experiment, benzene was added to a solution inoculated with Pseudomonas aeruginosa bacteria in order to achieve a potential or maximum biodegradation rate . In subsequent experiments, benzene was applied to a solution containing sandy soil and various PAC contents with and without inoculating P . aeruginosa . Benzene concentrations in the soil-free experiments decreased with time with two characteristic rates . A two-stage exponential decay model adequately represented the observed solution concentration pattern with time . Sorption experiments in bacteria-free soil also decreased monotonically, with the extent of sorption increasing as PAC content increased . The sorption data were represented well with a two-stage irreversible sorption model . A third set of experiments in the presence of both soil and bacteria showed more rapid concentration loss from solution than the set of experiments with bacteria-free soil . A model combining sorption and degradation greatly overestimated the loss when the rate coefficient from the bacteria-free experiments was used . Satisfactory agreement between model predictions and observed values was obtained when the degradation rate coefficients were decreased by factors ranging from 3 to 10, depending on the amount of PAC present . Model predictions of the percentage benzene mass remaining in the soil after 25 d of degradation ranged from 72 to 97%, depending on the PAC content, compared to only 2.5% remaining in soil-free solution.

Curr Opin Pulm Med, 2003 Nov, 9(6), 492 - 7
Pseudomonas acquisition in young patients with cystic fibrosis: pathophysiology, diagnosis, and management; Rosenfeld M et al.; PURPOSE OF REVIEW: To summarize the pathophysiology of, risk factors for, and outcomes of early Pseudomonas aeruginosa (Pa) infection in CF; to review the results of trials of early intervention and to describe treatment options for early Pa infection . RECENT FINDINGS: Chronic lower airway Pa infection is associated with significant morbidity and mortality among CF patients . However, first acquisition of Pa does not appear to cause an immediate and rapid decline in lung function . Early Pa isolates are generally non-mucoid, antibiotic-sensitive, and present at low density, suggesting a possible "window of opportunity" for early intervention . SUMMARY: Anti-pseudomonal therapy for early infection results in transient Pa eradication, but re-infection with Pa appears inevitable despite early aggressive treatment . There are no controlled trials demonstrating clinical benefit in young children . There is a critical need for further investigation of the clinical outcomes associated with early intervention, the long-term safety profile, and the optimal drug regimen.

Braz J Infect Dis, 2003 Aug, 7(4), 234 - 5 Epub 2003 Dec 08.
An outbreak of conjunctivitis caused by multiresistant Pseudomonas aeruginosa in a Brazilian newborn intensive care unit; Brito DV et al.; We report an outbreak of conjunctivitis due to Pseudomonas aeruginosa involving seven infants admitted in the Neonatal Intensive Care Unit (NICU) of the Uberlandial Federal University Hospital between March and September 2001 . Three infants developed systemic complications (01 sepsis and 02 pneumonias) . Ten isolates were obtained from conjunctival cultures and all were resistant to ceftazidime and aminoglycosides . Fast identification of the organism and treatment with imipenem were important in containing the outbreak of P . aeruginosa.

Folia Microbiol (Praha), 2003, 48(4), 529 - 33
Lack of efflux mechanism in a clinical isolate of Pseudomonas aeruginosa highly resistant to beta-lactams and imipenem; Kadry AA; An isolate of Pseudomonas aeruginosa from cystic fibrosis was highly resistant to beta-lactams and beta-lactamase inhibitors . The resistant determinants of clinical isolate to imipenem, ceftazidim, cefriaxone and cefepime were conjugally nontransferable . The slow or nonenzymically mediated breakdown of imipenem and other broad-spectrum beta-lactams suggested the resistance of P . aeruginosa isolate to these drugs which may be attributed to both permeability and efflux . Impaired penetration of imipenem and other beta-lactams through the membrane was detected by a diminished expression of outer-membrane proteins of approximate molar mass of 46 and 39 kDa, matched to OprD and OprF, respectively . Efflux resistance mechanism for meropenem and beta-lactams has been ruled out since the isolate failed to express outer-membrane protein of approximately 50 kDa which is matched to the OprM protein channel . Thus, reduced permeability in the clinical isolate is the main mechanism conferring resistance against beta-lactams including imipenem.

J Lab Clin Med, 2003 Sep, 142(3), 158 - 65
In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative; Lin WP et al.; Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the alpha-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity . Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both . The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa . Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule . The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration . Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA . However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.

J Clin Microbiol, 2003 Oct, 41(10), 4892 - 3
Serial granulocyte transfusions as a treatment for sepsis due to multidrug-resistant Pseudomonas aeruginosa in a neutropenic patient; Lin YW et al.; The emergence of multidrug-resistant Pseudomonas aeruginosa (MRPA) has become a major clinical problem . We successfully treated MRPA sepsis in a neutropenic patient undergoing peripheral blood stem cell transplantation with serial granulocyte transfusions . Granulocyte transfusion should be considered as a treatment for severe infection in patients with neutropenia.

Am J Health Syst Pharm, 2003 Oct 1, 60(19), 1962 - 70
Effect of fluoroquinolone expenditures on susceptibility of Pseudomonas aeruginosa to ciprofloxacin in U.S . hospitals; Bhavnani SM et al.; The effect of fluoroquinolone use on the susceptibility of Pseudomonas aeruginosa to fluoroquinolones in U.S . hospitals was studied . Benchmarking surveys were sent annually to pharmacists practicing in U.S . hospitals from 1993 to 1999 . Data collected included hospital characteristics, antimicrobial expenditures and use, antimicrobial stewardship activities, and bacterial susceptibilities . Antimicrobial expenditures were normalized for the number of occupied beds (OBs) per year . General linear modeling and repeated-measures mixed-effects modeling were used to determine factors predictive of P . aeruginosa susceptibility to fluoroquinolones . A total of 174 hospitals provided data for fluoroquinolone expenditures and susceptibility of P . aeruginosa; the median number of years of data was 3 (range, 1-6), representing 416 hospital years . Community hospitals contributed a majority of the data . Median fluoroquinolone expenditures increased gradually from $230 per OB in 1993 to $400 per OB in 1998 . A 55% increase to $620 per OB occurred in 1999, largely because of increased spending on levofloxacin . Susceptibility to ciprofloxacin was commonly used to assess fluoroquinolone susceptibility . The median susceptibility of P . aeruginosa to ciprofloxacin decreased from 84% to 71% . Increasing expenditures for ofloxacin and levofloxacin, but not ciprofloxacin, were associated with decreasing P . aeruginosa susceptibility to ciprofloxacin . In the final multivariable model, each study year after 1993 and every increase in ofloxacin expenditure of $100 per OB were associated with decreases in P . aeruginosa susceptibility . Data from a benchmarking survey of U.S . hospitals for 1993-1999 revealed increases in levofloxacin expenditures, total fluoroquinolone expenditures, expenditures for nonfluoroquinolone antipseudomonal antimicrobials, and total antimicrobial expenditures in 1999 . Increases in expenditures for levofloxacin and ofloxacin were associated with a significant decrease in P . aeruginosa susceptibility to ciprofloxacin.

Crit Care Med, 2003 Oct, 31(10), 2444 - 9
Decreased mortality and infectious morbidity in adult burn patients given enteral glutamine supplements: a prospective, controlled, randomized clinical trial; Garrel D et al.; OBJECTIVE: Enteral glutamine supplements have been shown to reduce infectious morbidity in trauma patients, but their effect on burn patients is not known . The objective of this study was to measure the impact of enteral glutamine supplementation on infectious morbidity, length of care, and the immune system in burn patients . DESIGN: Double-blinded, randomized clinical trial . SETTING: Burn center . PATIENTS: Forty-five adults with severe burns . INTERVENTIONS: Patients were randomized to receive either glutamine or an isonitrogenous control mixture until complete healing occurred . Length of care, incidence of positive blood culture, and mortality were recorded . Phagocytosis by circulating polymorphonuclear cells was measured every 3 days . MEASUREMENTS AND MAIN RESULTS: Patient characteristics were similar in both groups . Four patients were excluded from the analysis, because three of them died within 72 hrs and the fourth could not receive enteral nutrition and amino acid supplements for the first 10 days . Of the remaining 41 patients, length of care in the survivors was not different between groups (0.9 vs . 1.0 days/percent total body surface area for glutamine vs . control, respectively), positive blood culture was three times more frequent in control than in glutamine treatment (4.3 vs . 1.2 days/patient, p <.05), and Pseudomonas aeruginosa was detected in six patients on control and zero on glutamine (p <.05) . Phagocytosis by polymorphonuclear cells was not different between groups . Mortality rate was significantly lower in glutamine than in control: intention to treat, two vs . 12 (p <.05); per protocol analysis, zero vs . eight (p <.01) . CONCLUSIONS: Enteral glutamine supplementation in adult burn patients reduces blood infection by a factor of three, prevents bacteremia with P . aeruginosa, and may decrease mortality rate . It has no effect on level of consciousness and does not appear to influence phagocytosis by circulating polymorphonuclear cells.

J Immunol, 2003 Oct 15, 171(8), 4416 - 24
Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation; Ojielo CI et al.; Bone marrow transplantation (BMT) is an important therapeutic option for a variety of malignant and nonmalignant disorders . Unfortunately, BMT recipients are at increased risk of infection, and in particular, pulmonary complications occur frequently . Although the risk of infection is greatest during the neutropenic period immediately following transplant, patients are still vulnerable to pulmonary infections even after neutrophil engraftment . We evaluated the risk of infection in this postengraftment period by using a well-established mouse BMT model . Seven days after syngeneic BMT, B6D2F(1) mice are no longer neutropenic, and by 3 wk, they demonstrate complete reconstitution of the peripheral blood . However, these mice remain more susceptible throughout 8 wk to infection after intratracheal administration of Pseudomonas aeruginosa; increased mortality in the P . aeruginosa-infected BMT mice correlates with increased bacterial burden in the lungs as well as increased systemic dissemination . This heightened susceptibility to infection was not secondary to a defect in inflammatory cell recruitment to the lung . The inability to clear P . aeruginosa in the lung correlated with reduced phagocytosis of the bacteria by alveolar macrophages (AMs), but not neutrophils, decreased production of TNF-alpha by AMs, and decreased levels of TNF-alpha and IFN-gamma in the bronchoalveolar lavage fluid following infection . Expression of the beta(2) integrins CD11a and CD11c was reduced on AMs from BMT mice compared with wild-type mice . Thus, despite restoration of peripheral blood count, phagocytic defects in the AMs of BMT mice persist and may contribute to the increased risk of infection seen in the postengraftment period.

J Hosp Infect, 2003 Oct, 55(2), 137 - 40
Efficacy of some neutralizers in suspension tests determining the activity of disinfectants; Espigares E et al.; The ability of six mixtures to neutralize glutaraldehyde, o-phthalaldehyde and peracetic acid was tested using four reference strains: Pseudomonas aeruginosa CIP A22, Escherichia coli CIP 54127, Staphylococcus aureus CIP 53154, and Enterococcus faecium CIP 5855 . Glutaraldehyde was the hardest to neutralize, and peracetic acid the easiest . The most effective mixture was Tween 80 with sodium bisulphate, sodium thioglycolate, lecithin and cysteine, and the least effective was Tween 80, lecithin and histidine . The efficacy of the neutralizers may indicate a propensity loss of activity from interfering substances when disinfectants are used in practice.

J Hosp Infect, 2003 Oct, 55(2), 98 - 107
Development of bacterial resistance to several biocides and effects on antibiotic susceptibility; Walsh SE et al.; The aims of this study were to investigate the development of bacterial resistance to eugenol, thymol, trichlorocarbanalide (TCC), didecyldimethylammonium chloride (DDDMAC) and C10-16-alkyldimethyl, N-oxides (ADMAO) and subsequent effects on antibiotic susceptibility . An agar minimum inhibitory concentration (MIC) method was used to assess the activity of the biocides against standard bacterial strains and laboratory mutants . A range of techniques including disk diffusion and gradient plate experiments were used to attempt to develop bacterial 'resistance' or tolerance to the biocides . The mutants produced were examined for cross-resistance to the other biocides and to antibiotics via disk diffusion and gradient plate MIC methods . Outer membrane proteins of the mutants were extracted and examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . Escherichia coli triclosan-resistant mutants were not cross-resistant to eugenol, thymol, TCC, DDDMAC and ADMAO . Mutants with elevated MICs to DDDMAC (E . coli and Pseudomonas aeruginosa), thymol (E . coli) and eugenol (E . coli) were isolated, but all remained sensitive to higher concentrations of the agents . Bacteria with elevated MICs to TCC and ADMAO were not obtained . Some low-level cross-resistance between DDDMAC, eugenol and thymol was observed with the E . coli gradient plate mutants, as well as reduced susceptibility to antibiotics, most notably chloramphenicol . The lack of cross-resistance of the triclosan mutants suggested that the mode of action of triclosan is not shared with the other biocides studied . SDS-PAGE results indicated that the DDDMAC P . aeruginosa mutant had a reduced amount (or absence) of one outer membrane protein in comparison with the standard strain . In conclusion, under laboratory conditions, bacterial exposure to thymol, eugenol and DDDMAC can lead to reduced susceptibility between selected biocidal agents and antibiotics, more specifically, chloramphenicol . However, further studies are required to determine if this is of clinical significance.

Curr Pharm Des, 2003, 9(26), 2131 - 54
Prodrugs in genetic chemoradiotherapy; Patterson AV et al.; Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility . Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes . Gene-Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect) . This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed . In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization . Some, but not all GDEPT approaches have been analysed in combination with radiotherapy . Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N . Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E . coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart . Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.

Am J Physiol Lung Cell Mol Physiol, 2004 Feb, 286(2), L268 - 74 Epub 2003 Oct 03.
DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS; Tesfaigzi Y et al.; Exposure of pulmonary airways to environmental toxins and allergens may cause proliferation of airway epithelial cells and mucous cell metaplasia (MCM); however, it is unclear to what extent proliferating cells differentiate into mucus-storing cells and contribute to MCM . Our previous studies demonstrated that Bcl-2, an inhibitor of apoptosis with cell cycle regulatory functions, is expressed in metaplastic mucous cells . The purpose of the present study was to investigate the number of metaplastic mucous cells that are derived from proliferating epithelial cells and whether Bcl-2 has a role in cell cycle entry in these cells . Rats were intratracheally instilled with 100 microg of LPS from Pseudomonas aeruginosa in 500 microl of saline, and proliferating airway cells were labeled with bromodeoxyuridine (BrdU) by implanting a subcutaneous osmotic pump 24 h before instillation . The volume of stored mucosubstance and the number of mucous cells were increased 10- and 3-fold, respectively, from 24-48 h after instillation . The number of total epithelial cells per millimeter of basal lamina increased, and the number of serous cells per millimeter of basal lamina decreased during this time . Approximately 50% of Alcian blue-periodic acid Schiff-stained mucous cells were labeled with BrdU at 48 h after instillation, suggesting that one-half of the secretory cells were derived from proliferating cells . Furthermore, 50% of the Bcl-2-positive mucous cells were BrdU negative and therefore derived from nonproliferating, preexisting cells . Our findings demonstrate that preexisting and proliferating cells differentiate into mucous cells and compose LPS-induced metaplasia and that Bcl-2 does not have cell cycle regulatory function in these cells.

Am J Respir Cell Mol Biol, 2004 May, 30(5), 605 - 12 Epub 2003 Oct 03.
Airway epithelial integrity is protected by a long-acting beta2-adrenergic receptor agonist; Coraux C et al.; Airway epithelial integrity may be impaired by bacterial exoproducts, which are able to degrade tight junction-associated proteins such as zonula occludens 1 (ZO-1) . We have investigated the protective effect of salmeterol, a long-acting beta(2)-adrenergic agonist, on Pseudomonas aeruginosa-induced alteration of the epithelial junctional barrier . We demonstrate in human airway epithelial cells (HAEC) that salmeterol induces a time-dependent increase in ZO-1 protein, although no significant change in ZO-1 transcripts was observed . When HAEC cultures were exposed to P . aeruginosa (PAO1) supernatants, apical expression of ZO-1 protein was maintained in salmeterol-pretreated HAEC cultures, whereas it disappeared after PAO1 exposure in cultures not pretreated with salmeterol . Western blot experiments showed that the 220-kD ZO-1 protein was decreased after PAO1 incubation but was still present in salmeterol-pretreated HAEC extracts . The functional activity of ZO-1 protein was monitored by measuring transepithelial resistance and analyzing the diffusion of a low molecular weight tracer through the intercellular spaces . After PAO1 incubation, the epithelial integrity of HAEC was impaired, as shown by a decrease in transepithelial resistance and increased paracellular permeability, but was not significantly altered after salmeterol preincubation . These results demonstrate that salmeterol may contribute to the protection of the airway epithelium barrier against bacterial virulence factors.

J Bacteriol, 2003 Oct, 185(20), 6112 - 8
Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa; Matias VR et al.; High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure . The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques . Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material . Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel . Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted . Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space . It is possible that the protoplast has some capacity to float freely within the periplasm.

J Bacteriol, 2003 Oct, 185(20), 5976 - 83
Mechanism of Pseudomonas aeruginosa RhlR transcriptional regulation of the rhlAB promoter; Medina G et al.; Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits . We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer . Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter . RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P . aeruginosa virulence-associated traits.

Respir Res . 2003;4(1):9 . Epub 2003 Sep 08.
Gammadelta T lymphocytes from cystic fibrosis patients and healthy donors are high TNF-alpha and IFN-gamma-producers in response to Pseudomonas aeruginosa; Raga S et al.; BACKGROUND: Gammadelta T cells have an important immunoregulatory and effector function through cytokine release . They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity . On the other hand, they have been implicated in airway inflammation . METHODS: The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-gamma and TNF-alpha production by gammadelta and alphabeta T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA) . Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine . Proliferative responses, activation markers and receptor usage of gammadelta T cells were also evaluated . RESULTS: The highest production of cytokine was of TNF-alpha and IFN-gamma, gammadelta being better producers than alphabeta . No differences were found between patients and controls . The Vgamma9delta2 subset of gammadelta T cells was preferentially expanded . CD25 and CD45RO expression by the alphabeta T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes . CONCLUSION: Our results support the hypothesis that gammadelta T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients . They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease.

Diagn Microbiol Infect Dis, 2003 Oct, 47(2), 427 - 30
Left-sided endocarditis caused by Pseudomonas aeruginosa: successful treatment with meropenem and tobramycin; Gavin PJ et al.; Medical treatment alone is rarely successful in left-sided infective endocarditis caused by Pseudomonas aeruginosa . We report the cure of such a case with high-dose meropenem in combination with tobramycin.

Pediatr Pulmonol, 2003 Nov, 36(5), 405 - 12
Pseudomonas aeruginosa and cystic fibrosis: correlation between exoenzyme production and patient's clinical state; Lanotte P et al.; In this study, we investigated the correlation between the production by Pseudomonas aeruginosa isolates of four exoenzymes (protease, elastase, neuraminidase, and phospholipase C (PLC)) and the clinical state of cystic fibrosis (CF) patients . We studied 212 P . aeruginosa isolates from 22 CF patients chronically infected with this bacterium . Patients were classified into three clinical groups according to a modified Shwachman-Kulczycki-Khaw (SKK) scoring system . The production of enzymes by isolates from patients in the three populations was analyzed and compared using four statistical tests: chi-square, Mann-Whitney U, principal component analysis, and discriminant analysis . Isolates from patients with excellent or good clinical status (group I, SKK score >/=71) had higher elastase and neuraminidase activities than isolates from the other patients . In contrast, PLC activity, a common characteristic of CF isolates, was higher in isolates from patients with poor or weak clinical status (group III, SKK score </=55) . PLC also appeared to be the best parameter for differentiating between groups I and III . Enzyme production was highly variable in group II isolates (SKK score, 56-70) . Our results suggest that P . aeruginosa isolates from patients with good clinical status produce large amounts of neuraminidase, and that PLC production may be involved in the decrease in pulmonary function .

JAMA, 2003 Oct 1, 290(13), 1749 - 56
Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial; Saiman L et al.; CONTEXT: Treatment strategies for cystic fibrosis (CF) lung disease include antibiotics, mucolytics, and anti-inflammatory therapies . Increasing evidence suggests that macrolide antibiotics might be beneficial in patients with CF . OBJECTIVE: To determine if an association between azithromycin use and pulmonary function exists in patients with CF . DESIGN AND SETTING: A multicenter, randomized, double-blind, placebo-controlled trial conducted from December 15, 2000, to May 2, 2002, at 23 CF care centers in the United States . PARTICIPANTS: Of the 251 screened participants with a diagnosis of CF, 185 (74%) were randomized . Eligibility criteria included age 6 years or older, infection with Pseudomonas aeruginosa for 1 or more years, and a forced expiratory volume in 1 second (FEV1) of 30% or more . Participants were stratified by FEV1 (> or =60% predicted vs <60% predicted), weight of less than 40 kg vs 40 kg or more, and CF center . INTERVENTION: The active group (n = 87) received 250 mg (weight <40 kg) or 500 mg (weight > or =40 kg) of oral azithromycin 3 days a week for 168 days; placebo group (n = 98) received identically packaged tablets . MAIN OUTCOME MEASURES: Change in FEV1 from day 0 to completion of therapy at day 168 and determination of safety . Secondary outcomes included pulmonary exacerbations and weight gain . RESULTS: The azithromycin group had a mean 0.097-L (SD, 0.26) increase in FEV1 at day 168 compared with 0.003 L (SD, 0.23) in the placebo group (mean difference, 0.094 L; 95% confidence interval {CI}, 0.023-0.165; P =.009) . Nausea occurred in 17% more participants in the azithromycin group (P =.01), diarrhea in 15% more (P =.009), and wheezing in 13% more (P =.007) . Participants in the azithromycin group had less risk of experiencing an exacerbation than participants in the placebo group (hazard ratio, 0.65; 95% CI, 0.44-0.95; P =.03) and weighed at the end of the study an average 0.7 kg more than participants receiving placebo (95% CI, 0.1-1.4 kg; P =.02) . CONCLUSION: Azithromycin treatment was associated with improvement in clinically relevant end points and should be considered for patients with CF who are 6 years or older and chronically infected with P aeruginosa.

