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,