Cesk Slov Oftalmol, 2003 Sep, 59(5), 352 - 8
{Acanthamoeba keratitis after use of soft contact lenses--case report}; Ziak P et al.; The case history of a 39-year patient suffering from a deep inflammation of cornea and not responding to conventional antibiotic treatment is presented . The patient was using soft contact lenses during the period of initial symptoms; moreover, he was bathing in thermal bathing pool . A cultivation examination of smears from the area of corneal defect revealed the presence of Acanthamoeba lugdunensis in combination with bacterial infection by Pseudomonas aeruginosa . The available data indicate that it is the first case of acanthamoeba karatitis (AK) after the application of contact lenses in Slovakia . A long-term local treatment with propamidin isethionate (Brolene gtt, ung.) resulted in healing up . The subsequent vision after 16 months since the initial symptoms proved to be 6/12 (0.5) . The healing of the centrally localized defect changed the curvature of cornea with consequent hypermetropic shift . The defect completely corrected the patient's myopia (-8.5) . The paper describes present possibilities of AK therapy.

EMBO J, 2003 Oct 1, 22(19), 4957 - 67
Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas; Schoehn G et al.; Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients . Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon') . Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon . PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers . Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD . Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities . Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.

Am J Physiol Lung Cell Mol Physiol, 2004 Apr, 286(4), L717 - 26 Epub 2003 Sep 26.
Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection; Van Heeckeren AM et al.; In cystic fibrosis (CF) there is an excessive inflammatory response to lung infections with Pseudomonas aeruginosa, which causes significant morbidity and mortality . Mice deficient in the cystic fibrosis conductance transmembrane regulator homolog (Cftr) have exaggerated production of proinflammatory cytokines in epithelial lining fluid and increased mortality in response to chronic bronchopulmonary infection with mucoid P . aeruginosa, compared with infected wild-type littermates . Whether delivery of CFTR to CF airways by an adenoviral vector (Ad2/CFTR-16) decreases cytokine production and mortality in response to chronic bronchopulmonary infection with mucoid P . aeruginosa was tested . CF mice {stock Cftrtm1Unc-TgN(FABPCFTR)#Jaw} were anesthetized with isoflurane and inoculated intranasally with either Ad2/CFTR-16, diluent (sucrose), or empty vector (Ad2/EV) . Two weeks later, mice were anesthetized with 2.5% Avertin and inoculated transtracheally with P . aeruginosa-laden agarose beads (PA M57-15) . The cumulative 10-day survival of mice pretreated with Ad2/CFTR-16 was significantly higher compared with mice pretreated with sucrose but not significantly higher than mice pretreated with Ad2/EV . After adjusting for differences in experiment, we found weight loss at 3 days for mice treated with Ad2/CFTR-16 to be significantly less than for the sucrose- or Ad2/EV-treated groups . However, cytokine responses were similar in all groups 3 days after infection . In conclusion, the observed survival advantage of adenoviral delivery of CFTR to the CF lung may be due either to CFTR expression or possibly to proinflammatory effects of the adenoviral vector, or both.

Cent Eur J Public Health, 2003 Sep, 11(3), 129 - 31
Influence of growth media on potential virulence factors of Pseudomonas aeruginosa; Hostacka A; Potential virulence factors of three Pseudomonas aeruginosa strains after growth in three complex media (CM) and in one mineral medium (MM) were evaluated . Cell surface hydrophobicity demonstrated by adherence of bacteria to xylene as well as enzymatic activity (elastase, protease, lipase) of the strains grown in CM varied with composition of CM and with strain . All strains cultivated in CM showed higher hydrophobicity and higher elastase, protease and lipase (with the exception of one strain) activity in comparison with bacteria incubated in MM . Even no production of elastase was detected in the strains after growth in MM . Motility of bacteria was affected by culture media the least . In vitro composition of growth media influenced some potential virulence factors of P . aeruginosa.

Arch Microbiol, 2003 Nov, 180(5), 374 - 9 Epub 2003 Sep 26.
Transcriptome analysis of the Pseudomonas aeruginosa response to iron; Palma M et al.; To successfully infect humans, Pseudomonas aeruginosa (Pa) must overcome the low iron availability in host tissues . A transcriptome comparison was carried out between iron-starved cells of Pa treated with iron and untreated controls . The present study is the first global analysis of the early transcriptional response of exponentially growing Pa to iron . Approximately 1.3% of the Pa genes displayed > or = 5.0-fold changes in mRNA levels in iron-treated cells . Treatment affected the mRNA levels of many genes required for iron acquisition as well as several genes with relevance to virulence previously known to be regulated by iron . More importantly, the analysis permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron . These genes are good candidates for mutagenesis studies aimed at identifying novel functions relevant to iron metabolism in Pa . Some of these genes encode predicted siderophore receptors, iron transport systems, TonB-dependent receptors, regulatory proteins, and proteins relevant to virulence . Notably, 49 genes encode hypothetical or conserved hypothetical proteins of unknown function, suggesting that they are involved directly or indirectly in iron metabolism or metabolic adaptation to different iron-availability conditions.

APMIS, 2003 Sep, 111(9), 891 - 7
Cytokine and surface receptor diversity of NK cells in resistant C3H/HeN and susceptible BALB/c mice with chronic Pseudomonas aeruginosa lung infection; Calum H et al.; The purpose of the present study was to investigate whether NK cells from resistant C3H/HeN mice and susceptible BALB/c mice showed different release of cytokines and expression of surface molecules during chronic P . aeruginosa lung infection using alginate-embedded P . aeruginosa mimicking the infection in cystic fibrosis . Lung cell suspensions were depleted of lymphocytes by magnetic cell sorting . The concentrations of IFN-gamma, IL-1beta and GM-CSF were estimated by ELISA at day 1 and 2 after infection . Non-infected mice were used as controls . Flow cytometry was used to estimate the surface expression of the LFA-1 and Fc receptors on NK cells . At day 2, IFN-gamma levels increased in C3H/HeN mice but decreased in BALB/c mice . The GM-CSF levels increased only in the C3H/HeN mice at day 1 and 2 . Surface expression of LFA-1 on the NK cells was higher in C3H/HeN mice at day 1 and 2 . In contrast, the expression of Fc receptors was significantly lower on NK cells in C3H/HeN mice at day 1 and 2 . In conclusion, the present results show phenotypic differences in NK cells in the two mice strains in chronic P . aeruginosa lung infection, indicating different modulating effects in the Th1/Th2 balance.

Afr J Med Med Sci, 2001 Sep, 30(3), 221 - 3
Behavioural pattern of malignant otitis external: 10-year review in Ibadan; Lasisi OA et al.; Malignant externa otitis is a rapidly progressive infection of the external ear canal, mastoid and the base of the skull caused by Pseudomonas aeruginosa in elderly diabetics and other immunosuppressive conditions . Thirteen cases of malignant externa otitis seen in the E.N.T . Dept University College Hospital, Ibadan between 1988 and 1997 were reviewed . The mean age was 62 years and the mean duration of diabetes was 14 years . The most frequent symptoms were otalgia 13 (100%) and otorrhoea 12 (92%) . The complications include multiple cranial neuropathy 11 (85%), meningitis (31%), brain abscess (8%), and infratemporal abscess 1 (8%) . There were 8 deaths (62%) showing that this is still a dangerous condition in our environment . The problems identified were late presentation of cases and inavailability of facilities for prompt control and monitoring of patients . It is hoped that the outlook of the disease can be improved if there are corrected.

Pol J Vet Sci, 2003, 6(3 Suppl), 3 - 5
Echinacea purpurea stimulates cellular immunity and anti-bacterial defence independently of the strain of mice; Bany J et al.; One of the major functions of the immune system is anti-bacterial defence mediated among others by non-specific immunity (macrophages, granulocytes) . Echinacea purpurea extracts are widely used in prophylaxis and therapy of various infections, mainly the respiratory tract, in animals and humans . The aim of this work was to evaluate the effect of prophylactic use of Echinacea purpurea extract on the development of Pseudomonas aeruginosa infection in various strains of mice and on some parameters of non-specific and also specific cellular immunity . Mice expressed various, depending on the strain used, susceptibility to infection . Echinacea feeding resulted in diminishing of bacteria number in livers of C57Bl/6 (susceptible strain) as well as B6C3F1 (relative resistant strain) mice . Echinacea feeding of the second relative resistant strain (BALB/c x C3H) F1 resulted in stimulation of granulocytes chemiluminescent and lymphocytes proliferative response.

Invest Ophthalmol Vis Sci, 2003 Oct, 44(10), 4247 - 54
Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin; Zhang J et al.; PURPOSE . Flagellin is the major structural protein of the flagella of Gram-negative bacteria and is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms . In this study, we sought to determine whether flagellin induces an inflammation response in cultured human corneal epithelial (HCE) cells and to determine the underlying mechanisms . METHODS . Flagellin was purified from Pseudomonas aeruginosa (PA) strain PAO1 with ammonium sulfate gradient precipitation and lipopolysaccharide in flagellin preparation was removed by ion exchange chromatography . Purified flagellin was used to challenge HUCL, a telomerase-immortalized HCE cell line, and primarily cultured HCE cells . Inhibitory (I)kappaB-alpha phosphorylation and degradation were detected by Western blot . Interleukin (IL)-6 and -8 expression in mRNA levels and secretion were assessed using RT-PCR and enzyme-linked immunosorbent assay, respectively . TLR5 localization in human cornea was analyzed by immunohistochemistry using anti-TLR5 antibody . Anti-flagellum antiserum and anti-TLR5 antibody were used for functional blocking of flagellin stimulation and TLR5 activation . RESULTS . Exposure of both HUCL and primary HCE cells to purified PA flagellin (250 ng/mL) resulted in IkappaB-alpha phosphorylation and degradation in a time-dependent manner . Concomitant with NF-kappaB activation, transcriptional expression and subsequent secretion of IL-6 and -8 in these cells were also induced by flagellin . Toll-like receptor (TLR)-5, an innate immunity receptor for flagellin, was expressed in HUCL cells and located at the cell surface of the basal and wing, but not in superficial, cells of human corneal epithelium . Presence of flagellum- or TLR5-antisera in culture medium attenuated flagellin-induced IkappaB-alpha phosphorylation and degradation as well as IL-6 and -8 production . CONCLUSIONS . Flagellin of Gram-negative pathogens such as PA contributes to the inflammatory responses of corneal epithelium in a TLR5-NF-kappaB signaling pathway-dependent manner.

Br J Ophthalmol, 2003 Oct, 87(10), 1238 - 40
Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis; Lomholt JA et al.; AIM: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance . METHODS: Ciprofloxacin susceptibility was tested by an agar dilution method; genomic DNA corresponding to the quinolone target genes gyrA and parC, and the regulatory genes mexR and nfxB controlling drug efflux systems, was amplified by PCR and sequenced; multilocus enzyme electrophoresis was performed to examine the genetic relation among resistant strains . RESULTS: Three out of 90 keratitis isolates (3.3%), one from the United Kingdom and two from India, exhibited MIC values of 16 mg/l or 32 mg/l . The UK isolate had a mutation in gyrA (Thr83Ile), whereas the two Indian isolates showed mutations in both gyrA (Thr83Ile) and parC (Ser87Leu) . The remaining isolates from keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l . Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains . CONCLUSIONS: The vast majority of eye isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant . Mutations in more than one target gene were associated with higher MIC values.

Mol Microbiol, 2003 Oct, 50(1), 205 - 17
Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa; McPhee JB et al.; The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions . We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides . This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation . Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated . Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides . The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers . Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system . The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P . aeruginosa infections.

J Enzyme Inhib Med Chem, 2003 Jun, 18(3), 259 - 63
Zinc complexes of benzothiazole-derived Schiff bases with antibacterial activity; Chohan ZH et al.; Reaction of 2-acetamidobenzaldehyde with 2-amino-, 2-amino-4-methyl-, 2-amino-4-methoxy-, 2-amino-4-chloro-, 2-amino-6-nitro- and 2-amino-6-methylsufonylbenzothiazole afforded a series of Schiff bases . These compounds have been used for complexation reactions to obtain Zn(II) chelates having the same metal ion but different anions of the type {Zn(L)2}Xn {L = Schiff base derivative, X = SO4, NO3, C2O4 and CH3CO2 and n = 1 or 2} These complexes (Table I) have been characterized by physical, spectral, and analytical data . The Schiff bases act tridentately and their metal complexes were proposed to possess an octahedral geometry . To evaluate the antibacterial role of the anion, these compounds have been screened for antibacterial properties against pathogenic strains such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3202 - 7
Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates; Sobel ML et al.; MexXY is an aminoglycoside-inducible multidrug transporter shown to contribute to intrinsic and acquired aminoglycoside resistance in laboratory isolates of Pseudomonas aeruginosa . To assess its contribution to aminoglycoside resistance in 14 clinical isolates demonstrating a panaminoglycoside resistance phenotype unlikely to be explained solely by aminoglycoside modification, expression of mexXY by these isolates was examined by reverse transcription-PCR . Elevated levels of mexXY expression were evident for most strains compared with those detected for an aminoglycoside-susceptible control strain, although there was no correlation between mexXY levels and the aminoglycoside MICs for the resistant strains, indicating that if MexXY was playing a role, other factors were also contributing . Deletion of mexXY from 9 of the 14 isolates resulted in enhanced susceptibilities to multiple aminoglycosides, confirming the contribution of this efflux system to the aminoglycoside resistance of these clinical isolates . Still, the impact of MexXY loss varied, with some strains clearly more or less dependent on MexXY for aminoglycoside resistance . Expression of mexXY also varied in these strains, with some showing high-level expression of the efflux genes independent of aminoglycoside exposure (aminoglycoside-independent hyperexpression) and others showing hyperexpression of the efflux genes that was to a greater or lesser degree aminoglycoside dependent . None of these strains carried mutations in mexZ, which encodes a negative regulator of mexXY expression, or in the mexZ-mexXY intergenic region . Thus, mexXY hyperexpression in aminoglycoside-resistant clinical isolates occurs via mutation in one or more as yet unidentified genes.

J Am Chem Soc, 2003 Oct 1, 125(39), 11842 - 52
The hydroxide complex of Pseudomonas aeruginosa heme oxygenase as a model of the low-spin iron(III) hydroperoxide intermediate in heme catabolism: 13C NMR spectroscopic studies suggest the active participation of the heme in macrocycle hydroxylation; Caignan GA et al.; 13C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe(III)-OH), where OH(-) has been used as a model of the OOH(-) ligand to gain insights regarding the elusive ferric hydroperoxide (Fe(III)-OOH) intermediate in heme catabolism at ambient temperatures . Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe(III)-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale . The most abundant population exhibits a spin crossover between S = (1)/(2) and S = (3)/(2) spin states, and the two less abundant populations exhibit pure, S = (3)/(2) and S = (1)/(2), (d(xy)())(1) electronic configurations . We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle . This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH(-), is likely reinforced by the flexibility of the distal pocket in HO . These findings suggest that if the ligand field strength of the coordinated OOH(-) in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.

Chemotherapy, 2003 Sep, 49(5), 237 - 42
Effect of antibiotics on cell surface hydrophobicity of bacteria causing orthopedic wound infections; Kustos T et al.; BACKGROUND: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant . Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors . Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants . METHODS: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test . Results were compared to the hydrophobicity of three Hungarian standard bacterial strains . The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin)--applied most often in our Department for prophylaxis and treatment of patients--were analyzed . RESULTS: The cell surface hydrophobicity of certain strains showed considerable changes after antibiotic treatment . These alterations indicated the decrease in hydrophobicity . Supra-inhibitory concentrations (2x minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5x MIC) levels . CONCLUSIONS: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains . These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients .

Biochemistry, 2003 Sep 30, 42(38), 11334 - 46
Interaction of a peptide from the receptor-binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: changes in backbone dynamics induced by binding; Campbell AP et al.; The C-terminal receptor-binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P . aeruginosa infections . We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein . The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond . The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) . The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135) . These turns have been implicated in cross-reactive antibody recognition . (15)N NMR relaxation experiments of the (15)N-labeled recombinant PAK pilin peptide in complex with an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus, were performed in order to probe for changes in the mobilities and dynamics of the peptide backbone as a result of antibody binding . The major results of these studies are as follows: binding of Fab leads to the preferential ordering of the first turn over the second turn in each isomer, binding of Fab partially stabilizes peptide backbone regions undergoing slow (microsecond to millisecond) exchange-related motions, and binding of Fab leads to a greater loss in backbone conformational entropy at pH 7.2 versus pH 4.5 . The biological implications of these results will be discussed in relation to the role that fast and slow backbone motions play in PAK pilin peptide immunogenicity and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P . aeruginosa strains.

Infect Immun, 2003 Oct, 71(10), 6035 - 44
Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection; McMorran B et al.; Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses . An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria . We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P . aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors . Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 h of infection and could be classified into four different expression patterns . These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones) . In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos . ExoU-dependent gene expression was mediated in part by early and transient activation of the AP1 transcription factor complex . In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Infect Immun, 2003 Oct, 71(10), 5785 - 93
The Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone accelerates apoptosis in macrophages and neutrophils; Tateda K et al.; Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa . Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C(12)-HSL) and N-butyryl-L-homoserine lactone (C(4)-HSL) . Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C(12)-HSL, for modulation of the host immune system . Here we show the specific ability of 3-oxo-C(12)-HSL to induce apoptosis in certain types of cells . When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C(12)-HSL, but when they were incubated not C(4)-HSL, significant loss of viability was observed in a concentration (12 to 50 micro M)- and incubation time (1 to 24 h)-dependent manner . The cytotoxic activity of 3-oxo-C(12)-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2 . Cells treated with 3-oxo-C(12)-HSL revealed morphological alterations indicative of apoptosis . Acceleration of apoptosis in 3-oxo-C(12)-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression) . Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C(12)-HSL, the HSL backbone, and side chain length are required for maximal activity . These data suggest that Pseudomonas 3-oxo-C(12)-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C(12)-HSL-induced cytotoxicity in macrophages and neutrophils . Our data suggest that the quorum-sensing molecule 3-oxo-C(12)-HSL has critical roles in the pathogenesis of P . aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.

Infect Immun, 2003 Oct, 71(10), 5565 - 75
Mucin degradation mechanisms by distinct Pseudomonas aeruginosa isolates in vitro; Aristoteli LP et al.; Pseudomonas aeruginosa has emerged as an important causative agent of bacterial keratitis, a rapidly progressive ocular condition that may result in blindness . Secretory mucin forms the main constituent of the precorneal tear film, a three-layer film on the ocular surface protecting the underlying corneal epithelium from potential pathogens . The purpose of the present study was to compare mucin degradation mechanisms between ocular P . aeruginosa strains . Mucin degradation was assessed by agarose electrophoresis, lectin blotting, and size exclusion chromatography . The results indicate that certain P . aeruginosa strains (Paer12, ATCC 15442, 6294, and Paer25) had depleted mucin from the culture supernatant and that this was contingent on the inherent ability of these isolates to produce proteases . Non-protease-producing strains (Paer1 and Paer3) did not appreciably degrade mucin . Further, galactosidase, N-acetylglucosaminidase, and N-acetylgalactosaminidase activities were detected in some strains, suggesting the operation of further mechanisms of mucin degradation by P . aeruginosa . Mucin degradation by P . aeruginosa also seemed to be for the acquisition of nutrients, as a growth advantage was observed in mucin-depleting strains over nondepleting strains in the long term . It is postulated that the degradation of mucin serves to collapse the mucin barrier and its associated network containing antibacterial tear components and to provide energy for sustained bacterial growth.

Am J Respir Cell Mol Biol, 2003 Oct, 29(4), 432 - 8
Murine complement interactions with Pseudomonas aeruginosa and their consequences during pneumonia; Younger JG et al.; Complement is necessary for defense against lung infection with Pseudomonas aeruginosa in mice . We studied in vitro interactions between complement and P . aeruginosa and in vivo effects of complement depletion to better understand this relationship . In vitro, P . aeruginosa strain UI-18 was resistant to killing by mouse serum . However, C3 opsonized the organism (via the alternative and mannose binding lectin {MBL} pathways), and C5 convertase activity on the bacterial surface was demonstrated . In vivo, compared with normal mice, complement-deficient mice experienced higher mortality and failed to sterilize their bronchoalveolar space within 24 h of inoculation . These changes did not seem to be a result of decreased inflammation because complement-deficient mice had normal neutrophil recruitment, greater lung myeloperoxidase content, and, by 24 h, a 35-fold higher level of the CXC chemokine KC . Lung static pressure-volume curves were abnormal in infected animals but were significantly more so in complement deficient mice . These data indicate that although P . aeruginosa is resistant to serum killing, C3 opsonization and C5 convertase assembly occur on its surface . This interaction in vivo plays a central role in host survival beyond just recruitment and activation of phagocytes and may serve to limit the inflammatory response to and tissue injury resulting from bacterial infection.

Anesth Analg, 2003 Oct, 97(4), 1133 - 6, table of contents
Prepared endotracheal tubes: are they a potential source for pathogenic microorganisms?
Rasic NF, Friesen RM, Anderson B, Hoban SA, Olson N, Kress J.
Prepared endotracheal tubes (PETTs) are frequently used for unanticipated difficult intubation, but their storage time is highly variable and institution-dependent . We sought to determine first, if open, unused PETTs are a potential source of pathogenic microorganisms, and second, if PETTs can provide a medium for bacterial survival after deliberate contamination . A stylet was inserted into a 7-mm ETT, and this system was ethylene oxide sterilized . The PETTs were placed in 20 different locations and sampled 8 times in a 4-wk period . Growth was determined after 48-h incubation, and the microorganism was identified . In Phase 2, the PETT (n = 40) was swabbed with a fresh suspension of H . influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecium, or a negative control . Nonvirulent bacteria were cultured from 13 of 160 (8.1%) samples and from 15 of 320 (4.7%) samples in Phases 1 and 2, respectively . No PETT grew the same bacteria more than once . In Phase 2, after 24 h, only E . faecium was recovered . Based on this study, the pathogenic potential of PETTs is very small, and they can be safely used for up to 1 mo . This practice could translate to significant cost reduction for operating room budgets . IMPLICATIONS: Prepared endotracheal tubes (PETTs) are back-up airway equipment to be used in the case of a difficult intubation . A short PETT shelf life because of unknown safe storage time results in significant budget costs . This blinded, controlled study examined the pathogenic potential of PETTs in the operating room environment.

J Microbiol Methods, 2003 Oct, 55(1), 231 - 40
Genomic typing of Pseudomonas aeruginosa isolates by comparison of Riboprinting and PFGE: correlation of experimental results with those predicted from the complete genome sequence of isolate PAO1; Botes J et al.; The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P . aeruginosa PAO1 . The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII . When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE . These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P . aeruginosa PAO1 . Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates . This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence.

J Microbiol Methods, 2003 Oct, 55(1), 201 - 11
A new enzymatic method for the detachment of particle associated soil bacteria; Bockelmann U et al.; A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths . Many soil microorganisms are closely associated with the organic matrix of soil particles . They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates . To characterize the EPS, a prestaining of the soil samples with different lectins was performed . Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa . Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer . The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining . With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer . Efficiency of the technique was assessed by scanning electron microscopy . These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.

J Microbiol Methods, 2003 Oct, 55(1), 105 - 8
Simple co-agglutination assay for rapid identification of Pseudomonas aeruginosa; Sciortino CV Jr; A new co-agglutination assay for the identification of Pseudomonas aeruginosa from culture was developed and evaluated . A total of 232 P . aeruginosa isolates, and 36 oxidase-positive, Gram-negative clinical isolates were tested . The sensitivity of the assay was 96%, and the specificity was 91.9% . The assay took <15 min to perform.

Biochim Biophys Acta, 2003 Sep 23, 1651(1-2), 1 - 4
Approaching the speed limit for Greek Key beta-barrel formation: transition-state movement tunes folding rate of zinc-substituted azurin; Pozdnyakova I et al.; Azurin is a blue-copper protein with a beta-barrel structure of Greek Key topology . In vitro, copper can be substituted with zinc without change in protein structure . We here analyze the kinetic folding behavior of zinc-substituted Pseudomonas aeruginosa azurin . Our findings can be summarized in three key conclusions: first, zinc remains strongly bound to the polypeptide upon unfolding, suggesting that the cofactor may bind to the protein before polypeptide folding in vivo . Second, the semi-logarithmic plot of folding and unfolding rates for zinc-substituted azurin as a function of denaturant concentration exhibits curvature due to a changing transition-state structure . Third, the extrapolated folding speed in water for zinc-substituted azurin is similar to that of other proteins with the same topology, implying that there is a speed limit that can be modulated by stability-driven transition-state movement for formation of beta-barrel structures with Greek Key topology.

Intensive Care Med, 2003 Nov, 29(11), 1981 - 8 Epub 2003 Sep 10.
A 7-year study of severe hospital-acquired pneumonia requiring ICU admission; Valles J et al.; OBJECTIVE: To examine the characteristics, prognostic factors, and outcome of patients with severe hospital-acquired pneumonia admitted to the ICU . DESIGN AND SETTING: Prospective observational clinical study in two medical-surgical ICUs with 16 and 20 beds PATIENTS AND PARTICIPANTS: During a 7-year period all hospitalized patients requiring admission to either ICU for hospital-acquired pneumonia were followed up . MEASUREMENTS AND RESULTS: We diagnosed 96 episodes of severe hospital-acquired pneumonia, and in 67 cases a causal diagnosis was made . Most episodes were late-onset pneumonia . Gram-negative micro-organisms were isolated in 51% of episodes diagnosed, and Pseudomonas aeruginosa was the most frequent pathogen isolated (24%) . Clearly significant variations happened between hospitals, particularly affecting the incidence of Aspergillus spp . and Legionella pneumophila . Forty-nine patients developed septic shock (51%) . Fifty-one patients died (53%) . Aspergillosis and pneumonia due to P . aeruginosa were associated with the highest mortality . Septic shock (OR: 14.27) and chronic obstructive pulmonary disease (OR: 6.11) were independently associated with a poor prognosis . CONCLUSIONS: Patients with severe hospital-acquired pneumonia admitted to the ICU present high mortality . The presence of septic shock and chronic obstructive pulmonary disease in conjunction with specific microorganisms are associated with a poor prognosis . Local epidemiological data combined with a patient-based approach may allow a more accurate therapy decision making.

Med Hypotheses, 2003 Oct, 61(4), 431 - 4
Treatment of post-burns bacterial infections by Fenton reagent, particularly the ubiquitous multiple drug resistant Pseudomonas spp; Ahmad SI et al.; Post-burn microbial infections are a major problem in burns, and in cases of third degree burns, the survival of patients can depend not only upon the severity but also upon the extent and the type of infections . If proper measures are not employed, patients may suffer from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs . Infection by multiple drug resistant (MDR) bacteria can create further complexity to the treatment . It is proposed that a combination of diluted hydrogen peroxide (H(2)O(2)) and ferrous sulphate (FeSO(4)), which generates hydroxyl radicals (*OH) via Fenton reaction, can effectively be used for the treatment of post-burns bacterial infections . It should be particularly useful for the ubiquitous opportunistic pathogen, Pseudomonas aeruginosa, known to be notoriously resistant to various antibiotics . This reactive oxygen species (ROS)-induced inactivation of the bacterial skin infections may be of particular importance in Third World countries where the incidence of burns and post-burns infections by MDR bacteria (due to the indiscriminate use of antibiotics, lack of stringent safety regulations and proper hygiene) may be more prevalent and where cocktails of antibiotics may be less affordable . Also, since the putative lack of development of bacterial resistance to *OH is not known, it provides an added advantage to the treatment . Finally, although this work addresses the control of bacterial infections in burns cases, it is envisaged that this ROS-induced chemotherapy may also be useful in combating other kinds of skin infections particularly those resisting antibiotic treatment.

Int J Antimicrob Agents, 2003 Sep, 22(3), 262 - 4
The in-vitro antimicrobial effect of non-antibiotics and putative inhibitors of efflux pumps on Pseudomonas aeruginosa and Staphylococcus aureus; Hendricks O et al.; The anti-microbial activity of six non-antibiotics (one amino-ethylchloride, three phenothiazines, two tricyclic antidepressives) were tested on 20 clinical isolates of Pseudomonas aeruginosa, one clinical isolate of Klebsiella pneumoniae, 2 ATTC strains and 14 clinical isolates of Staphylococccus aureus, using the plate dilution method . The effects on P . aeruginosa were independent of antibiotic resistance pattern and the species Stenotrophomonas maltophilia was found to be the most susceptible to the non-antibiotics, with MIC values as low as 20 mg/l for some of the substances . The 16 S . aureus strains tested were all particularly susceptible to the anti-microbial effects of the putative inhibitors of efflux pumps thioridazine and trifluoperazine with MIC values of < or =16 mg/l independently of the methicillin resistance profile of the strains . Because phenothiazines are well known to inhibit efflux pumps our results may indicate the existence of such pumps . Current works in progress are attempts at reversing the antibiotic resistance of selected bacterial strains using specific non-antibiotics and their stereo-chemical isomers.

Int J Antimicrob Agents, 2003 Sep, 22(3), 254 - 61
Phenylpiperidine selective serotonin reuptake inhibitors interfere with multidrug efflux pump activity in Staphylococcus aureus; Kaatz GW et al.; Structural variants of phenylpiperidine selective serotonin reuptake inhibitors (P-SSRIs) inhibited the function of two unique Staphylococcus aureus multidrug efflux pumps . The most active compound was the paroxetine isomer NNC 20-7052, which had an IC(50) for ethidium, acriflavine, and pyronin Y efflux of 9, 53, and 18% of its MIC, respectively, against the NorA pump . The unbalanced effect of NNC 20-7052 on the efflux of different substrates suggests the possibility that P-SSRIs function by a physical interaction with NorA . Under the conditions employed pump inhibition partially extended to the resistance-nodulation-division (RND) pump AcrAB-TolC, but not to the Pseudomonas aeruginosa RND pumps MexAB-OprM or MexCD-OprJ.

J Bacteriol, 2003 Oct, 185(19), 5807 - 14
Inter- and intraclonal diversity of the Pseudomonas aeruginosa proteome manifests within the secretome; Wehmhoner D et al.; The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain . Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro . In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones . The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant . Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system . Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly . We concluded that the secretome expression is a sensitive measure of P . aeruginosa strain variation.

Ophthalmology, 2003 Sep, 110(9), 1714 - 7
Endophthalmitis caused by Pseudomonas aeruginosa; Eifrig CW et al.; OBJECTIVE: To investigate the clinical settings and treatment outcomes for endophthalmitis caused by Pseudomonas aeruginosa . DESIGN: Retrospective, noncomparative, consecutive case series . METHODS: The medical records were reviewed of all patients treated for P . aeruginosa endophthalmitis at a single institution between January 1, 1987, and December 31, 2001 . MAIN OUTCOME MEASURES: Final visual acuity and rate of enucleation or evisceration . RESULTS: The study included 28 eyes of 28 patients with a median age of 75 years (range, 5-93 years) . The clinical setting of endophthalmitis included: cataract surgery (n = 9), corneal ulcer (n = 7), penetrating keratoplasty (n = 5), bleb associated (n = 2), glaucoma drainage implant (n = 2), pars plana vitrectomy (n = 1), iris cyst removal (n = 1), and trauma (n = 1) . In acute-onset postoperative cases (n = 10), the median interval between surgery and presentation with endophthalmitis was 4 days (range, 1-26 days) . The median duration of symptoms was 1 day, and all patients were treated on the day of diagnosis . Eleven patients (39%) had hand motions or better vision in the infected eye at the time of initial diagnosis . Because of no light perception visual acuity, necrosis of cornea and sclera, and intractable pain, 7 eyes (25%) underwent evisceration or enucleation as initial treatment; of the remaining 21 eyes, intravitreal antibiotics were administered in all cases and intravitreal dexamethasone was administered in 15 cases (71%) . Pars plana vitrectomy was performed in 12 patients (43%) . The organism was sensitive to the initial antibiotics administered in all but 2 cases . Final visual acuity was 5/200 or better in 2 of 28 eyes (7%) . Nineteen patients (68%) had a final visual acuity outcome of no light perception, and no patient achieved a final visual acuity of better than 20/400 . Overall, 18 of the 28 eyes (64%) were either eviscerated or enucleated . CONCLUSIONS: Endophthalmitis caused by P . aeruginosa is associated with poor visual outcomes despite prompt treatment with intravitreal antibiotics to which the organisms were sensitive.

J Biol Chem, 2003 Nov 28, 278(48), 47400 - 7 Epub 2003 Sep 15.
Identification and characterization of a novel translational repressor of the steroid-inducible 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni; Xiong G et al.; Comamonas testosteroni 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR) is a key enzyme in the degradation of steroid compounds in soil and may therefore play a significant role in the bioremediation of hormonally active compounds in the environment . The enzyme is also involved in the degradation of the steroid antibiotic fusidic acid . In addition, 3 alpha-HSD/CR mediates the carbonyl reduction of non-steroidal aldehydes and ketones . Because the gene of 3 alpha-HSD/CR (hsdA) is inducible by steroids, we were interested in the mode of its molecular regulation . Recently, we could identify the first molecular determinant in procaryotic steroid signaling, i.e . a repressor protein (RepA), which acts as a negative regulator by binding to upstream operator sequences of hsdA, thereby blocking hsdA transcription . In this work, we identified and cloned a second novel regulator gene that we named repB . The gene locates 932 bp downstream from hsdA on the C . testosteroni chromosome with an orientation opposite to that of hsdA . The open reading frame of repB consists of 237 bp and translates into a protein of 78 amino acids that was found to act as a repressor that regulates hsdA expression on the translational level . Northern blot analysis, UV-cross linking, gel-shift assays, and competition experiments proved that RepB binds to a 16-nucleotide sequence downstream of AUG at the 5' end of the 3 alpha-HSD/CR mRNA, thereby blocking hsdA translation . Testosterone, on the other hand, was shown to specifically bind to RepB, thereby yielding the release of RepB from the 3 alpha-HSD/CR mRNA such that hsdA translation could proceed . Data bank searches with the RepB primary structure yielded a 46.2% identity to the regulator of nucleoside diphosphate kinase, a formerly unknown protein from Escherichia coli that can restore a growth defect in alginate production in Pseudomonas aeruginosa . In conclusion, the induction of hsdA by steroids in fact is a derepression where steroidal inducers bind to two repressor proteins, RepA and RepB, thereby preventing blocking of hsdA transcription and translation, respectively.

J Environ Biol, 2003 Apr, 24(2), 211 - 2
Antimicrobial activities of Eusteralis deccanensis and E . quadrifolia essential oils; Thoppil JE et al.; Antimicrobial activity of the essential oils of Eusteralis deccanensis and E . quadrifolia were investigated on Bacillus subtilis, B . megaterium, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Rhizopus oryzae, Aspergillus niger and Colletotrichum musae . Both the oils possess growth inhibitory activity against most of the microorganisms tested.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11618 - 23 Epub 2003 Sep 12.
Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells; Shao MX et al.; Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands {e.g., transforming growth factor type alpha (TGF-alpha)} and EGFR phosphorylation are implicated in mucin production in airway epithelial cells . Tumor necrosis factor alpha-converting enzyme (TACE) is reported to cleave precursor of TGF-alpha, with release of soluble mature TGF-alpha in various epithelial tissues . We hypothesized that TACE increases the shedding of TGF-alpha, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells . To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli {lipopolysaccharide (LPS) and supernatant from the Gram-negative bacterium Pseudomonas aeruginosa (PA sup)} . PMA, PA sup, and LPS increased MUC5AC gene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-alpha-neutralizing Ab, implicating ligand (TGF-alpha)-dependent EGFR phosphorylation in mucin production . These stimuli induced release of soluble TGF-alpha, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-alpha protease inhibitor-1 and tissue inhibitor of metalloprotease-3 . We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE . Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF-alpha shedding, EGFR phosphorylation, and mucin production . From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligand-EGFR cascade in response to various stimuli.

Am J Respir Crit Care Med, 2003 Dec 15, 168(12), 1471 - 5 Epub 2003 Sep 11.
Inflammatory and microbiologic markers in induced sputum after intravenous antibiotics in cystic fibrosis; Ordonez CL et al.; Induced sputum has been used to study airway inflammation . We sought to determine whether markers of infection and inflammation in induced sputum were a useful and safe outcome measure in cystic fibrosis . We hypothesized that bacterial density and inflammatory content of induced sputum would decrease after antibiotic therapy . Induced sputum was assayed for bacterial density, cell count, and differential and inflammatory markers before and after treatment with intravenous antibiotics . Fifty-five of the 72 subjects enrolled (mean age +/- SD 18.2 +/- 7.9 years) completed the study . FEV1 increased by an average 0.3 +/- 0.3 L (10.4 +/- 8.7% predicted FEV1), p<0.0001; density of Pseudomonas aeruginosa and Staphylococcus aureus decreased by 2.4 +/- 3.1 log10 cfu/g (p<0.0005) and 4.0 +/- 2.3 log10 cfu/ml (p<0.0001), respectively; neutrophil count decreased by 0.4 +/- 0.6 log10 cells/ml (p<0.0001), interleukin-8 concentration by 0.5 +/- 1.3 log10 pg/ml (p<0.05), and neutrophil elastase by 0.4 +/- 0.7 log10 microg/ml (p<0.005) . Seven of 127 (6%) sputum induction procedures showed a decrease in FEV1 of 20% or more . We conclude that markers in induced sputum may be useful, noninvasive outcome measures to assess response to therapies in cystic fibrosis studies.

J Appl Microbiol, 2003, 95(4), 874 - 82
Intrinsic and acquired resistance to quaternary ammonium compounds in food-related Pseudomonas spp; Langsrud S et al.; AIMS: To determine the sensitivity of a strain used for disinfectants testing (Pseudomonas aeruginosa ATCC 15442) and food-associated isolates to benzalkonium chloride and didecyl dimethylammonium chloride (DDAC) . To determine whether the increase in bacterial resistance after adaptation to DDAC can be associated with phenotypic changes . To test the activity of alternative disinfectants to eliminate resistant Pseudomonas spp . METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 was among the most resistant strains tested using a bactericidal suspension test . Growth of a sensitive Ps . fluorescens in gradually higher concentrations of DDAC resulted in stable higher resistance and to some cross-resistance to several antibacterial agents, with the exception of disinfectants containing chloramine T, glutaraldehyde or peracetic acid . It was shown by microscopy that adaptation was followed by loss of flagella, and slime formation . Removal of the slime by sodium dodecyl sulphate resulted in partial loss of the acquired resistance . CONCLUSIONS: Pseudomonas spp . may adapt to survive against higher concentrations of quaternary ammonium compounds (QACs), but resistant strains can be eliminated with chemically unrelated disinfectants . SIGNIFICANCE AND IMPACT OF THE STUDY: The work supports the rotation of disinfectants in food processing environments for avoiding the development of bacterial resistance to QACs . The alternating disinfectants should be chosen carefully, because of possible cross-resistance.

Rev Esc Enferm USP, 2003 Mar, 37(1), 90 - 6
{Antimicrobial activity of paraformaldehyde tablets reproducing their use conditions in Brazilian health institutions}; Graziano KU et al.; The sterilizing activity of Parafolmaldehyde Tablets reproducing the conditions of use in the Brazilian health Institutions (without the heating, without increment of the relative humidity, in 0.5%, even for a long period of exhibition of 12 hours) was evaluated "in vitro" by microbiologic monitoring, according to the methodology of the AOAC, officially adopted by the Brazilian Health Ministry for registration of that category of products . The results of the experiments refuted the sterilizing action of Parafolmaldehyde Tablets in this conditions . Thus, it was evaluated the disinfectant action of the product using the vegetative bacteria by Use Dilution Method, preconized for AOAC and adapted for gaseous products, against the tests microorganisms standardized Staphylococcus aureus (ATCCn . 6538), Samonella choleraesuis (ATCCn . 10708) and Pseudomonas aeruginosa (ATCC n . 15442) . In these experiments, the results of the cultures showed 100% negative against all the tested bacteria inferring indications of disinfectant activity of high level action.

Diagn Microbiol Infect Dis, 2003 Sep, 47(1), 365 - 72
Antimicrobial spectrum of activity for meropenem and nine broad spectrum antimicrobials: report from the MYSTIC Program (2002) in North America; Rhomberg PR et al.; The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program provides susceptibility data for participating medical centers where carbapenems are utilized . The activity of meropenem and nine broad-spectrum antimicrobial agents were assessed against 3,047 bacterial isolates collected during 2002 from 16 North American sites . The overall rank order of susceptibility of the 10 antimicrobial agents tested against Gram-negative isolates was: meropenem (98%) > imipenem (97%) > cefepime (95%) > tobramycin (93%) > piperacillin/tazobactam = gentamicin (92%) > ceftazidime (91%) > ciprofloxacin (87%) > aztreonam (86%) > ceftriaxone (74%) . These results and those from previous years, demonstrate the continued excellent potency and spectrum of activity for meropenem . The utility of meropenem against Pseudomonas aeruginosa isolates has increased steadily with a rise in percent susceptibility each year from 78.2% in 1999 to a present rate of 93.1% susceptible . Conversely, we showed the susceptibility for ciprofloxacin against these same P . aeruginosa isolates has decreased from 82.9 to 72.3% susceptible over four years . Many medical centers have observed a decreased activity of some aminoglycosides, cephalosporins and fluoroquinolones due to increases in rates of extended-spectrum beta-lactamases, Amp C and other resistance mechanisms . Carbapenem resistance remains rarely documented and these beta-lactamase-stable agents appear to be an alternative treatment option for serious community-acquired or nosocomial infections in high risk patient populations.

Biomed Environ Sci, 2003 Jun, 16(2), 163 - 72
Characterization of phenol biodegradation by Comamonas testosteroni ZD4-1 and Pseudomonas aeruginosa ZD4-3; Chen YX et al.; OBJECTIVE: To investigate the characteristic and biochemical mechanism about the phenol biodegradation by bacterial strains ZD 4-1 and ZD 4-3 . METHODS: Bacterial strains ZD 4-1 and ZD 4-3 were isolated by using phenol as the sole source of carbon and energy, and identified by 16S rDNA sequence analysis . The concentrations of phenol and total organic carbon (TOC) were monitored to explore the degradation mechanism . The biodegradation intermediates were scanned at 375 nm by using a uv-vis spectrophotometer . The enzyme assays were performed to detect the activities of dioxygenases . RESULTS: Bacterial strains ZD 4-1 and ZD 4-3 were identified as Comamonas testosteroni and Pseudomonas aeruginosa by 16S rDNA sequence analysis, respectively . The growth of the two strains was observed on a variety of aromatic hydrocarbons . The strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and meta-pathways, respectively . In addition, the results of enzyme assays showed that the biodegradation efficiency of phenol by meta-pathways was higher than that by ortho-pathways . Finally, the results of induction experiment indicated that the catechol dioxygenases, both catechol 1,2-dioxygenase (C120) and catechol 2,3-dioxygenase (C230), were all inducible . CONCLUSION: The strains ZD 4-1 and ZD 4-3 metabolize phenol through ortho-pathways and meta-pathway, respectively . Furthermore, the biodegradation efficiency of phenol by meta-pathways is higher than that by ortho-pathways.

J Basic Microbiol, 2003, 43(5), 407 - 13
Evaluation of a new agar medium containing cetrimide, kanamycin and nalidixic acid for isolation and enhancement of pigment production of Pseudomonas aeruginosa in clinical samples; Kodaka H et al.; A new selective agar medium (CKNA), containing cetrimide 0.3 g/l, kanamycin sulfate 50 mg/l, and nalidixic acid 5 mg/l, was developed for the isolation of Pseudomonas aeruginosa . It was compared to Nalidixic Acid Cetrimide agar (NAC), Pseudomonas Aeruginosa Selective Agar (PASA) and Pseudosel(TM) agar (CET) using 1,148 clinical specimens . The sensitivities rates of P . aeruginosa with CKNA, NAC, PASA, and CET were 88.2%, 81.3%, 79.2%, and 84.0%, respectively . The specificities of CKNA, NAC, PASA, and CET were 99.2%, 98.4%, 99.2%, and 99.7%, respectively.

Protein Expr Purif, 2003 Sep, 31(1), 155 - 60
A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species; Bergendahl V et al.; The use of antibodies for protein purification is a powerful technique but the release of the target protein in its active form is often difficult . So called "polyol-responsive" monoclonal antibodies (PR-MAbs) have a feature that allows elution of the antigen under very gentle conditions, so that even multi-subunit proteins can be released in their active form . In this work a PR-MAb, 8RB13, was isolated that can purify RNA polymerase (RNAP) from many different bacterial species . High specificity towards RNAP with a broad species cross-reactivity was achieved by immunization with RNAP from Escherichia coli and screening with Bacillus subtilis RNA polymerase . The isolated MAb could detect the beta-subunit of RNA polymerase from 10 out of 12 species tested on a Western blot indicating its potential for purification of core RNAP from these organisms . Representatively, four of these species E . coli, B . subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor were subjected to immunoaffinity purification yielding RNA polymerases that were active in in vitro transcription and seemed to be primarily core polymerase, lacking sigma-subunits.

J Chemother, 2003 Aug, 15(4), 315 - 22
Current and future perspectives for levofloxacin in severe Pseudomonas aeruginosa infections; Marchetti F et al.; The question of whether levofloxacin includes Pseudomonas aeruginosa in its spectrum of clinical activity is discussed by reviewing the major findings on this issue, mainly those published in Italy . The in vitro activity of levofloxacin against P . aeruginosa is now documented on thousands of strains worldwide . The pharmacodynamic properties of levofloxacin allow for the treatment of pseudomonal infections . The levofloxacin clinical results extrapolated from published studies document the efficacy of levofloxacin in the treatment of infections sustained by P . aeruginosa.

Anesthesiology, 2003 Sep, 99(3), 652 - 5
Bacterial reduction by cell salvage washing and leukocyte depletion filtration; Waters JH et al.; BACKGROUND: Blood conservation techniques are being increasingly used because of the increased cost and lack of availability of allogeneic blood . Cell salvage offers great blood savings opportunities but is thought to be contraindicated in a number of areas (e.g., blood contaminated with bacteria) . Several outcome studies have suggested the safety of this technique in trauma and colorectal surgery, but many practitioners are still hesitant to apply cell salvage in the face of frank bacterial contamination . This study was undertaken to assess the efficacy of bacterial removal when cell salvage was combined with leukocyte depletion filtration . METHODS: Expired packed erythrocytes were obtained and inoculated with a fixed amount of a stock bacteria (Escherichia coli American Type Culture Collections {ATCC} 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, or Bacteroides fragilis ATCC 25285) in amounts ranging from 2,000 to 4,000 colony forming units/ml . The blood was processed via a cell salvage machine . The washed blood was then filtered using a leukocyte reduction filter . The results for blood taken during each step of processing were compared using a repeated-measures design . RESULTS: Fifteen units of blood were contaminated with each of the stock bacteria . From the prewash sample to the postfiltration sample, 99.0%, 99.6%, 100%, and 97.6% of E . coli, S . aureus, P . aeruginosa, and B . fragilis were removed, respectively . DISCUSSION: Significant but not complete removal of contaminating bacteria was seen . An increased level of patient safety may be added to cell salvage by including a leukocyte depletion filter when salvaging blood that might be grossly contaminated with bacteria.

Pneumonol Alergol Pol, 2003, 71(1-2), 5 - 11
{Microflora colonizing pleural drains after thoracic surgery}; Korona-Glowniak I et al.; The aim of this paper was to analyse aerobic and anaerobic bacterial flora colonizing pleural drains in 32 patients with lung tumor undergoing pulmonary resection and receiving antimicrobial prophylaxis . Fluid from pleural drains was taken up on the day of operation and on the 3-rd or 4-th day after the operation . Significant number of aerobic bacteria, mainly methicillin-resistant coagulase-negative staphylococci and multidrug resistant Gram-negative rods, were isolated from 30 (46.9%) specimens . Lower number of anaerobic bacteria were found in 24 (37.5%) specimens . Colonization of the pleural drains does not mean infection, (in only one patient isolated Pseudomonas aeruginosa could be the etiologic agent of infection) however knowledge about bacterial species found in drain fluid in a local population and drug susceptibility of isolated strains allows to propose effective antimicrobial prophylaxis.

J Clin Microbiol, 2003 Sep, 41(9), 4312 - 7
Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis; Qin X et al.; Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity . We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P . aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers . Of 50 P . aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers . For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P . aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers . A single false-positive P . aeruginosa result was seen with oprI primers . The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group . Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time . Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P . aeruginosa and for non-P . aeruginosa gram-negative isolates.

Am J Respir Crit Care Med, 2003 Dec 15, 168(12), 1462 - 70 Epub 2003 Sep 04.
Modulation of bacterial growth by tumor necrosis factor-alpha in vitro and in vivo; Lee JH et al.; Tumor necrosis factor-alpha (TNF-alpha) plays an important role in innate immunity . Recent in vitro studies have shown that TNF-alpha may also serve as a growth factor for some bacteria . We examined the physiologic relevance of this phenomenon both in vitro and in vivo . Recombinant mouse TNF-alpha increased in vitro proliferation of Escherichia coli but not Pseudomonas aeruginosa in a concentration-dependent manner, and this effect was attenuated by anti-TNF-alpha antibodies . However, in vivo, TNF-alpha gene-deficient (TNF-alpha-/-) mice showed higher mortality than wild-type (TNF-alpha+/+) mice after inoculation of intranasal bacteria . An impaired bacterial clearance in TNF-alpha-/- mice was associated with decreased systemic concentrations of chemokine macrophage inflammatory protein-2, reduced pulmonary neutrophil recruitment, and depressed expression of neutrophil CD11b and CD16/CD32, suggesting that the effect of TNF-alpha on E . coli growth was outweighed by the recruited neutrophils . We also demonstrated that neutropenic TNF-alpha+/+ mice had approximately 100-fold higher E . coli counts in their lungs than TNF-alpha-/- mice, although survival rates in both groups were similar . We conclude that TNF-alpha augments E . coli growth in vitro and in vivo . However, in vivo, this effect becomes only apparent in neutropenic animals . The relevance of these findings for immune compromised patients remains to be investigated.

Org Biomol Chem, 2003 Jul 21, 1(14), 2461 - 7
Application of the Barton photochemical reaction in the synthesis of 1-dethia-3-aza-1-carba-2-oxacephem: a novel agent against resistant pathogenic microorganisms; Hakimelahi GH et al.; Racemic 7-(phenylacetamido)-1-dethia-3-aza-1-carba-2-oxacephem 3 was synthesized and found to possess antibacterial activity against Staphylococcus aureus FDA 209P, Escherichia coli ATCC 39188, Pseudomonas aeruginosa 1101-75 and Klebsiella pneumoniae NCTC 418 as well as the beta-lactamase producing organisms E . coli A9675 and P . aeruginosa 18S-H and the methicillin-resistant organism S . aureus 95 . Formation of the carbacephem 3 originated from the Barton photochemical reaction in the conversion of 8 to 10 . Intramolecular cyclization of syn-oximino beta-lactam 10 afforded 7-azido-2-oxa-3-azacephem 11, which was reduced and acylated to 12 . Enzymatic removal of the methyl group from 12 gave the target molecule 3.

Clin Infect Dis, 2003 Sep 15, 37(6), 745 - 51 Epub 2003 Aug 23.
Pseudomonas aeruginosa bacteremia: risk factors for mortality and influence of delayed receipt of effective antimicrobial therapy on clinical outcome; Kang CI et al.; Among the nosocomial pathogens, Pseudomonas aeruginosa is recognized as a major cause of morbidity and mortality . Data on 136 patients with P . aeruginosa bacteremia were retrospectively analyzed to evaluate risk factors for mortality . The median age of the patients was 55 years (range, 15-85 years), 78.7% of the cases were hospital-acquired, and the 30-day mortality rate was 39% (53 of 136 patients) . Multivariate analysis demonstrated that risk factors for mortality included severe sepsis, pneumonia, delay in starting effective antimicrobial therapy, and an increasing APACHE II score (all P values <.05) . In 123 of the 136 patients (excluding 13 patients treated with inadequate definitive antibiotics), 30-day mortality was 27.7% (13 of 47 patients) in the group of patients who received initially effective empirical antimicrobial therapy, and 43.4% (33 of 76) in the group of patients who received delayed effective antimicrobial therapy (P=.079) . There was a trend toward higher mortality as the length of delay increased . Delay in starting effective antimicrobial therapy for P . aeruginosa bacteremia tended to be associated with higher mortality.

Int J Pharm, 2003 Sep 16, 263(1-2), 61 - 8
Antimicrobial properties of silver-containing wound dressings: a microcalorimetric study; O'Neill MA et al.; The studies reported here have been undertaken to assess the potential use of isothermal microcalorimetry in studying the antimicrobial efficacy of wound dressings that contain antimicrobial agents . The microcalorimetric technique allows non-invasive and non-destructive analysis to be performed directly on a test sample, regardless of whether it is homogeneous or heterogeneous in nature . Microcalorimetry is an established procedure that offers quantitative measurements and has the distinct advantage over traditional antimicrobial test methodologies in that calorimetric measurements are made continuously over real-time, thus the dynamic response of microorganisms to an antimicrobial agent is observed in situ . The results described in this paper are for interaction of two silver-containing wound care products AQUACEL Ag Hydrofiber (ConvaTec, Deeside, UK) and Acticoat 7 with SILCRYST (Smith and Nephew Healthcare, UK) with the wound pathogenic organisms Staphylococcus aureus and Pseudomonas aeruginosa . Both dressings are shown, microcalorimetrically, to have the capacity to kill these common wound pathogens within 1-2 h of contact . A dose-response study was conducted with the AQUACEL Ag dressing . Microcalorimetry is shown to be rapid, simple and effective in the study of the antimicrobial properties of gel forming wound dressings.

J Antimicrob Chemother, 2003 Oct, 52(4), 668 - 74 Epub 2003 Sep 01.
Treatment and outcome of Pseudomonas aeruginosa bacteraemia: an antibiotic pharmacodynamic analysis; Zelenitsky SA et al.; OBJECTIVES: To conduct a retrospective study of antibiotic pharmacodynamics in the treatment of Pseudomonas aeruginosa bacteraemia, and to identify pharmacodynamic indices associated with clinical cure . METHODS: Cases of P . aeruginosa bacteraemia were identified, and information related to patient demographics, clinical status, antibiotic treatment and clinical outcome were documented . Anti-pseudomonal therapy was assessed, and concentration versus time profiles were constructed using measured levels for aminoglycosides, or population pharmacokinetic models for other antibiotics . P . aeruginosa isolates from all patients were retrieved and MICs for the anti-pseudomonal agents used to treat the episode of bacteraemia were determined . Patient- and treatment-related factors were tested for associations with clinical outcome using univariate and multivariate analyses . RESULTS: Fifty cases of P . aeruginosa bacteraemia were identified and 38 cases were included in the pharmacodynamic analysis . Eighty-seven percent of patients received an aminoglycoside or ciprofloxacin and 79% received piperacillin or ceftazidime . A majority of patients, 71%, were administered a combination of antibiotics . Treatment outcomes were documented as persistent infection in 21%, death within 2-30 days in 21% and clinical cure in 58% of cases . Peak/MIC (P=0.001) and AUC24/MIC (P=0.002) for aminoglycosides and ciprofloxacin were significant factors in univariate tests . Only peak/MIC was associated independently with treatment outcome (P=0.017) in logistic regression analysis . The predicted probability of cure was > or =90% when peak/MIC was at least 8 . CONCLUSION: Pharmacodynamic considerations including aggressive dosing with targeted peak/MICs for aminoglycosides and ciprofloxacin are strongly associated with clinical outcome and essential to the appropriate management of P . aeruginosa bacteraemia.

J Antimicrob Chemother, 2003 Oct, 52(4), 572 - 5 Epub 2003 Sep 01.
A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa; Li Y et al.; OBJECTIVES: Multidrug efflux pumps are thought to be involved in mediating multidrug resistance in Pseudomonas aeruginosa . Here we aim to characterize hitherto uncharacterized multidrug efflux pumps from P . aeruginosa . MATERIALS AND METHODS: We isolated a mutant, YM442, which showed elevated resistance to several antimicrobial agents from P . aeruginosa YM44 lacking four major multidrug efflux pumps, MexAB, MexCD-OprJ, MexEF-OprN and MexXY . We cloned genes responsible for the resistance from chromosomal DNA of YM442 using YM44 as host . RESULTS: We designated the genes mexVW . Introduction of a recombinant plasmid pTAJ2 carrying the mexVW into YM44 cells conferred resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine . Elevated ethidium bromide extrusion was observed with cells of YM442 and of YM44/pTAJ2 . An outer membrane protein OprM was able to cooperate with MexVW . Elevated expression of the mexV gene was observed with YM442 compared with YM44 . CONCLUSIONS: MexV (membrane fusion protein)-MexW (RND-type membrane protein)-OprM is a tripartite multidrug efflux pump . It is suggested that other outer membrane component(s) could cooperate with MexVW.

J Antimicrob Chemother, 2003 Oct, 52(4), 699 - 702 Epub 2003 Sep 01.
Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-beta-lactamase; Gales AC et al.; BACKGROUND: In Brazil, carbapenem use has been limited by high carbapenem-resistance rates among Pseudomonas aeruginosa isolates . OBJECTIVE: The main objective of this study was to evaluate the presence of an epidemic P . aeruginosa strain in unrelated Brazilian hospitals . We also aimed to search for the gene blaSPM, which encodes production of SPM, a novel metallo-beta-lactamase (MBL) . METHODS: A reference broth microdilution method was used for antimicrobial susceptibility testing . The isolates were typed by ribotyping and pulsed-field gel electrophoresis (PFGE) . A disc-approximation test using MBL inhibitors was employed to screen isolates for MBL production . PCR was used to search for the gene blaSPM . RESULTS: A total of 43 clinical isolates of carbapenem-resistant P . aeruginosa were collected from 12 hospitals . Colistin retained greatest activity in vitro . A single ribogroup included 17 P . aeruginosa isolates (39.5%) collected from seven unrelated hospitals located in five Brazilian states . Sixteen of these isolates showed an identical PFGE pattern, and 15 produced an SPM-1-like MBL . The remaining 26 isolates were grouped into 25 diverse ribogroups; none were MBL producers . CONCLUSIONS: The emergence and dissemination of an epidemic clone has contributed to the high carbapenem resistance rates among P . aeruginosa isolates in Brazil . In addition, the production of SPM MBL has an important role in carbapenem resistance in this region . This is the first report of dissemination of an SPM-1-like-MBL-producing strain of P . aeruginosa among unrelated Brazilian hospitals.

Microbiology, 2003 Sep, 149(Pt 9), 2627 - 34
Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1; Kim EJ et al.; Pseudomonas aeruginosa PAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface . In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena . The physiological responses of two P . aeruginosa PAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations . In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed . The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient, kLa, significantly decreased under iron-limited conditions . The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration . The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way for P . aeruginosa PAO1 to adapt to growth conditions of iron limitation . Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation . The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions . These results have implications for lung infection of cystic fibrosis patients by P . aeruginosa in view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death.

J Bacteriol, 2003 Sep, 185(18), 5391 - 7
Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli; Park SJ et al.; The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene . However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated . Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E . coli strain . When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E . coli WB108, which is a derivative of E . coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E . coli WB101 . The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E . coli WB108 . Also, E . coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene . For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E . coli and purified . Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA . These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E . coli strain.

Biochemistry (Mosc), 2003 Aug, 68(8), 918 - 25
Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide; Bystrova OV et al.; The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy . It was found that two major products have the same inner core region and lipid A carbohydrate backbone (A) but different outer core regions (B and C) . The difference is in the position of a rhamnose residue, which is substituted with either an additional glucose residue (B) or a disaccharide remainder of the degraded O-polysaccharide (C) . The site and the configuration of the linkage between the O-polysaccharide and the core were determined and, together with published data, the structure of the so-called biological repeating unit of the O-antigen was defined (D) . The glycosidic linkage of the quinovosamine residue is beta when it links the O-polysaccharide to the core (C) and alpha when it connects the interior repeating units of the O-polysaccharide to each other (D) {Formula: see text} . In the structures shown Rha stands for rhamnose, Kdo for 3-deoxy-D-manno-oct-2-ulosonic acid, Hep for L-glycero-D-manno-heptose, GalNAcA for 2-acetamido-2-deoxygalacturonic acid, QuiN for 2-amino-2,6-dideoxyglucose (quinovosamine), DeltaHexNA for 2-amino-2-deoxy-D-threo-hex-4-enuronic acid; all monosaccharides are in the pyranose form and have the D configuration, except for Rha and GalNAcA that have the L configuration . In C, the remainder of the degraded O-polysaccharide is shown in bold type.

Epidemiol Infect, 2003 Aug, 131(1), 799 - 804
Bacterial infection in exacerbated COPD with changes in sputum characteristics; Monso E et al.; We examined the risk factors for bacterial exacerbation, defined as the presence of pathogenic bacteria in sputum, in 90 chronic obstructive pulmonary disease (COPD) patients with an exacerbation and changes in sputum characteristics . Smoking, alcohol, lung function, body mass index, medical visits and treatments were the independent variables assessed using multivariable logistic regression modelling (OR, 95% CI) . A bacterial exacerbation was diagnosed in 39 (43.3%) of 90 patients . Bacterial exacerbations were more prevalent among current smokers (OR 3.77, 95% CI 1.17-12.12), in patients with poor compliance with inhalation therapy (OR 3.25, 95% CI 1.18-8.93) and with severe lung function impairment (FEV1 OR 0.96, 95% CI 0.93-1.00) . Prior use of antibiotics was a risk factor for Pseudomonas aeruginosa infection (OR 6.06, 95% CI 1.29-28.44) and influenza vaccination appeared to have a protective effect against this infection (OR 0.15, 95% CI 0.03-0.67) . We conclude that severe impairment of lung function, smoking and poor compliance with therapy are risk factors for bacterial infection in COPD, and P . aeruginosa should be suspected in patients who have been treated with antibiotics and in those not vaccinated against influenza.

Thorax, 2003 Sep, 58(9), 794 - 6
Survey of resistance of Pseudomonas aeruginosa from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents; Pitt TL et al.; BACKGROUND: Respiratory infection with Pseudomonas aeruginosa is very common in patients with cystic fibrosis (CF) but antimicrobial resistance rates of CF isolates across the UK are largely unknown . METHODS: The susceptibility of 417 CF patient isolates of P aeruginosa from 17 hospitals to six commonly prescribed antibiotics were examined . Isolates were tested by an agar break point dilution method and E-tests according to British Society of Antimicrobial Chemotherapy guidelines . Genotyping of isolates was performed by XbaI DNA macrorestriction and pulsed field gel electrophoresis . RESULTS: 38% of isolates were susceptible to all of the agents tested; almost half were resistant to gentamicin compared with ceftazidime (39%), piperacillin (32%), ciprofloxacin (30%), tobramycin (10%), and colistin (3%) . Approximately 40% were resistant to two or more compounds with ceftazidime in combination with gentamicin, piperacillin or ciprofloxacin being the most common cross resistances . Resistance rates were generally similar to those reported recently from the USA and Germany . A selection of resistant isolates proved to be predominantly genotypically distinct by XbaI DNA macrorestriction but six pairs from three centres had similar genotypes . CONCLUSIONS: The level of resistance to front line antipseudomonal agents, with the exception of colistin, is disturbingly high . The prudent use of antimicrobial drugs and closer monitoring of accumulation of resistant strain populations should be actively considered.

Phytochemistry, 2003 Sep, 64(2), 561 - 5
Composition and antimicrobial activity of the essential oils from invasive species of the Azores, Hedychium gardnerianum and Pittosporum undulatum; Medeiros JR et al.; The compositions of the essential oils from the leaves and flowers of Hedychium gardnerianum and from the leaves of Pittosporum undulatum growing on San Miguel Island (Azores) were investigated, and the compounds were identified by GC-MS analyses . The oils in the leaves and flowers of H . gardnerianum were rich in alpha-pinene, beta-pinene and alpha-cadinol, whereas that from P . undulatum was found to contain monoterpenes, sesquiterpenes, diterpenes and alkanes, of which the sesquiterpenes, calamenene (41.4%), farnesol (10.9%), spathulenol (5.6%) and beta-selinene (5.2%) and the diterpene (8beta,13beta)-kaur-16-ene (10.7%) were the major components . Their potential antimicrobial activities were tested against Staphylococcus aureus, S . epidermis and Pseudomonas aeruginosa, and those with the highest activities against S . aureus and S . epidermis were from H . gardnerianum; none had activity against P . aeruginosa . Additionally, the essential oils from Pittosporum undulatum had good antithrombin activity whereas that from H . gardnerianum did not.

J Immune Based Ther Vaccines . 2003 Aug 13;1(1):2.
Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosa-induced acute lung injury in a rat model; Faure K et al.; BACKGROUND: The effects of the murine monoclonal anti-PcrV antibody Mab166 on acute lung injury induced by Pseudomonas aeruginosa were analyzed in a rat model . METHODS: Lung injury was induced by the instillation of P . aeruginosa strain PA103 directly into the left lungs of anesthetized rats . One hour after the bacterial instillation, rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments were administered intratracheally directly into the lungs . The degree of alveolar epithelial injury, amount of lung edema, decrease in oxygenation and extent of lung inflammation by histology were evaluated as independent parameters of acute lung injury . RESULTS: These parameters improved in rats that had received intratracheal instillation of either rabbit polyclonal anti-PcrV IgG, murine monoclonal anti-PcrV IgG Mab166 or Mab166 Fab-fragments in comparison with the control group . CONCLUSION: Mab166 and its Fab fragments have potential as adjuvant therapy for acute lung injury due to P . aeruginosa pneumonia.

Invest Ophthalmol Vis Sci, 2003 Sep, 44(9), 3892 - 8
Contribution of ExsA-regulated factors to corneal infection by cytotoxic and invasive Pseudomonas aeruginosa in a murine scarification model; Lee EJ et al.; PURPOSE: The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa . In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells . Invasive P . aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia . In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated . METHODS: After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P . aeruginosa or with isogenic mutants in exsA-related genes . Inocula of 10(3) to 10(6) bacteria/5 micro L were used, and at least five animals were assigned to each experimental group . Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems . RESULTS: For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease . Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (>3-log increase) and disease severity to wild-type levels . Complementation with exoT alone increased colonization ( approximately 3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections . Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins . CONCLUSIONS: ExsA contributed to corneal virulence of only cytotoxic P . aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity . This differs from cytotoxic P . aeruginosa virulence in the lung, which is ExoU-dependent.

Invest Ophthalmol Vis Sci, 2003 Sep, 44(9), 3795 - 801
Expression of human beta-defensins in conjunctival epithelium: relevance to dry eye disease; Narayanan S et al.; PURPOSE: The goals of this study were to investigate whether beta-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human beta-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells . METHODS: RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3 . Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa . RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively . RESULTS: hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects' samples, whereas hBD-1 and -3 were detected in all subjects tested . RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1beta, IL-1beta and tumor necrosis factor-alpha (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3) . hBD-2 expression was upregulated from 4 hours of treatment with IL-1beta (at 10 ng/mL; (n = 2-3) and at a concentration of 0.1 ng/mL IL-1beta (24-hour treatment; n = 2-3) . Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data . CONCLUSIONS: hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye . Because cytokines such as IL-1beta and TNF-alpha induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate dry eye is mediated by proinflammatory cytokine activity.

Mem Inst Oswaldo Cruz, 2003 Jun, 98(4), 549 - 52 Epub 2003 Aug 18.
Antibacterial xanthones from Kielmeyera variabilis mart . (Clusiaceae); Pinheiro L et al.; The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w) . The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 g/ml . When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 g/ml against B . subtilis . Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains . None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa . Viable cells of S . aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture . It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved

J Athl Train, 2002 Mar, 37(1), 51 - 54
The Bactericidal And Cytotoxic Effects Of Antimicrobial Wound Cleansers; Rabenberg VS et al.; OBJECTIVE: Wound care is a part of daily activity for many athletic trainers . Knowing which cleansers are effective against the bacteria that most commonly cause infection and whether they are toxic to healthy cells enables athletic trainers to make educated decisions on which cleanser to use . We compared the bactericidal effectiveness and cytotoxicity to human fibroblast cells of 4 cleansers at various dilutions . DESIGN AND SETTING: A 4 x 4 factorial design was used for the cytotoxicity testing . The independent variables were type and dilution of cleanser . The dependent variable was cell viability of the human fibroblast cells . We used a 2 x 3 x 4 x 4 factorial design for the bacterial testing . The independent variables were type and dilution of bacteria and type and dilution of cleanser . The dependent variable was the bactericidal action of the cleanser on the bacteria . SUBJECTS: Human foreskin samples were used to obtain a line of fibroblast cells . Bacterial samples were obtained from an athletic training clinic, isolated from swabs of a whirlpool water supply valve (Pseudomonas aeruginosa) or skin surface (Staphylococcus aureus) . MEASUREMENTS: We obtained bactericidal measurements by testing isolated Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria . Minimum and maximum concentrations were identified according to bactericidal effectiveness . Cytotoxicity measurements were obtained from spectrophotometer readings of a neutral red assay for fibroblast cell viability . Final dilutions tested were determined by pilot testing . RESULTS: At the 1:5 dilution of product in sterile 0.9% saline, both Cinder Suds and Nitrotan and hydrogen peroxide were different from the control with regard to Pseudomonas aeruginosa . At the 1:10 dilution, both Betadine and hydrogen peroxide were different from the control with regard to Pseudomonas aeruginosa . These 2 cleansers were also different from each other . At the 1:10 dilution, only Betadine was not different from the control for the cytotoxicity testing . CONCLUSIONS: Betadine was both effective against bacteria and not harmful to human fibroblast cells at a 1:10 dilution of a commercially purchased solution.

Arch Dis Child Fetal Neonatal Ed, 2003 Sep, 88(5), F434 - 5
Contamination of a milk bank pasteuriser causing a Pseudomonas aeruginosa outbreak in a neonatal intensive care unit; Gras-Le Guen C et al.; An environmental investigation and a cohort study were carried out to analyse an outbreak of infection caused by a serotype O10 Pseudomonas aeruginosa in a neonatal intensive care unit . Thirty one cases of infection were recorded, including four lethal ones . The outbreak was stopped by eradicating the environmental sources: a contaminated milk bank pasteuriser and bottle warmer.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2997 - 3001
Novel Pseudomonas aeruginosa gene that suppresses tolerance to carbapenems; Taniguchi K et al.; A biapenem-tolerant mutant of Pseudomonas aeruginosa was isolated by Tn1737KH insertion . The survival of the mutant 3 h after the addition of biapenem was about 1000 times greater than that of the wild type . The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2990 - 2
Functional cloning and characterization of a multidrug efflux pump, mexHI-opmD, from a Pseudomonas aeruginosa mutant; Sekiya H et al.; We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps . The genes responsible for the resistance were mexHI-opmD . Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes . Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2756 - 64
Effectiveness of combination antimicrobial therapy for Pseudomonas aeruginosa bacteremia; Chamot E et al.; It remains controversial whether combination therapy, given empirically or as definitive treatment, for Pseudomonas aeruginosa bacteremia is associated with a better outcome than monotherapy . The aim of the present study was to compare the rates of survival among patients who received either combination therapy or monotherapy for P . aeruginosa bacteremia . We assembled a historical cohort of 115 episodes of P . aeruginosa bacteremia treated with empirical antipseudomonal therapy between 1988 and 1998 . On the basis of susceptibility testing of the bacteremic P . aeruginosa isolate, we defined categories of empirical treatment, including adequate combination therapy, adequate monotherapy, and inadequate therapy, as well as corresponding categories of definitive therapy . Neither the adequacy of the empirical treatment nor the use of combination therapy predicted survival until receipt of the antibiogram . However, the risk of death from the date of receipt of the antibiogram to day 30 was higher for both adequate empirical monotherapy (adjusted hazard ratio {aHR}, 3.7; 95% confidence interval {CI}, 1.0 to 14.1) and inadequate empirical therapy (aHR, 5.0; 95% CI, 1.2 to 20.4) than for adequate empirical combination therapy . Compared to adequate definitive combination therapy, the risk of death at 30 days was also higher with inadequate definitive therapy (aHR, 2.6; 95% CI, 1.1 to 6.7) but not with adequate definitive monotherapy (aHR, 0.70; 95% CI, 0.30 to 1.7) . In this retrospective analysis the use of adequate combination antimicrobial therapy as empirical treatment until receipt of the antibiogram was associated with a better rate of survival at 30 days than the use of monotherapy . However, adequate combination antimicrobial therapy given as definitive treatment for P . aeruginosa bacteremia did not improve the rate of survival compared to that from the provision of adequate definitive monotherapy.

Adv Biochem Eng Biotechnol, 2003, 83, 117 - 40
Application of proteomics to Pseudomonas aeruginosa; Nouwens AS et al.; The recent completion of the Pseudomonas Genome Project, in conjunction with the Pseudomonas Community Annotation Project (PseudoCAP) has fast-tracked our ability to apply the tools encompassed under the term 'proteomics' to this pathogen . Such global approaches will allow the research community to answer long-standing questions regarding the ability of Pseudomonas aeruginosa to survive diverse habitats, its high intrinsic resistance to antibiotics and its pathogenic nature towards humans . Proteomics provides an array of tools capable of confirming the expression of Open Reading Frames (ORF), the relative levels of their expression, the environmental conditions required for this expression and the sub-cellular location of the encoded gene-products . Since proteins are important cellular effectors, the biological questions we pose can be defined in terms of changes in protein expression detectable by separation to purity using two-dimensional gel electrophoresis (2-DGE) and relation to gene sequences via mass spectrometry . As such, we can compare strains with well-characterized phenotypic differences, growth under a variety of stresses, protein interactions and complexes and aid in defining proteins of unknown function . While the complete genome has only recently been finished, a number of studies have already utilized this information and examined various protein gene-products using proteomics . This review summarizes the application of proteomics to P . aeruginosa and highlights potential areas of future research, including overcoming the traditional technical limitations associated with 2-DGE . More focused approaches that target sub-cellular fractions ('sub-proteomes') prior to 2-DGE can provide further functional information . A review of current and previous proteomic projects on P . aeruginosa is presented, as well as theoretical considerations of the importance of sub-proteomic approaches to enhance these investigations.

Klin Lab Diagn, 2003 Jul, (7), 52 - 3
{A case of the long-term carrier state of Pseudomonas aeruginosa on the hand skin of a nurse in a resuscitation department}; Belokrysenko SS et al.; A case of a three-month carrier-state of P . aeruginosa in the hands' skin of a nurse in a newborns reanimation department is described . The carrier state did not seize after the nurse was isolated from work and was related with colonization of the dermal tissue, therefore, it could be detected only after the hands' skin was treated with a detergent solution . The carrier state was arrested after a chemotherapy course with cyprofloxacyn administered both locally and perorally.

Infect Immun, 2003 Sep, 71(9), 5389 - 93
Twitching motility contributes to the role of pili in corneal infection caused by Pseudomonas aeruginosa; Zolfaghar I et al.; Twitching motility is a form of surface-associated bacterial movement mediated by type IV pili of Pseudomonas aeruginosa . Others have shown that pilT and pilU mutants, which are piliated but defective in twitching motility, display reduced cytotoxic capacity towards epithelial cells in vitro . Although these mutants efficiently infected lungs in vivo, they were defective in dissemination to the liver . In this study the role of twitching motility in P . aeruginosa epithelial cell invasion and corneal disease pathogenesis was explored . pilU and pilT mutants of P . aeruginosa strain PAK were compared to a nonpiliated pilA mutant and to wild-type bacteria in their ability to associate with and to invade corneal epithelial cells in vitro and to cause disease in a murine model of corneal infection . As expected, the pilA mutant demonstrated reduced association and invasion of corneal epithelial cells (P < 0.05 in both cases) . The pilT mutant, but not the pilU mutant, was less invasive than wild-type PAK was (P < 0.05 versus P = 0.43), while both pilU and pilT mutants exhibited association levels similar to those of the wild type (P = 0.31 and 0.52, respectively) . In vivo, all mutants were markedly attenuated in virulence and showed reduced ability to colonize the cornea at 4 and 48 h (all P values < 0.02) . Thus, twitching motility contributed to the role of pili in corneal disease but was not involved in the role of pili in adherence to or invasion of corneal epithelial cells.

Infect Immun, 2003 Sep, 71(9), 5296 - 305
Characterization of Pseudomonas aeruginosa exoenzyme S as a bifunctional enzyme in J774A.1 macrophages; Rocha CL et al.; Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families . The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P . aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity . Distinct and coordinated functions were identified for both domains . The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation . Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation . The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays . The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation . The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.

Carbohydr Res, 2003 Sep 1, 338(18), 1895 - 905
Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12 with a randomly O-acetylated core region; Bystrova OV et al.; The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and analyses by ESI FT-MS and NMR spectroscopy . The structures of the lipopolysaccharide core and the O-polysaccharide repeating unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established . The core was found to be randomly O-acetylated, most O-acetyl groups being located on the terminal rhamnose residue of the outer core region.

Rinsho Shinkeigaku, 2003 May, 43(5), 258 - 64
{Two cases of hypertrophic cranial pachymeningitis associated with infection in the external auditory canal and paranasal sinus}; Tajima Y et al.; We here present two cases of hypertrophic cranial pachymeningitis exhibiting unique multiple cranial neuropathies, both of which were associated with otic and paranasal infections . Case 1: A 76-year-old woman developed headache after undergoing surgical dilatation of the external auditory canal, with subsequent development of a bacterial infection . Neurological examination reveled only bilateral hearing disturbance . MRI and CT scans demonstrated thickening of the dura mater and inflammatory granulation around the left cerebellar tentorium . Based on a diagnosis of hypertrophic pachymeningitis associated with previous infection, antibiotics were administered, followed by oral prednisolone therapy . This treatment relieved the headache and improved the MRI findings . However, 2 months later, the headache became worse and impaired movement of the soft palate, atrophy of the left side of the tongue, and atrophy of the sternocleidomastoideus muscle were noted . MRI revealed aggravated inflammatory changes around the left cerebellar tentorium and their expansion into the jugular foramen . Occlusive changes in the transverse and sigmoid sinuses were also seen . Case 2: A 78-year-old man developed bilateral visual loss, right frontal headache, and bilateral restriction of eye movement . He had been treated for phemphigus with prednisolone and azathioprine . MRI showed hypertrophic dura mater spreading continuously from the frontal base and ethmoid and frontal sinuses to the falx and right frontal lobe . Since Pseudomonas aeruginosa was cultivated in biopsy specimens from the dura mater, antibiotic agents were administered . The clinical symptoms resolved and MRI findings gradually improved.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2003 Aug, 96(2), 136 - 40
Necrotizing stomatitis: report of 3 Pseudomonas aeruginosa-positive patients; Barasch A et al.; Necrotizing oral lesions have been described in immunosuppressed patients, usually in association with gingival and periodontal pathoses . The etiology of these lesions has not been completely elucidated . We present 3 patients with a type of necrotizing stomatitis in which clinical patterns appear distinct from the periodontal forms of the disease . The lesions yielded bacterial cultures positive for Pseudomonas aeruginosa and reverted to no growth in 2 patients after proper antibiotic therapy . We propose that P aeruginosa may be responsible for selected necrotizing oral lesions with a clinical presentation lacking typical necrotizing periodontal disease and that this condition may represent the intraoral counterpart of ecthyma gangrenosum . In such cases, bacterial culture of the lesion becomes imperative because the disease does not respond to typical periodontal and antimicrobial therapy.

Exp Dermatol, 2003 Aug, 12(4), 418 - 25
Supernatants of Pseudomonas aeruginosa induce the Pseudomonas-specific antibiotic elafin in human keratinocytes; Meyer-Hoffert U et al.; Elafin is a skin-derived serine-protease inhibitor . It is thought to be important to prevent human leukocyte elastase-mediated tissue damage and might play an important role in maintaining the integrity of the human epidermis . Recent studies have provided evidence for an antimicrobial activity of elafin against P . aeruginosa . As gram-negative infections typically occur in barrier-disrupted skin we were interested to determine whether supernatants of the gram-negative bacteria P . aeruginosa and Escherichia coli were capable of inducing elafin expression . Supernatants of various P . aeruginosa strains stimulated elafin mRNA-expression and protein release, whereas supernatants of E . coli did not induce elafin expression . In non-differentiated cells the relative increase of elafin mRNA was much higher (100-fold) than in differentiated cells (sixfold), although the latter exhibited higher constitutive mRNA-expression (150-fold) . However, concentrations of secreted elafin were similar in differentiated and non-differentiated cells after stimulation . We could not confirm a bactericidal effect against P . aeruginosa as described previously but observed that its growth was inhibited as demonstrated for different strains in liquid cultures . Growth of E . coli was not affected by elafin . In conclusion, the data presented in this paper suggest that elafin represents an innate immune response factor induced by secreted products of P . aeruginosa . Besides its elastase inhibitory potency elafin is an antimicrobial agent against P . aeruginosa.

Burns, 2003 Sep, 29(6), 547 - 51
Serovar determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (IRAN); Shahcheraghi F et al.; The serovars and drug susceptibility patterns of 265 isolates of Pseudomonas aeruginosa cultured from burn patients during 2001-2002 at Motahari and Tohid Hospital were determined . Distribution of serovars was different at two hospitals . Most of the isolates at Tohid Hospital belonged to serovar 0:1, but 21 and 13% of them were untypeable or polyagglutinable, respectively . Serovar 0:11 was the most prevalent serovar at Motahari Hospital . All the strains were multi resistant to tetracyclin, carbenicillin, amikacin, ceftazidime, sulfamethoxazol, ciprofloxacin, tobramycin, kanamycin, cefotaxime and gentamicin . Further analysis of the strains by plasmid profiling demonstrated that 95% of the isolates carried two megaplasmids . However, there was not any correlation between the serotyping and presence of plasmids . Changes in the drug susceptibility patterns and beta-lactamase production of some cured derivatives were observed after the strains lost their plasmids . The emergence of multi-drug resistant strains of P . aeruginosa is a serious concern in burn patients who are hospitalized in Tehran.

Int J Antimicrob Agents, 2003 Aug, 22(2), 164 - 7
Effects on adhesiveness and hydrophobicity of sub-inhibitory concentrations of netilmicin; Furneri PM et al.; The effect of sub-inhibitory concentrations (SICs) of netilmicin on bacterial hydrophobicity and adhesiveness to conjunctival cells was investigated . One strain each of Pseudomonas aeruginosa, Pseudomonas spp., Staphylococcus aureus and S . epidermidis was investigated for its susceptibility to netilmicin, its adherence to conjunctival cells and to the effect of hydrocarbon hexadecane before and after treatment with SIC of netilmicin . All of the bacteria tested were susceptible to netilmicin except for Pseudomonas spp . which showed intermediate resistance . Netilmicin-treated Pseudomonas strains exhibited a lower level of hydrophobicity towards n-hexadecane compared with non-treated strains, while netilmicin-treated S . epidermidis and S . aureus showed a slight increase of hydrophobicity . Adherence of the two Pseudomonas strains to conjunctival cells was significantly reduced after growth in the presence of netilmicin, while the adherence of the two staphylococci was only slightly reduced.

Biochem Biophys Res Commun, 2003 Sep 5, 308(4), 922 - 6
Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa; Narita S et al.; The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method . A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1 . The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells . Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies . The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).

Auris Nasus Larynx, 2003 Aug, 30(3), 253 - 7
Types and causes of otorrhea; Bardanis J et al.; INTRODUCTION: Otorrhea is a common symptom and sign of patients seeking examination in an ENT Department of a General Hospital . The objective of this review is to assess the cause of otorrhea according to the type of it . METHODS: Retrospective review of 306 ears discharging some kind of fluid of 296 patients, who appeared in the ENT Department of our Hospital over a 58-month period . RESULTS: The most common type of otorrhea was the purulent one: 276 ears among 306 discharging ears (90%) . The most common cause of purulent otorrhea was otitis externa: 156 ears among 276 ears discharging purulent fluid (56%) . The germ most commonly isolated in the cultures of purulent aural discharge was Pseudomonas aeruginosa: 67 isolations of P . aeruginosa among 256 cultures (26%) . CONCLUSIONS: Otomicroscopic examination and accurate culture of purulent discharge are mandatory for the clinician to establish a correct diagnosis and suggest a proper therapy in every case of otorrhea.

Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1665 - 7 Epub 2003 Aug 19.
Purification, crystallization and preliminary diffraction studies of the Pseudomonas aeruginosa strain K122-4 monomeric pilin; Audette GF et al.; The monomeric pilin from Pseudomonas aeruginosa strain K122-4 has been crystallized and preliminary X-ray diffraction data have been collected . Pilin is the monomeric subunit of the type IV pilus, the dominant adhesin of the opportunistic pathogen P . aeruginosa . The K122-4 pilin crystallizes as a dimer in space group P1, with unit-cell parameters a = 40.19, b = 38.93, c = 37.22 A, alpha = 66.38, beta = 111.12, gamma = 93.74 degrees . Diffraction data were collected using a synchrotron-radiation source and were processed to 1.54 A d-spacing.

Biochem J, 2003 Sep 1, 374(Pt 2), 413 - 21
Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues; Amara AA et al.; The class II PHA (polyhydroxyalkanoate) synthases {PHA(MCL) synthases (medium-chain-length PHA synthases)} are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate . Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved . A threading model of the P . aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily . The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis . In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity . The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases . Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity . Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition . The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes . In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases . Inhibitor studies indicated that catalytic histidines reside in the active site . The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue . In the threading model this residue was found to be surface-exposed . No evidence for post-translational modification by 4-phosphopantetheine was obtained . Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the alpha/beta-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

Biochemistry, 2003 Aug 26, 42(33), 9946 - 51
Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase; Naught LE et al.; In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P . The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme . Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site . An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers . Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type . The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site . In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site . When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108 . (31)P NMR spectra of the S108A protein confirm that it is phosphorylated . Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.

Pflugers Arch, 2003 Oct, 447(1), 23 - 8 Epub 2003 Aug 13.
Pseudomonas aeruginosa activates Cl- channels in host epithelial cells; Ullrich S et al.; Exposure to Pseudomonas aeruginosa triggers the apoptotic cell death of Chang epithelial cells, and this depends on the expression of both the CD95 receptor and CD95 ligand . In lymphocytes CD95-mediated apoptosis is paralleled by the activation of outwardly rectifying Cl- channels . The present study was performed to explore whether P . aeruginosa-induced apoptosis of Chang epithelial cells is paralleled by activation of Cl- channels . According to whole-cell patch-clamp recordings, exposure of Chang epithelial cells to P . aeruginosa does lead to rapid activation of an outwardly rectifying Cl- -selective current . The current is inhibited by the Cl- channel blocker NPPB . Exposure of Chang epithelial cells to P . aeruginosa led to a significant decrease of cell membrane capacitance by 6%, pointing to a decrease in cell volume by 7% . Exposure to P . aeruginosa depolarized the mitochondrial membrane potential indicating apoptotic cell death . The decline of mitochondrial membrane potential was not significantly affected by NPPB . In conclusion, P . aeruginosa-induced apoptosis of Chang epithelial cells is paralleled by activation of Cl- channels . Activation of the channels participates in the alteration of cell volume but is not a prerequisite for P . aeruginosa-induced apoptosis.

Cochrane Database Syst Rev . 2003;(3):CD002203.
Macrolide antibiotics for cystic fibrosis; Southern KW et al.; BACKGROUND: The antibiotic treatment of chest infections which characterise cystic fibrosis (CF) has significantly improved prospects for people with CF . The nature of organisms causing these infections has restricted antibiotic choice . Pseudomonas aeruginosa, especially, is resistant to most oral antibiotics . There is evidence from the laboratory and from other disease processes that macrolide antibiotics, whilst not directly active against Pseudomonas aeruginosa, may have indirect actions against this organism . OBJECTIVES: We aimed to test the hypotheses that macrolide antibiotics:(1) improve clinical status compared to placebo or another antibiotic;(2) have no unacceptable adverse effects.If benefit was demonstrated, we aimed to assess the optimal type, dose and duration of macrolide therapy . SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group trials register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings.We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture macrolide antibiotics for unpublished or follow-up data (December 2002).Date of the most recent search of the Group's register: March 2003 . SELECTION CRITERIA: Published or unpublished randomised controlled trials of macrolide antibiotics compared to placebo, another class of antibiotic or another macrolide antibiotic . Studies comparing regimens of the same macrolide antibiotic at different doses will also be included . DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data and assessed study quality . Two groups were contacted for missing data, but these were unavailable for the review . MAIN RESULTS: Searches identified eleven studies, two were included in this review (101 participants) . One study enrolled adults and the other children (a significant number of whom were not colonised with Pseudomonas aeruginosa) . Both studies report small but significant changes in respiratory function (% change in FEV1) in favour of azithromycin . Meta-analysis at the two-month time point demonstrated a significant benefit with respect to percentage change in FVC (weighted mean difference 5.42 (1.77 to 9.07)) from azithromycin, but no difference with respect to percentage change of FEV1 . There were no significant adverse effects reported . REVIEWER'S CONCLUSIONS: The role of macrolides in the management of CF lung disease remains unclear and there are many unanswered questions . Two small randomised controlled trials have suggested short-term improvement in respiratory function with azithromycin . Until the results of further studies are available the widespread use of azithromycin in CF cannot be advocated and should be restricted to well-designed randomised controlled trials.

Cochrane Database Syst Rev . 2003;(3):CD001912.
Prophylactic antibiotics for cystic fibrosis; Smyth A et al.; BACKGROUND: Staphylococcus aureus causes pulmonary infection in young children with cystic fibrosis (CF) . Prophylactic antibiotics are widely prescribed in the hope of preventing infection with Staphylococcus aureus and lung damage . Antibiotics also have adverse effects and long-term use might lead to chronic infection with organisms like Pseudomonas aeruginosa . OBJECTIVES: To assess the effect of continuous oral antibiotic prophylaxis compared to no prophylaxis in people with CF, we tested these hypotheses . Prophylaxis: (1) improves clinical status, lung function and survival; (2) causes adverse effects (eg diarrhoea, skin rash, candidiasis); (3) leads to fewer isolates of common pathogens from respiratory secretions; (4) leads to the emergence of antibiotic resistance and the colonisation of the respiratory tract with organisms, e.g . Pseudomonas aeruginosa . SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group trials register, comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings . Companies manufacturing anti-staphylococcal antibiotics were approached for unpublished data.Most recent search of the Group's register: March 2003 . SELECTION CRITERIA: Randomised trials of continuous oral prophylactic antibiotics (given for at least one year) compared to intermittent antibiotic therapy given "as required," in people with CF of any disease severity . DATA COLLECTION AND ANALYSIS: The reviewers assessed trials for eligibility and methodological quality and extracted data . MAIN RESULTS: Four studies, totalling 303 participants (139 boys) aged zero to seven years on enrollment, were included . Fewer children receiving anti-staphylococcal antibiotic prophylaxis had one or more isolates of Staphylococcus aureus . There was no significant difference between groups in infant or conventional lung function tests . We found no significant effect on nutrition, hospital admissions, additional courses of antibiotics or adverse effects . There is no significant difference in the number of isolates of Pseudomonas aeruginosa between groups, though there was a trend towards a lower cumulative isolation rate of Pseudomonas aeruginosa in the prophylaxis group, at two and three years and a trend towards a higher rate from four to six years . However, as the duration of the studies reviewed has been of six years or less, conclusions cannot be drawn about the long-term effects of prophylaxis . REVIEWER'S CONCLUSIONS: Anti-staphylococcal antibiotic prophylaxis leads to fewer children having isolates of Staphylococcus aureus, when commenced early in infancy and continued up to six years of age . The clinical importance of this finding is uncertain . Further research may establish whether the trend towards more children with CF with Pseudomonas aeruginosa, after four to six years of prophylaxis, is a chance finding . Future work should explore whether choice of prophylactic antibiotic or duration of treatment might influence infection with Pseudomonas aeruginosa.

Cochrane Database Syst Rev . 2003;(3):CD001021.
Nebulised anti-pseudomonal antibiotics for cystic fibrosis; Ryan G et al.; BACKGROUND: Persistent infection by Pseudomonas aeruginosa contributes to lung damage, resulting in illness and death in people with cystic fibrosis (CF) . Nebulised antibiotics are commonly used to treat this infection . OBJECTIVES: To examine the evidence that nebulised anti-pseudomonal antibiotic treatment in people with CF reduces frequency of exacerbations of infection, improves lung function, quality of life and survival . To examine adverse effects of nebulised anti-pseudomonal antibiotic treatment . SEARCH STRATEGY: Trials were identified from the Cochrane Cystic Fibrosis and Genetic Disorders Group clinical trials register . Companies that marketed nebulised anti-pseudomonal antibiotics were contacted for information on unpublished trials.Most recent search of the Group's trials register: August 2002 . SELECTION CRITERIA: Trials were selected if, nebulised anti-pseudomonal antibiotics treatment was used for four weeks or more in people with CF, allocation to treatment was randomised or quasi-randomised, and there was a placebo or a no placebo control group or another nebulised antibiotic comparison . DATA COLLECTION AND ANALYSIS: For the first version of this review, two reviewers independently selected and judged the quality of, the trials to be included in the review . One reviewer extracted data from these trials and performed all tasks for the updated version of the review . MAIN RESULTS: Out of 33 trials identified, there were 11, with 873 participants, that met the inclusion criteria . Ten trials with 758 participants compared a nebulised anti-pseudomonal antibiotic with placebo or usual treatment . One of these trials accounted for 68% of the total participants and seven of these trials used a cross-over design . Tobramycin was studied in four trials and follow up ranged from 1 to 32 months . Lung function, measured as forced expired volume in one second (FEV1) was better in the treated group than in control group in nine of these . Resistance to antibiotics increased more in the antibiotic treated group than in placebo group . Tinnitus and voice alteration were more frequent with tobramycin than placebo . One short-term trial of one month, with 115 participants, compared tobramycin and colistin, and showed a trend towards greater improvement in FEV1 in the tobramycin group . REVIEWER'S CONCLUSIONS: Nebulised anti-pseudomonal antibiotic treatment improves lung function . However, more evidence, from longer duration trials, is needed to determine if this benefit is maintained as well as to determine the significance of development of antibiotic resistant organisms . There is insufficient evidence for recommendations about type of drug and dose regimens.

Am J Respir Crit Care Med, 2003 Nov 1, 168(9), 1100 - 8 Epub 2003 Aug 13.
Bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis; Farrell PM et al.; Although early diagnosis of cystic fibrosis (CF) can lead to nutritional benefits, there has been uncertainty about pulmonary outcomes . Using a randomized controlled trial with unique unblinding/surveillance, we evaluated patients with CF who received similar treatment after being assigned to an early diagnosis (screened) group or to a standard diagnosis (control) group . When the youngest patient was 7 years of age, we compared outcomes using pulmonary function data and quantitative chest radiology . In the screened group (56 patients), diagnosis was made at a younger age of 12.4 weeks, compared with the diagnosis in control group (47 control patients) at the age of 95.8 weeks, but included a significantly greater proportion of patients with deltaF508 genotypes and pancreatic insufficiency . The first chest radiograph showed significantly fewer abnormalities in the screened group; but, over time, the two groups converged, and after 10 years of age the screened patients showed worse chest X-ray scores associated with earlier acquisition of Pseudomonas aeruginosa . No differences were detected in any measure of pulmonary dysfunction, which was generally mild in each group . Although CF neonatal screening provides a potential opportunity for better pulmonary outcomes, it appears that respiratory infections and pancreatic status are the dominant factors in pulmonary prognosis.

AJNR Am J Neuroradiol, 2003 Aug, 24(7), 1310 - 6
Central skull base osteomyelitis in patients without otitis externa: imaging findings; Chang PC et al.; BACKGROUND AND PURPOSE: Skull base osteomyelitis typically arises as a complication of ear infection in older diabetic patients, involves the temporal bone, and has Pseudomonas aeruginosa as the usual pathogen . Atypical skull base osteomyelitis arising from the sphenoid or occipital bones without associated external otitis occurs much less frequently and initially may have headache as the only symptom . The purpose of this study was to review the clinical and MR imaging features of central skull base osteomyelitis . METHODS: We retrospectively reviewed MR images obtained in six patients with central skull base osteomyelitis . No patient had predisposing external otitis or osteomyelitis of the temporal bone . RESULTS: All of our patients presented with headache, no external ear pain, and cranial nerve deficits . Five of six patients had a predisposition to infection, and the erythrocyte sedimentation rate was elevated in the five patients in whom it was checked . In each case, the diagnosis was delayed until MR imaging demonstrated central skull base abnormality, and the diagnosis was then confirmed with tissue sampling . The most consistent imaging findings were clival bone marrow T1 hypointensity and preclival soft tissue infiltration . Five of six patients were cured with no recurrence of skull base infection over a 2-4-year follow-up period . CONCLUSION: In the setting of headache, cranial neuropathy, elevated erythrocyte sedimentation rate, and abnormal clival imaging findings, central skull base osteomyelitis should be considered as the likely diagnosis . Early tissue sampling and appropriate treatment may prevent or limit further complications such as intracranial extension, empyema, or death.

Pest Manag Sci, 2003 Aug, 59(8), 872 - 82
Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26; Lee JY et al.; The bacterial strain GC-B26, which showed strong antifungal and anti-oomycete activity against some plant pathogens, was isolated from a grassland soil in Korea . Based on morphological, physiological and biochemical characteristics, GC-B26 was identical to Pseudomonas aeruginosa (Schroeter) Migula . The antibiotic G26A, active against Phytophthora capsici Leonian and Colletotrichum orbiculare (Berk & Mont) van Arx, was isolated from the culture filtrates of Ps aeruginosa strain GC-B26 using various chromatographic procedures . The EI mass and UV spectral results indicated that G26A is an analogue of phenazines, having molecular formula C13H8N2O2 (M+, m/z 224.0664) . On the basis of NMR spectral data, G26A was confirmed as phenazine-1-carboxylic acid . C orbiculare, P capsici and Pythium ultimum Trow were most sensitive to G26A, with MIC values of approximately 5 microg ml(-1) . However, no antimicrobial activity was found against yeasts and bacteria, even at a concentration of over 100 microg ml(-1) . Treatment with the antibiotic gave highly significant protective activity against the development of Phytophthora disease on pepper and anthracnose on cucumber plants . The disease control efficacy was only slightly less than that of the commercial fungicides metalaxyl and chlorothalonil.

Am J Hum Genet, 2003 Sep, 73(3), 591 - 600 Epub 2003 Aug 12.
Extensive normal copy number variation of a beta-defensin antimicrobial-gene cluster; Hollox EJ et al.; Using a combination of multiplex amplifiable probe hybridization and semiquantitative fluorescence in situ hybridization (SQ-FISH), we analyzed DNA copy number variation across chromosome band 8p23.1, a region that is frequently involved in chromosomal rearrangements . We show that a cluster of at least three antimicrobial beta-defensin genes (DEFB4, DEFB103, and DEFB104) at 8p23.1 are polymorphic in copy number, with a repeat unit >/=240 kb long . Individuals have 2-12 copies of this repeat per diploid genome . By segregation, microsatellite dosage, and SQ-FISH chromosomal signal intensity ratio analyses, we deduce that individual chromosomes can have one to eight copies of this repeat unit . Chromosomes with seven or eight copies of this repeat unit are identifiable by cytogenetic analysis as a previously described 8p23.1 euchromatic variant . Analysis of RNA from different individuals by semiquantitative reverse-transcriptase polymerase chain reaction shows a significant correlation between genomic copy number of DEFB4 and levels of its messenger RNA (mRNA) transcript . The peptides encoded by these genes are potent antimicrobial agents, especially effective against clinically important pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, and DEFB4 has been shown to act as a cytokine linking the innate and adaptive immune responses . Therefore, a copy number polymorphism involving these genes, which is reflected in mRNA expression levels, is likely to have important consequences for immune system function.

J Biol Chem, 2003 Oct 17, 278(42), 41326 - 32 Epub 2003 Aug 12.
In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors; Phillips RM et al.; A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown . Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoU-expressing strain of P . aeruginosa is blocked by various inhibitors of cytosolic (cPLA2) and Ca2+ -independent (iPLA2) phospholipase A2 enzymes . In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A2 enzymes or by a number of inhibitors of signal transduction pathways . This suggests that phospholipase A2 inhibitors may represent a novel mode of treatment for acute P . aeruginosa infections . We find that 300-600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion . We also show that ExoU interacts in vitro with an inhibitor of cPLA2 and iPLA2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA2 and iPLA2 enzymes . Mutation of either the serine or the aspartate renders ExoU non-cytotoxic . Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.

Med J Aust, 2003 Aug 18, 179(4), 185 - 90
Effectiveness of ototopical antibiotics for chronic suppurative otitis media in Aboriginal children: a community-based, multicentre, double-blind randomised controlled trial; Couzos S et al.; OBJECTIVES: To compare the effectiveness of ototopical ciprofloxacin (0.3%; CIP) with framycetin (0.5%), gramicidin, dexamethasone (FGD) eardrops (5 drops twice daily for 9 days) together with povidone-iodine (0.5%) ear cleaning as treatments for chronic suppurative otitis media (CSOM) in Aboriginal children . DESIGN AND PARTICIPANTS: Aboriginal community-controlled, community-based, multicentre, double-blind, randomised controlled trial in eight Aboriginal Community Controlled Health Services across northern Australia, involving 147 Aboriginal children with CSOM . MAIN OUTCOME MEASURES: Resolution of otorrhoea (clinical cure), proportion of children with healed perforated tympanic membrane (TM) and improved hearing, 10-21 days after starting treatment . RESULTS: 111 children aged 1-14 years (CIP, 55; FGD, 56) completed treatment . CSOM cures occurred in 64% (CIP, 76.4%; FGD, 51.8%), with a significantly higher rate in the ciprofloxacin group (P = 0.009, absolute difference of 24.6% {95% CI, 15.8%-33.4%}) . TM perforation size and the level of hearing impairment did not change . Pseudomonas aeruginosa was the most common bacterial pathogen (in 47.6%), while respiratory pathogens were rare (in 5.7%) . CONCLUSIONS: Twice-daily ear cleaning and topical ciprofloxacin is effective at community-level in achieving cure for CSOM . Healthcare providers to Aboriginal children with CSOM should be given special access to provide ototopical ciprofloxacin as first-line treatment.

J Med Microbiol, 2003 Sep, 52(Pt 9), 731 - 40
Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection; Song Z et al.; Pseudomonas aeruginosa is an opportunistic respiratory pathogen that accounts for most of the morbidity and mortality in cystic fibrosis (CF) patients . In CF-affected lungs, the bacteria undergo conversion from a non-mucoid to a non-tractable mucoid phenotype, due to overproduction of alginate . The effect of alginate production on pathogenicity was investigated by using an acute lung infection mouse model that compared a non-mucoid P . aeruginosa strain, PAO1, to its constitutive alginate-overproducing derivative, Alg(+) PAOmucA22, and an alginate-defective strain, Alg(-) PAOalgD . Bacterial suspensions were instilled into the left bronchus and examined 24 and 48 h post-infection . The highest bacterial loads and the most severe lung pathology were observed with strain Alg(-) PAOalgD at 24 h post-infection, which may have been due to an increase in expression of bacterial elastase by the mutant . Significantly lower lung and spleen bacterial loads were found in the two non-mucoid (PAO1 and Alg(-) PAOalgD) groups, compared to the mucoid Alg(+) PAOmucA22 group, between 24 and 48 h post-infection . The positive correlation between lung bacteriology and lung macroscopic pathology in the Alg(+) PAOmucA22 group suggests that alginate production not only impedes pulmonary clearing, but also results in severe lung damage . Positive correlations between IL12 levels and lung macroscopic pathology, and between IL12 and IFN-gamma levels in the Alg(+) PAOmucA22 group, suggested a possible contribution of these pro-inflammatory cytokines to tissue damage . No significant differences were found between the three groups in lung cytokine responses at 24 or 48 h post-infection . However, on comparison within each group at 24 and 48 h post-infection, a significant increase in the pro-inflammatory cytokine IFN-gamma was observed . Higher ratios of IFN-gamma/IL4 and IFN-gamma/IL10, but lower IL10 levels, were also found in all three groups . These results indicate a Th1-predominated immune response in these animals . Such cytokine responses could have aided the clearance of non-mucoid P . aeruginosa, but were not sufficient to alleviate infection by the mucoid variants . Alginate production may promote survival and persistence of this pathogenic micro-organism in the lung.

J Mol Biol, 2003 Aug 22, 331(4), 861 - 70
Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa; Loris R et al.; The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose . All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains . The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose . In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential . In the fucose complex, no such defects are observed . Instead, C6 makes favourable interactions with a small hydrophobic patch . Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.

J Dairy Sci, 2003 Jul, 86(7), 2276 - 82
Pseudomonas aeruginosa lectin PA-IIL as a powerful probe for human and bovine milk analysis; Lesman-Movshovich E et al.; Milk composition exhibits species-specific differences depending on genetic, evolutionary, and environmental factors . In addition, commercial milk preparations are also changed by industrial manipulations, including severe heat processing . Cow milk, used as human food, provides important nutrients but lacks some essential components that are present in raw human milk . The present study, which was aimed at comparing infant breastfeeding to cow-based formula nourishment, shows major differences between the human and the commercial cow milk glycans detectable by the lectins PA-IL (galactose-binding) and PA-IIL (fucose and mannose-binding) isolated from the cells of human pathogen Pseudomonas aeruginosa . More than 40 human milk samples, several cow milks, and bovine milk-based infant formulas, were examined using these two lectins . For purposes of comparison, the plant lectins Concanavalin A (Con A), which binds mannose, and Ulex europaeus 1st lectin (UEA-I), which binds fucose, were also used . The most prominent difference was revealed using PA-IIL, which displayed a unique high sensitivity to the human milk fucosylated compounds . PA-IL and UEA-I also exhibited preferential sensitivity to the human milk but considerably lower than that of PA-IIL . Con A was inhibited by human and the other milk preparations examined to the same extent . These findings indicate the superb applicability of PA-IIL for rapid and reliable comparative investigation of milk glycans from human and cow, indicating which glycans could be added to infant formulas in order to enrich them, as well as for verification and quality control of otherwise improved bovine milk-based infant formulas.

Acta Odontol Latinoam, 1999, 12(2), 83 - 8
Quality control of decontaminating agents; Arancegui N et al.; The present study evaluates the efficiency of the following decontaminating agents for the multiresistant, locally circulating bacterium Pseudomonas aeruginosa: glutaraldehyde 2%--makes A and B-, glutaraldehyde-formaldehyde; povidone-iodine-makes A, B and C-; sodium hypochloride; chloroxylenol--makes A and B-; and lapire chloride . The 9027 ATCC strain was used as a standard . A modification of the method of Kelsey and Sykes (1) was used to evaluate decontaminating efficiency . Highly satisfactory results were obtained with glutaraldehide 2% A and B, glutaraldehyde-formaldehyde and sodium hypochlorite . The results for povidone-iodine A, B and C were satisfactory but were unsatisfactory for chloroxylenol and lapirium chloride.

Pediatr Res, 2003 Nov, 54(5), 756 - 61 Epub 2003 Aug 06.
Impact of intravenous antibiotic therapy on total daily energy expenditure and physical activity in cystic fibrosis children with Pseudomonas aeruginosa pulmonary exacerbation; Beghin L et al.; Resting energy expenditure (REE) increases during pulmonary exacerbation by Pseudomonas aeruginosa in cystic fibrosis (CF) patients, and decreases after i.v . anti-Pseudomonas aeruginosa antibiotic therapy (IVAT) . However, the impact of IVAT on total energy expenditure (TEE) is unknown . The aim of this study was to assess the changes in TEE and its main components after IVAT administered at home . Body composition measured by skinfold thickness and bio-impedance analysis, energy intake (EI) assessed by a weekly diary, REE measured by indirect calorimetry (IC), TEE assessed by a technique using 24-h heart-rate monitoring method and physical activity (PA) monitored using an activity diary (AD) were assessed in 16 patients (9 boys and 7 girls) aged 12.1 +/- 2.3 y (range, 7.1-14.6 y), before and after 28 +/- 4 d including a 14-d IVAT course . After IVAT, weight increased significantly by 1.9% (32.1 +/- 7.5 versus 32.7 +/- 7.6 kg; p < 0.05), while fat mass and fat free mass increased non significantly . EI increased by 4.6% (10,797 +/- 3039 versus 11320 +/- 3074 kJ/d; p < 0.05) . TEE was not affected by IVAT (7014 +/- 1929 versus 7081 +/- 1478 kJ/d) whereas REE decreased by 4.1% (5295 +/- 909 versus 5093 +/- 837 kJ/d; p < 0.05), resulting in 9.3% increase in PA assessed by AD converted to metabolic equivalent tasks (MET) (37.0 +/- 3.1 versus 40.7 +/- 4.5 MET; p < 0.05) . The improvement in nutritional status after IVAT is not related to a decrease in TEE, but probably to an increase in EI and a decrease of REE after IVAT . After IVAT, the reduction in REE is probably compensated by an increase in PA in CF patients.

Microbiology, 2003 Aug, 149(Pt 8), 2291 - 9
Mutation of lasA and lasB reduces Pseudomonas aeruginosa invasion of epithelial cells; Cowell BA et al.; Pseudomonas aeruginosa is an opportunistic bacterial pathogen implicated in a variety of devastating conditions . Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions . ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of P . aeruginosa . This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells . The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated P . aeruginosa invasion . Mutation of lasA and/or lasB reduced P . aeruginosa invasion, which was not fully restored by extracellularly added LasB, P . aeruginosa conditioned medium containing LasA and LasB, or EGTA pretreatment of cells . This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity . Upon mutation of lasA and/or lasB, steady-state levels of ExoS and ExoT were increased in culture medium of P . aeruginosa grown under conditions stimulatory for these toxins . The increase in ExoS was significantly correlated with reduced invasion . In vitro experiments showed that purified LasB degraded recombinant ExoS . Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells . By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

Microbiology, 2003 Aug, 149(Pt 8), 2005 - 13
rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids; Deziel E et al.; Pseudomonas aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids . Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood . Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths . RhlB is the catalytic protein of the rhamnosyltransferase . rhlA is indispensable for rhamnolipid synthesis, but its function is unknown . Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P . aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis . In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties . P . aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents . Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants . Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility.

J Clin Microbiol, 2003 Aug, 41(8), 3712 - 8
Evaluation of the Osiris expert system for identification of beta-lactam phenotypes in isolates of Pseudomonas aeruginosa; Bert F et al.; Osiris is a video zone size reader for disk diffusion tests featuring a built-in extended expert system (EES) . The efficacy of the EES for the identification of the beta-lactam susceptibility phenotypes of Pseudomonas aeruginosa isolates was evaluated . Thirteen beta-lactams were tested in four laboratories by the disk diffusion test with 53 strains with well-characterized resistance mechanisms, including the production of 12 extended-spectrum beta-lactamases (ESBLs) . The plates were read with the Osiris system and the results were interpreted with the ESS, and then the phenotype identified by the EES was compared to the resistance mechanism . The strains were also screened for the presence of ESBL production by a double-disk synergy test by placing the strains between an extended-spectrum cephalosporin-containing disk and a clavulanic acid-containing disk at distances of 30, 20, 15, and 10 mm from each other . Overall, the EES accurately identified the phenotypes of 88.2% of the strains and indicated an association with several mechanisms for 3.8% of the strains . No phenotype was identified in four strains with low levels of penicillinase production . Misidentifications were observed for two penicillinase-producing strains: one strain with partially derepressed cephalosporinase production and one strain overexpressing the MexA-MexB-OprM efflux system . The production of only four ESBLs was detected by the standard synergy test with a 30-mm distance between the disks . The production of five further ESBLs was identified by reducing the distance to 20 mm, and the production of the last three ESBLs was detected only at a distance of 15 or 10 mm . Our results indicate that the Osiris EES is an effective tool for the identification of P . aeruginosa beta-lactam phenotypes . A specific double-disk synergy test with reduced disk distances is necessary for the detection of ESBL production by this organism.

J Clin Microbiol, 2003 Aug, 41(8), 3526 - 31
Single-nucleotide-polymorphism mapping of the Pseudomonas aeruginosa type III secretion toxins for development of a diagnostic multiplex PCR system; Ajayi T et al.; We mapped the coding single nucleotide polymorphisms in four toxin genes-exoS, exoT, exoU, and exoY-of the Pseudomonas aeruginosa type III secretion system among several clinical isolates . We then used this information to design a multiplex PCR assay based on the simultaneous amplification of fragments of these genes . Eight strains of known genotype were used to test our multiplex PCR method, which showed 100% sensitivity and specificity in this small sample size . This assay appears to be promising for the rapid and accurate genotyping of the presence of these genes in clinical strains of P . aeruginosa.

Lett Appl Microbiol, 2003, 37(3), 201 - 6
Decontamination of dental unit water systems with hydrogen peroxide; Zanetti F et al.; AIMS: Transmission of microbial pathogens to patients from water in dental units is a concern . To reduce this risk, the decontaminating efficiency of hydrogen peroxide was evaluated . METHODS AND RESULTS: Three percent hydrogen peroxide diluted 1 : 4 in distilled water (contact time 15 min) was used daily to disinfect the waterlines of a pilot unit previously contaminated with Pseudomonas aeruginosa or Staphylococcus aureus . The behaviour of the test bacteria was seen to differ over time . Staph . aureus numbers slowly decreased until only low numbers were recovered, after which the levels remained stable . Ps . aeruginosa abatement was more rapid and the density of the bacteria reached a peak when the circuit was empty . CONCLUSIONS: Staph . aureus and Ps . aeruginosa treated with hydrogen peroxide fell from 6 to 4 log . SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of dental unit waterlines with hydrogen peroxide was seen to be able to keep the number of the bacteria under control, as long as the treatment was repeated daily.

J Pediatr Hematol Oncol, 2003 Aug, 25(8), 665 - 7
Infection-associated hemophagocytic syndrome due to Pseudomonas aeruginosa in preterm infants; Aygun C et al.; Infection-associated hemophagocytic syndrome due to bacteria is rare and its appearance in preterm babies is uncommon . The signs, symptoms, laboratory values, and clinical status of three preterm babies with infection-associated hemophagocytic syndrome due to Pseudomonas aeruginosa are described and differences between preterm and infant cases are discussed in this report.

Biochem Biophys Res Commun, 2003 Aug 22, 308(2), 234 - 9
Characterisation of CadR from Pseudomonas aeruginosa: a Cd(II)-responsive MerR homologue; Brocklehurst KR et al.; cadR from Pseudomonas aeruginosa encodes a transcriptional regulatory protein which responds to Cd(II)>>Zn(II)>Hg(II) at its cognate promoter PcadA . CadR will also act to induce transcription at the Escherichia coli ZntR cognate promoter, PzntA, however, the induction profile is altered to Hg(II)>Cd(II)>Zn(II) . Two separate single base pair deletions within PzntA result in further alteration of relative specificity in metal-ion induction profile for CadR . This demonstrates that the operator/promoter sequence can play a role in defining optimal ligand response and that for these regulators specificity is not solely a function of the regulatory protein.

Chin Med Sci J, 2000 Jun, 15(2), 83 - 8
Effects of Radix Angelicae sinensis and shuanghuanglian on a rat model of chronic Pseudomonas aeruginosa pneumonia; Song ZJ et al.; OBJECTIVE: To study the effect of two kinds of Chinese herbal medicine, Radix angelicae sinensis (RAS) ({Chinese characters: see text}) and Shuanghuanglian (SHL) ({Chinese characters: see text}) on chronic Pseudomonas aeruginosa (PA) lung infection in a rat model mimicking cystic fibrosis (CF) . METHODS: Rats were divided into RAS, SHL and control groups . All rats were challenged intratracheally with alginate embedded PA and the treatments with herbal medicine started on the same day of challenge . The drugs were administered subcutaneously once a day for ten days and the control group was treated with sterile saline . The rats were sacrificed two weeks after challenge . RESULTS: Significantly improved lung bacterial clearance (P < 0.05, P < 0.01) and milder macroscopic lung pathology (P < 0.005) were found in the two treated groups compared to the control group . In the SH treated group, the neutrophil percent in the peripheral blood leukocytes (P < 0.05), the anti-PA IgG level in serum (P < 0.05), the incidence of lung abcesses (P < 0.005) and the incidence of acute lung inflammation (P < 0.05) were significantly lower than in the control group . The RAS treatment reduced fever (P < 0.05), decreased the incidence of lung abcesses (P < 0.005) and lung mast cell number (P < 0.05), and lowered anti-PA IgG1 level in serum (P < 0.05) when compared to the control group . The anti-PA bacterial activity test in SHL was weakly positive whereas in RAS it was negative . CONCLUSION: The treatment with both herbal medicines could increase the resistance of the rats against PA lung infection and they therefore might be potential promising drugs for stimulation of the immnune system in CF patients with chronic PA lung infection.

Chin Med Sci J, 1999 Mar, 14(1), 57 - 60
Role of outer membrane proteins in imipenem diffusion in Pseudomonas aeruginosa; Lian Z et al.; The present study identified the properties of porins in the outer membrane in Pseudomonas aeruginosa, and showed the role of outer membrane in determining imipenem diffusion in Pseudomonas aeruginosa . The molecular weight of the major outer membrane protein was analyzed by SDS-PAGE . The purification of the porins in Pseudomonas aeruginosa was achieved by DEAE ion-exchange HPLC . The purified outer membrane proteins were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and were tested by the liposomes swelling method for the diffusion of imipenem . The permeability assay showed that OprC (70 kD), OprD2 (46 kD), and OprE (43 kD) were the channel-forming proteins . But only OprD2 was thought to be the likely route of imipenem diffusion.

J Infect Dis, 2003 Aug 15, 188(4), 512 - 8 Epub 2003 Jul 23.
Genotypic and phenotypic analysis of type III secretion system in a cohort of Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association between O serotypes and exo genes; Berthelot P et al.; The type III secretion system (TTSS) of Pseudomonas aeruginosa was characterized genetically and phenotypically in 92 epidemiologically unrelated bacteremic strains . Four groups of strains (TTSS types) were defined according to the level of type III protein secretion and kinetics of cytotoxicity . Type 1 strains (n=26) were highly and rapidly cytotoxic and secreted ExoU, type 2 strains (n=48) exhibited slower cytotoxic rates and expressed ExoS but not ExoU, type 3 strains (n=14) were poorly cytotoxic, and type 4 strains (n=4) were not cytotoxic . Type 3 and 4 strains did not have detectable secretion phenotype; however, some type 4 strains were able to reach a level of cytotoxicity similar to that of type 1 and type 2 strains when complemented in trans by a functional exsA gene . A statistically significant association (P<.001) was found between TTSS types and detection of the mutually exclusive exoU and exoS genes . In addition, 24 of 25 serotype O:1, O:10, and O:11 strains contained exoU, whereas 54 of 55 serotype O:3, O:4, O:6, O:12, and O:16 strains contained exoS (P<.001) . Our results demonstrate correlations among exoU or exoS genotype, TTSS phenotype, and O serotype in bacteremic P . aeruginosa isolates.

Laryngoscope, 2003 Aug, 113(8), 1394 - 400
Bacteria and granulation tissue associated with Montgomery T-tubes; Schmal F et al.; OBJECTIVES: Although complications (infection, development of granulation tissue) of silicone Montgomery T-tubes have been reported, the microbiological consequences and the origin of granulation tissue have not yet been evaluated . STUDY DESIGN: A prospective trial . METHODS: Twenty-three Montgomery T-tubes from 10 patients were analyzed with regard to the development of granulation tissue, bacterial growth (including genotyping with polymerase chain reaction), and results of sensitivity testing . Furthermore, stent sterilization (n = 6) was investigated . RESULTS: Granulation tissue occurred with 74% of the stents, and all specimens showed signs of infection but no foreign body reaction . The predominant organisms were Staphylococcus aureus (35%) and Pseudomonas aeruginosa (17%) . The differences between groups with and without granulation tissue were significant for P aeruginosa . Polymerase chain reaction fingerprinting of the S aureus obtained from 15 stents (n = 3 patients) revealed a total of seven different genotypes . Whereas two of these patients harbored six different genotypes of S aureus, the third patient was persistently colonized by S aureus over a 15-month period with the identical genotype . Susceptibility testing showed most commonly (65%) sensitivity to a combination of amoxicillin-clavulanate and ofloxacin . After sterilization, 92% of analyzed stent segments showed no bacterial growth . CONCLUSIONS: Granulation tissue commonly occurred next to the silicone (subglottic area, stoma) where S aureus and P aeruginosa were commonly isolated . A combination of mechanical irritation and bacterial infection seems to account for the development of granulation tissue . Polymerase chain reaction fingerprinting showed both prolonged persistence and a change of colonizing strains after multiple stent replacements . A combination of amoxicillin-clavulanate and ofloxacin is the most effective antibiotic therapy . Sterilization of the cost-intensive silicone stents is feasible, and reuse in the same patient is justifiable from economic aspects.

Ann Fr Anesth Reanim, 2003 Jun, 22(6), 505 - 9
{Pseudomonas aeruginosa epidemiology in intensive care units: importance of cross-transmission}; Bertrand X et al.; OBJECTIVES: To update the local epidemiological data of Pseudomonas aeruginosa in intensive care units (ICU) by assessing the colonisation incidence rate and the level of cross-transmission . METHODS: Study carried out in both adult ICUs of the university-hospital of Besancon during a 2 years period . Clinical and surveillance specimens were screened for P . aeruginosa . Pulsed-field-gel-electrophoresis was used as genotyping method to evaluate the rate of cross-transmission . RESULTS: During the study, 314 patients were positive for P . aeruginosa (incidence rate of 19.1 patients per 100 admitted patients) . One hundred sixty-six of these patients were detected with a clinical specimen and 148 with a screening specimen . Seventy-seven patients were colonised upon admission in the intensive care unit and 237, negative on admission, became positive during their stay . Of the ICU-acquired cases, the mean length of stay before P . aeruginosa colonisation was acquired was 15.7 days . Genotyping revealed that 53.5% of P . aeruginosa colonisation was acquired via cross-transmission (respectively 48.1% in the medical ICU and 59.2% in the surgical ICU); the other cases probably originated from endogenous sources . CONCLUSION: The incidences of P . aeruginosa colonisation upon admission and during hospitalisation are consistent with other french and european studies . Although we probably over-estimated the rate of cross-transmission, our results demonstrate that cross-transmission may be a major cause of P . aeruginosa dissemination in ICUs.

Carbohydr Res, 2003 Aug 12, 338(17), 1801 - 6
Structure of the biological repeating unit of the O-antigen of Pseudomonas aeruginosa immunotype 4 containing both 2-acetamido-2,6-dideoxy-D-glucose and 2-acetamido-2,6-dideoxy-D-galactose; Bystrova OV et al.; A phosphorylated core-lipid A backbone oligosaccharide that carries a disaccharide remainder of the first O-antigen repeating unit was derived by strong alkaline degradation following mild hydrazinolysis of the lipopolysaccharide of Pseudomonas aeruginosa immunotype 4 (serogroup O-1) . The structure of the oligosaccharide was determined using ESI MS and NMR spectroscopy and it was demonstrated that 2-acetamido-2,6-dideoxy-D-glucose is the first monosaccharide of the O-polysaccharide that is linked to the LPS core . These data define the structure of the biological repeating unit of the O-antigen.

Am Fam Physician, 2003 Jul 15, 68(2), 309 - 12
Necrotizing (malignant) external otitis; Handzel O et al.; Necrotizing (malignant) external otitis, an infection involving the temporal and adjacent bones, is a relatively rare complication of external otitis . It occurs primarily in immunocompromised persons, especially older persons with diabetes mellitus, and is often initiated by self-inflicted or iatrogenic trauma to the external auditory canal . The most frequent pathogen is Pseudomonas aeruginosa . Patients with necrotizing external otitis complain of severe otalgia that worsens at night, and otorrhea . Clinical findings include granulation tissue in the external auditory canal, especially at the bone-cartilage junction . Facial and other cranial nerve palsies indicate a poor prognosis; intracranial complications are the most frequent cause of death . Diagnosis requires culture of ear secretions and pathologic examination of granulation tissue from the infection site . Imaging studies may include computed tomographic scanning, technetium Tc 99m medronate bone scanning, and gallium citrate Ga 67 scintigraphy . Treatment includes correction of immunosuppression (when possible), local treatment of the auditory canal, long-term systemic antibiotic therapy and, in selected patients, surgery . Family physicians and others who provide medical care for immunocompromised patients should be alert to the possibility of necrotizing external otitis in patients who complain of otalgia, particularly if they have diabetes mellitus and external otitis that has been refractory to standard therapy . Susceptible patients should be educated to avoid manipulation of the ear canal (i.e., they should not use cotton swabs to clean their ears) and to minimize exposure of the ear canal to water with a high chloride concentration . Appropriate patients should be referred to an otolaryngologist.

Virology, 2003 Jul 20, 312(1), 49 - 59
The genome of bacteriophage phiKMV, a T7-like virus infecting Pseudomonas aeruginosa; Lavigne R et al.; The complete DNA sequence of a new lytic T7-like bacteriophage phiKMV is presented . It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa . The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand . Despite absence of homology at the DNA level, 11 of the 48 phiKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins . Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins . Surprisingly, the major capsid protein completely lacks sequence homology to any known protein . Also, the strong virulence toward many clinical P . aeruginosa isolates and a short replication time make phiKMV attractive for phage therapy or a potential source for antimicrobial proteins.

Biotechnol Lett, 2003 Jun, 25(12), 959 - 62
Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid; Kim Y et al.; Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb) . Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l(-1) . Presence of the plasmid in P . aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host . Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P . aeruginosa transformant.

J Antimicrob Chemother, 2003 Sep, 52(3), 457 - 63 Epub 2003 Jul 29.
Characterization of the onset and consequences of pneumonia due to fluoroquinolone-susceptible or -resistant Pseudomonas aeruginosa; Paladino JA et al.; OBJECTIVES: This study was conducted to identify and compare the microbiological and clinical outcomes among hospitalized adults with pneumonia caused by fluoroquinolone-susceptible or -resistant strains of Pseudomonas aeruginosa . Antibiotic regimens used prior to, as well as those used to treat, the infections were characterized . PATIENTS AND METHODS: This non-randomized multicentre study included 100 consecutively identified patients with pneumonia caused by fluoroquinolone-susceptible (n = 50) or fluoroquinolone-resistant (n = 50) strains of P . aeruginosa . Medical records were examined for demographic, clinical and treatment variables including antibiotics received in the 30 days before the index respiratory or blood culture; AUICs were calculated for each patient using reported or derived MICs . Multivariate logistic and linear regressions were used to identify factors associated with successful clinical and microbiological outcomes . RESULTS: The study population was primarily elderly, frequently in a critical care unit, with low serum albumin and with a high probability of failure and mortality . Patients with pneumonia caused by fluoroquinolone-resistant P . aeruginosa were more likely to have received antibiotics within 7 days before the infection (P = 0.027); the antibiotic regimen was more likely to be of a weak potency (mean AUIC of 58 versus 169, P = 0.001) and to include levofloxacin (P < 0.0001) than what was administered to patients who became infected with a fluoroquinolone-susceptible strain . Regardless of susceptibility, a mean of between 2 and 3 weeks of directed antibiotic therapy was administered to each patient . CONCLUSIONS: Pneumonia caused by fluoroquinolone-resistant P . aeruginosa is frequently associated with prior exposure to levofloxacin . Treatment of P . aeruginosa pneumonia is difficult and usually consists of combination regimens with multiple modifications.

Biochem Soc Trans, 2003 Aug, 31(Pt 4), 815 - 8
Expression and anti-bacterial activity of human parotid secretory protein (PSP); Geetha C et al.; Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands . A related sequence (C20orf70) was identified on human chromosome 20 . The goal of this study was to determine if PSP is expressed in the human parotid gland . The cDNA for human PSP was amplified from a human parotid cDNA sample . A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy . Immunoaffinity chromatography suggested that PSP was expressed in human saliva . PSP is related to bactericidal/permeability-increasing protein (BPI) . To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells . The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP . These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.

Biochemistry, 2003 Aug 5, 42(30), 8945 - 56
Modeling domino effects in enzymes: molecular basis of the substrate specificity of the bacterial metallo-beta-lactamases IMP-1 and IMP-6; Oelschlaeger P et al.; Metallo-beta-lactamases can hydrolyze a broad spectrum of beta-lactam antibiotics and thus confer resistance to bacteria . For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported . IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime . Interestingly, this biochemical effect is caused by a residue remote from the active site . The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions . Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6 . At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time . However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation . To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme-substrate complexes . Stable structures were further heated to 200 and 300 K . By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency . The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes . The remote effect of residue 196 can be described by a domino effect: upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site.

Pharmacotherapy, 2003 Jul, 23(7), 916 - 24
The role of multidrug efflux pumps in the antibiotic resistance of Pseudomonas aeruginosa and other gram-negative bacteria . Insights from the Society of Infectious Diseases Pharmacists; Aeschlimann JR; Gram-negative bacteria remain clinically important pathogens in both hospital and community settings . Recent research indicates that efflux pumps play a prominent role in the multidrug resistance of Pseudomonas aeruginosa and many other gram-negative bacteria . Four multidrug efflux pump systems have been well characterized in P . aeruginosa: MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM . These efflux pumps have different substrate specificities, and their production and activity can be increased by many factors commonly present in infections (e.g., high inocula of bacteria, low pH, and stationary-phase growth) . Moreover, fluoroquinolone antibiotics can commonly select mutants that constitutively overproduce Mex-Opr efflux pump systems . Based on most recent studies, the prevalence of efflux pump overproduction in clinical strains of P . aeruginosa may range from 14-75% . The best treatment for infections caused by bacteria that overproduce efflux pumps is unknown, but pharmacodynamic optimization of antibiotics and the use of antibiotic combinations that are substrates for different pump systems may represent reasonable strategies until more data are available.

Nippon Geka Gakkai Zasshi, 2003 Jul, 104(7), 527 - 9
{Microbiological approach to antibiotic therapy against multidrug-resistant bacteria}; Kusachi S; Many severe infections that occur after gastrointestinal surgery are caused by multidrug-resistant bacteria such as methicillin-resistant staphylococcus aureus and Pseudomonas aeruginosa . (P . aeruginosa,) which has been isolated from such cases, is resistant to many beta-lactamantibiotics, including imipenem (IPM) . IPM-resistant P . aeruginosa lacks the outer membrane proteins (Opr) D, OprD2, OprC, and OprE1, which together normally form an opening in the outer membrane . Therefore IPM cannot pass through the outer membrane . But it is also known that meropenem, caftazidime, and ciprofloxacin are not restricted by this effect and that they can easily pass through the outer membrane . Therefore if we can identify these genes by using the polymerase chain reaction, appropriate antibiotics can be swiftly administered without waiting for the results of drug sensitivity tests.

Am J Otolaryngol, 2003 Jul-Aug, 24(4), 209 - 12
Has cerumen a protective role in recurrent external otitis?
Pata YS, Ozturk C, Akbas Y, Gorur K, Unal M, Ozcan C.
PURPOSE: We investigated the bactericidal activity of the cerumen in patients with recurrent otitis externa . Materials and methods: Cerumen samples were collected from 2 groups . Group A (n = 20) consisted of patients with recurrent otitis externa (2 or more acute otitis externa attack in the current year) and group B (n = 30) consisted of cerumen from a healthy population . We examined the bactericidal activity against the common microorganisms (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus) that encounter the recurrent otitis externa . RESULTS: A significant decrease in the count of S epidermidis was observed in group A (P <.05) and B (P <.01) was observed . A comparison of decreases and increases in the percentages of microorganisms between the groups A and B showed that there was a significant difference only in the count of E coli (P <or=.05) . The count of E coli decreased in cerumen suspension in group A, but it increased in the normal population . CONCLUSION: We did not confirm that cerumen from patients with recurrent otitis externa had less bactericidal activity than from a healthy population.

Invest Ophthalmol Vis Sci, 2003 Aug, 44(8), 3409 - 16
Analysis of Pseudomonas aeruginosa corneal infection using an oligonucleotide microarray; Huang X et al.; PURPOSE: To compare the early gene expression pattern of normal versus Pseudomonas aeruginosa-infected corneas in resistant (cornea heals) versus susceptible (cornea perforates) mice . METHODS: A microarray analysis of normal versus postinfection (PI) day 1 BALB/c and B6 corneas was performed with a murine gene microarray . Real-time RT-PCR was used to confirm the microarray pattern selectively . RESULTS: The 1257 regulated transcripts detected were organized into nine clusters by a self-organizing map (SOM) algorithm according to their different behavior in each mouse group . At least three groups of genes associated with a CD4(+) T-cell type-1 (Th1) immune response and three clusters linked with a type-2 T-cell (Th2) response were identified . Biological categorization revealed that the cornea of B6 mice showed a dominant type-1-like immune response profile, whereas BALB/c mice showed a dominant type-2-like profile . In addition, expression of several genes that promote apoptosis (e.g., caspase-9) was upregulated in BALB/c mouse cornea, whereas genes with apoptosis-inhibiting activity (e.g., BCL2) were significantly upregulated in B6 mouse cornea . The infected cornea of BALB/c mice also showed increased gene expression of factors associated with matrix remodeling and tissue repair (e.g., tissue inhibitor of matrix metalloproteinase {TIMP-2} and epidermal growth factor {EGF}) and/or bacterial killing (e.g., inducible nitric oxide synthase {iNOS}) . CONCLUSIONS: The data provide new insight into biological processes involved in Pseudomonas aeruginosa keratitis and confirm that B6 mice are Th1 and BALB/c mice are Th2 cytokine responsive to bacterial antigen early after challenge with P . aeruginosa.

Biotechnol Lett, 2003 Feb, 25(3), 235 - 9
Fur mutants of Pseudomonas aeruginosa PAO1: phenotypic characterization in terms of iron-dependent pattern of deregulation of pyoverdin synthesis; Johnova A et al.; Fur mutants (Mn(r)) of Pseudomonas aeruginosa Fe10 (FF13 and FF28) and PAO1 (FPA12) were evaluated for the pattern of deregulation of pyoverdin synthesis in iron-replete medium with Casamino acids {CAM(Fe)} or succinate {SM(Fe)} . With respect to siderophore synthesis, we found in CAM(Fe) medium two Fur phenotypes: FurA (full deregulation, FF13) and FurB (partial deregulation, FF28 and FPA12) . Fur mutants compared to parental strains grew with slower specific growth rates on SM(0) and CAM(0) media in a stirred bioreactor . Fur mutants grew in SM(0) with mu about half of that in CAM(0).

Biotechnol Lett, 2003 Jan, 25(1), 29 - 33
A facile reactor process for producing 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid conversion by Pseudomonas aeruginosa; Kuo TM et al.; Pseudomonas aeruginosa strain PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) . The bioconversion was scaled up in a 7-l bench-top, stirred-batch reactor to produce DOD for testing of potential industrial uses . Aeration was supplied continuously from the top through two ports on the headplate and periodically through a bottom sparger, in conjunction with the use of marine impellers for agitation . This unique aeration arrangement maintained the dissolved O2 concentration in the 40-60% range during the period of maximal bioconversion and it also avoided excessive medium foaming during the reaction . Furthermore, the level of dissolved O2 in the first 24 h of reaction played an important role in the initial rate of DOD production . DOD production reached a plateau after 72 h with a yield up to 100 g (or 50% recovery) from a total of 9 l medium from two reactors run simultaneously . The final culture broth was processed using newly adapted procedures in the pilot plant that included crystallization of DOD from ethyl acetate solution at -15 degrees C . The newly developed bioprocess will serve as a platform for the scale-up production of other value-added products derived from vegetable oils and their component fatty acids.

Paediatr Respir Rev, 2003 Sep, 4(3), 172 - 7
Respiratory virus infections in cystic fibrosis; Wat D et al.; Respiratory virus infections have pronounced and long-lasting effects on patients with cystic fibrosis (CF), resulting in significant declines in FVC, FEV(1) and Shwachman score, significantly increasing both the frequency and duration of hospitalisation . Deleterious effects on patients with CF have been reported for most viruses studied but the effects of respiratory syncytial virus and influenza appear the greatest . There is circumstantial evidence that respiratory virus infections may facilitate bacterial infections, particularly Pseudomonas aeruginosa.

Alcohol Clin Exp Res, 2003 Jul, 27(7), 1199 - 206
Effect of acute ethanol exposure on the dermal inflammatory response after burn injury; Faunce DE et al.; BACKGROUND: More than 100,000 people each year are admitted to U.S . hospitals for severe burn injury . Strikingly, ethanol use prior to injury is apparent in nearly 50% of burn patients, rendering them six times more likely to die from infection than patients not exposed to ethanol . We previously reported that the kinetics and magnitude of neutrophil chemokine production and subsequent accumulation of neutrophils in the lung was dramatically altered when ethanol exposure preceded injury . Here, we tested whether burn injury and ethanol exposure combined, altered susceptibility to infection, neutrophil chemoattractant production, and neutrophil accumulation at the site of the burn wound . METHODS: Male B(6)D(2)F1 mice were administered a dose of ethanol designed to achieve 90-100 mg/dl circulating levels and 30 min later subjected to a 15% total body surface area dorsal scald injury . Susceptibility to topically applied Pseudomonas aeruginosa was examined . At various times after injury, burn wound and normal tissues were collected for assessments of neutrophil counts, myeloperoxidase quantitation, and neutrophil chemoattractant (KC and MIP-2) production . RESULTS: Ethanol exposure prior to burn injury enhanced susceptibility to infection after burn and was associated with significantly elevated production of KC, but not MIP-2, at the wound site . Despite the enhanced elevation of KC, neutrophil accumulation in the wounds of ethanol exposed, burn injured mice did not differ from those that received burn injury alone . TNFalpha (a potent activator of neutrophils), however, was found to be significantly elevated in the wounds of mice that received only burn injury, but not in those that received injury in combination with prior ethanol exposure . CONCLUSION: In the presence of ethanol, neutrophils are adequately recruited to the site of burn injury, but their host defense functions are impaired, perhaps due to the lack of proinflammatory cytokines such as TNFalpha.

Am J Respir Cell Mol Biol, 2003 Aug, 29(2), 188 - 97
Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator; Cannon CL et al.; Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe . Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-kappa B and cellular desquamation within minutes of P . aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr . Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P . aeruginosa, we found that human bronchial epithelial cells expressing Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR . Mice with a WT Cftr allele had apoptotic cells in their lungs after P . aeruginosa infections, whereas mice homozygous for the Delta F508 or G551D Cftr alleles showed little apoptosis in response to acute infection . Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles . Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P . aeruginosa infection . Prompt apoptosis of infected epithelial cells may be critical for clearance of P . aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.

Antimicrob Agents Chemother, 2003 Aug, 47(8), 2615 - 8
In vitro activities of novel oxapenems, alone and in combination with ceftazidime, against gram-positive and gram-negative organisms; Jamieson CE et al.; Four novel oxapenem compounds (i.e., AM-112, AM-113, AM-114, and AM-115) were investigated for their beta-lactamase inhibitory activity against a panel of isolated class A, C, and D enzymes, which included expanded-spectrum beta-lactamase enzymes (ESBLs) . The oxapenems were potent beta-lactamase inhibitors . Activity varied within the group, with AM-113 and AM-114 proving to be the most active compounds . The 50% inhibitory concentrations for these agents were up to 100,000-fold lower than that of clavulanic acid against class C and D enzymes . As a group, the oxapenems were more potent than clavulanic acid against enzymes from all classes . The ability of these compounds to protect ceftazidime from hydrolysis by beta-lactamase-producing strains was evaluated by MIC tests that combined ceftazidime and each oxapenem in a 1:1 or 2:1 ratio . The oxapenems markedly reduced the MICs for ceftazidime against class C hyperproducing strains and strains producing TEM- and SHV-derived ESBLs . There was little difference between the activity of 1:1 and 2:1 combinations of ceftazidime and oxapenem . The oxapenems failed to enhance the activity of ceftazidime against derepressed AmpC-producing Pseudomonas aeruginosa strains.

Bioorg Med Chem, 2003 Aug 5, 11(16), 3475 - 85
CP0569, a new broad-spectrum injectable carbapenem . Part 1: synthesis and structure-activity relationships; Aihara K et al.; A series of 1beta-methylcarbapenems bearing an (imidazo{5,1-b}thiazolium-6-yl)methyl moiety, a 5,5-fused heterobicycle, at the C-2 position was synthesized and evaluated for in vitro antibacterial activities . CP0569 (1r) and its analogues showed potent antibacterial activities against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria, including Pseudomonas aeruginosa . Moreover, CP0569 (1r) exhibited stronger antibacterial activity against MRSA and higher resistance to renal dehydropeptidase-1 (DHP-1) than any currently marketed carbapenems, that is, imipenem (IPM), panipenem (PAPM), and meropenem (MEPM).

Acta Crystallogr D Biol Crystallogr, 2003 Aug, 59(Pt 8), 1499 - 501 Epub 2003 Jul 23.
Crystallization and preliminary X-ray analysis of alginate lyase, a member of family PL-7, from Pseudomonas aeruginosa; Yamasaki M et al.; Alginate lyase depolymerizes alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction . A protein PA1167 with a molecular mass of 25 kDa produced by Pseudomonas aeruginosa is an alginate lyase classified into polysaccharide lyase family PL-7 . The enzyme was crystallized at 293 K in a drop solution comprising 1.4 M sodium chloride, 0.1 M potassium sodium phosphate and 0.1 M 2-morpholinoethanesulfonate-sodium hydroxide pH 6.5 by means of the vapor-diffusion method . The crystals were monoclinic and belonged to space group P2(1), with unit-cell parameters a = 43.4, b = 70.3, c = 67.4 A, beta = 94.5 degrees . Diffraction data were collected to 2.0 A from a single crystal.

Chin Med J (Engl), 2003 May, 116(5), 676 - 8
Allogeneic peripheral blood stem cell transplantation in the treatment of severe aplastic anemia and severe infection; Wan L et al.; OBJECTIVE: To investigate the efficacy of allogeneic peripheral blood stem cell transplantation (PBSCT) in the treatment of severe aplastic anemia (SAA) and severe infection . METHODS: A patient with SAA and pseudomonas aeruginosa septicemia was treated with PBSCT from an HLA-identical sibling with cyclophosphamide (CY) and total body irradiation (TBI) for conditioning . The patient was infused with 20.3 x 10(8)/kg mononuclear cells including 61.0 x 10(6)/kg CD34(+) cells following the conditioning regimen . RESULTS: Twelve days after PBSCT, the absolute neutrophil count (ANC) of 1.0 x 10(9)/L was achieved, with platelet count > 50 x 10(9)/L at twenty days . The donor origin of engraftment was confirmed by polymerase chain reaction (PCR) analysis of short tandem repeats at the end of the first, sixth and twelfth month . The patient's body temperature dropped to normal level when her ANC reached 0.5 x 10(9)/L on day 10, and the bacterial culture of blood sample became negative subsequently . Symptoms and signs of acute or chronic graft versus host disease (GVHD) were not observed in 30 months after PBSCT . CONCLUSIONS: Hematopoiesis was reconstituted shortly after PBSCT . The combination of CY and TBI and the infusion of sufficient peripheral blood stem cells may contribute to the successful engraftment . PBSCT may be considered as the first choice when hematopoietic stem cell transplantation is needed for SAA patients complicated with severe infection.

Infect Immun, 2003 Aug, 71(8), 4614 - 22
Pseudomonas aeruginosa slime glycolipoprotein is a potent stimulant of tumor necrosis factor alpha gene expression and activation of transcription activators nuclear factor kappa B and activator protein 1 in human monocytes; Lagoumintzis G et al.; Pseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts . Production of tumor necrosis factor alpha (TNF-alpha), primarily by cells of monocytic lineage, is a crucial event in the course of these infections . During in vivo infections with P . aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released . In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-alpha production by human monocytes . To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated with P . aeruginosa LPS or GLP . GLP was found to be a more potent stimulus for TNF-alpha production (threefold higher) by human monocytes than LPS . Moreover, its effect was comparable to that of viable bacteria . Quantitative mRNA analysis revealed predominantly transcriptional regulation . Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-kappa B and activator protein 1 (AP-1) . NF-kappa B activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-kappa B through GLP . Moreover P . aeruginosa GLP induced the formation of AP-1 complex with delayed kinetics compared with NF-kappa B but much more efficiently than the homologous LPS . These results identify GLP as the most important stimulant for TNF-alpha production by human monocytes . Activation of NF-kappa B and AP-1 by P . aeruginosa GLP may be involved not only in TNF-alpha induction but also in many of the inflammatory responses triggered in the course of infection with P . aeruginosa.

Infect Immun, 2003 Aug, 71(8), 4421 - 31
Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone; Ritchie AJ et al.; N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule of Pseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression . Several reports have suggested that OdDHL can also directly modulate host immune responses . However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses . This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls . In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-gamma) tending to be more inhibited than interleukin-4 (IL-4) . In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-gamma in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice . Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.

Anesth Analg, 2003 Aug, 97(2), 409 - 11, table of contents
Ropivacaine 0.1% with sufentanil 1 microg/mL inhibits in vitro growth of Pseudomonas aeruginosa and does not promote multiplication of Staphylococcus aureus; Kampe S et al.; We investigated the effect of ropivacaine combined with sufentanil, a mixture frequently used for postoperative epidural analgesia, on the growth of Staphylococcus aureus and Pseudomonas aeruginosa at room temperature . Aliquots of suspension of S . aureus and P . aeruginosa in saline were transferred into test tubes containing either a mixture of ropivacaine 0.1% and sufentanil 1 microg/mL (R+S) or saline (SA), with the latter serving as control . At 0, 3, 6, 24, and 48 h after inoculation, 1 mL of each solution was spread over standard blood agar . The plates were incubated at 22 degrees C for 48 h, and the numbers of colony-forming units (cfu) were counted . The growth ratio for both bacterial strains was calculated as cfu time (t(n))/cfu baseline (t(0)) . The primary efficacy variable was the area under the curve (AUC) in (cfu t(n)/cfu t(0)) x time, based on the growth ratios . The AUC for P . aeruginosa was significantly less in R+S than in SA (P = 0.028) . Multiplication of P . aeruginosa (growth ratio >1) was observed for at least 6 h after inoculation in SA . Growth of P . aeruginosa was significantly less in R+S than in SA at 3 h (P = 0.043) and 24 h (P = 0.012) after inoculation . The AUC for S . aureus did not differ significantly between R+S and SA (P = 0.74) . Neither R+S nor SA promoted multiplication of S . aureus . Forty-eight hours after inoculation, growth of S . aureus was significantly less in R+S than in SA (P < 0.0001) . We conclude that R+S inhibited growth of P . aeruginosa and did not promote multiplication of S . aureus when compared with SA . IMPLICATIONS: This laboratory study demonstrated that compared with saline, ropivacaine 0.1% with 1 microg/mL of sufentanil inhibited growth of Pseudomonas aeruginosa and did not promote multiplication of Staphylococcus aureus at room temperature . With respect to bacterial infection with these two strains, the mixture seems to be safe for continuous epidural administration if prepared under aseptic conditions and after alcohol hand rub.

Bioorg Med Chem Lett, 2003 Aug 18, 13(16), 2755 - 8
Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa; Renau TE et al.; Conformational restriction of the ornithine residue of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in potentiating the activity of the fluoroquinolone levofloxacin via the inhibition of efflux pumps in Pseudomonas aeruginosa.

J Surg Res, 2003 Jun 1, 112(1), 49 - 58
Increased tissue oxygen extraction and acidosis with progressive severity of sepsis; Hart DW et al.; BACKGROUND: Lactic acidosis and increased production of CO(2) are common in septic shock . Presumably, both acidosis and CO(2) enhance the release of oxygen from hemoglobin . The purpose of this study was to assess the relationship of oxygen utilization, CO(2) production, acidosis, and hemoglobin oxygen (Hgb-O(2)) dissociation with progressive severity of sepsis to shock . MATERIALS AND METHODS: Femoral arterial and vein, hepatic vein, portal vein, and pulmonary artery catheters were placed in 16 anesthetized swine . Organ blood flow was determined by timed injections of colored microspheres . After baseline measurements, Pseudomonas aeruginosa was infused in eight animals . This bacterial slurry was continued inciting a progression of sepsis to shock . Eight animals served as instrumented controls . RESULTS: With sepsis and shock, there was a progressive decrease in pH and an increase in pCO(2) in plasma with all sampling sites (P < 0.01 septic shock versus baseline versus control) . Blood flow to the liver and intestines increased with sepsis (P < 0.01) but then returned to near baseline control values during shock . VO(2) and/or percent O(2) extraction increased with sepsis and septic shock for the whole body and for the liver, intestine and leg (P < 0.01) . There was a strong correlation between venous O(2) saturation, acidosis, and pCO(2) to percent O(2) extraction (r > 60; P < 0.0001) . However, calculated P(50) values for Hgb-O(2) dissociation remained unchanged . CONCLUSIONS: This study demonstrates that increased oxygen extraction in severe sepsis is related to a fall in tissue oxygen availability and not related to any allosteric change in Hgb-O(2) dissociation . Therefore, acidosis and hypercapnia do not have a demonstrable effect on altering oxygen availability during sepsis.

Am J Physiol Lung Cell Mol Physiol, 2003 Nov, 285(5), L1077 - 86 Epub 2003 Jul 18.
The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells; O'Malley YQ et al.; Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide . Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase . Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells . In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content . Pyocyanin also decreased catalase mRNA . Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined . Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase . These data suggest a direct effect of pyocyanin on catalase activity . Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity . Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity . Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide . We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme . Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.

Int J Med Microbiol, 2003 Jun, 293(2-3), 191 - 7
Effect of basic amino acids on susceptibility to carbapenems in clinical Pseudomonas aeruginosa isolates; Muramatsu H et al.; We evaluated effects of medium composition, including basic amino acid content and pH, on susceptibility to carbapenems such as imipenem, panipenem and meropenem, in clinical isolates of Pseudomonas aeruginosa . Susceptibility to carbapenems was reduced by basic amino acids in the medium, while susceptibilities to ceftazidime and aztreonam were not . Among carbapenems, susceptibility to panipenem was most sharply reduced by addition of basic amino acids to 1:16 Mueller-Hinton agar (MHA) . In 174 of 175 clinical isolates, MICs for carbapenems were affected to different degrees by medium composition . One isolate, in which MICs for carbapenems did not differ between MHA and 1:16 MHA, showed reduced production of porin (OprD) . Our results suggest that susceptibility to individual carbapenems, especially panipenem, is difficult to evaluate based on MICs for other carbapenems determined on MHA . For a better prediction of antibiotic efficacy, it may be important to evaluate the susceptibility for each carbapenem individually.

J Chemother, 2003 Jun, 15(3), 235 - 8
In vitro interaction of colistin and rifampin on multidrug-resistant Pseudomonas aeruginosa; Giamarellos-Bourboulis EJ et al.; The administration of colistin is considered as the last alternative for infections by multidrug-resistant isolates of Pseudomonas aeruginosa; however its use is limited due to its considerable toxicity and poor pharmacokinetics . In order to define the in vitro activity of colistin combined with rifampin, 28 isolates resistant to piperacillin, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, rifampin and colistin were tested . Seventeen of them that were found by PFGE to be genetically distinct were over time exposed to 2 microg/ml of colistin, to 2 microg/ml of rifampin and to their combination . Applied concentrations were selected to correspond to the mean serum level of the tested antimicrobials . Synergy between colistin and rifampin was found in four (23.5%), six (35.3%), seven (41.7%) and two (11.8%) isolates after 2, 4, 6 and 24 hours of growth, respectively . Bacterial re-growth was detected after 24 hours of exposure to the tested interaction . It is concluded that colistin and rifampin express a considerable in vitro synergistic effect on multidrug-resistant P . aeruginosa . The reported interaction should be tested in animal studies before introduction in clinical practice.

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