Pseudomonas aeruginosa

Jpn J Antibiot, 2002 Feb, 55(1), 61 - 6
{In vitro interaction of tazobactam/piperacillin combined with aminoglycosides against Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli AND Staphylococcus aureus}; Yamada H et al.; The in vitro combination effect of tazobactam/piperacillin (TAZ/PIPC) with aminoglycosides (amikacin (AMK) and isepamicin (ISP)) were investigated by the checkerboard dilution method against PIPC-resistant and TAZ/PIPC-susceptible Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Methicillin-sensitive Staphylococcus aureus (MSSA) . The following results were obtained . 1 . The combination of TAZ/PIPC with AMK showed synergistic effect for 66.7% of P . aeruginosa and 9.1% of K . pneumoniae and additive effect for 76.9% of E . coli and 74.1% of MSSA . The antagonistic effect of TAZ/PIPC with AMK was not demonstrated for all tested strains . 2 . The combination of TAZ/PIPC with ISP showed synergistic effect for 61.9% of P . aeruginosa and 22.7% of K . pneumoniae and additive effect for 84.6% of E . coli and 66.7% of MSSA . The antagonistic effect of TAZ/PIPC with ISP was not demonstrated for all tested strains . In conclusion, these results suggest that the combination therapies of TAZ/PIPC with aminoglycosides are useful for the clinical treatment of sepsis caused by above four species.

Am J Health Syst Pharm, 2002 Apr 15, 59(8 Suppl 3), S4 - 6
Assessing the impact of antimicrobial resistance; Gums JG; Trends in infectious disease mortality, contributing factors, and resistance patterns of commonly isolated microorganisms are described . Antimicrobial resistance was first reported in 1941 with sulfonamides . The frequency of antimicrobial resistance has increased in hospital and community settings, resulting in therapeutic failures, the use of increasingly costly and toxic antimicrobials, extended hospital stays, and increased morbidity, mortality, and health care costs . Retrospective analysis of infectious disease mortality in the United States reveals that resistance has increased despite the development of new antimicrobials . Factors contributing to antimicrobial resistance include patients' expectations of receiving an antimicrobial prescription after an office visit (even when one is not warranted), low vaccination rates among the elderly, international travel, and continuous exposure to small amounts of antimicrobials in the food supply . Analysis of susceptibility and resistance patterns of more than 10.3 million isolates found that 36%, 30%, and 31% of Escherichia coli isolates were resistant to ampicillin, ampicillin-sulbactam, and piperacillin, respectively . E . coli and Klebsiella pneumoniae exhibited reduced susceptibility to ceftazidime in certain regions of the country . Pseudomonas aeruginosa exhibited resistance to ciprofloxacin, imipenem, gentamicin, and ceftazidime . The occurrence of ciprofloxacin- and levofloxacin-resistant Staphylococcus aureus was similar, while the frequency of erythromycin-resistant S . aureus varied widely nationwide . Nationwide susceptibility to cefotaxime was significantly lower than that to ceftriaxone . Antimicrobial resistance is an increasing problem that contributes to morbidity, mortality, and increased health care costs . Analysis of susceptibility and resistance patterns of specific microorganisms is necessary to gain further insight into the causes of antimicrobial resistance and ways to reduce it.

J Bacteriol, 2002 May, 184(10), 2709 - 18
Caulobacter crescentus synthesizes an S-layer-editing metalloprotease possessing a domain sharing sequence similarity with its paracrystalline S-layer protein; Umelo-Njaka E et al.; Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface . The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously . In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts . The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence . We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis . A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants . The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity . Instead, there was a region of significant similarity to the N-terminal region of RsaA . We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to "scan" for modifications.

J Bacteriol, 2002 May, 184(10), 2620 - 5
The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene; Wang L et al.; Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E . coli clones . We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes . Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis . The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E . coli O113, respectively . We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes . Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby . It is thought that the E . coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage . Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes . Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.

J Bacteriol, 2002 May, 184(10), 2576 - 86
Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression; Diggle SP et al.; Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL) . In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL . To identify novel regulators responsible for this growth phase dependency, a P . aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis . A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes . While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS . MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production . This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL . Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

Kansenshogaku Zasshi, 2002 Mar, 76(3), 188 - 94
{Pneumonia associated with lung cancer in the elderly}; Kobashi Y et al.; We investigated the clinical characteristics separating pneumonia as a complication in elderly lung cancer patients into obstructive and non-obstructive pneumonia . Two hundred and five patients with pneumonia as a complication in elderly lung cancer patients were classified into two groups; 64 patients with obstructive pneumonia and 141 patients with non-obstructive pneumonia . Most of the patients in both groups were male . Concerning histological findings, while most of the patients with obstructive pneumonia had squamous cell carcinoma, those with non-obstructive pneumonia had the same proportion of squamous cell carcinoma as all elderly patients with lung cancer . Most of the patients with obstructive pneumonia were in good general condition including their nutritional condition, but the patients with non-obstructive pneumonia were in significantly poor condition . A low percent of microorganisms were isolated from the sputum obtained from the patients with non-obstructive pneumonia, but a high percentage were obtained from those with non-obstructive pneumonia . Frequent involvement of gram-negative bacilli such as Pseudomonas aeruginosa and Klebsiella pneumoniae or Staphylococcus aureus containing MRSA was also found in these patients . Regarding treatment, although carbapenem was used either alone or in combination therapy as the regimen of treatment for pneumonia as a complication in elderly lung cancer patients with both the obstructive and non-obstructive pneumonia patients, the efficacy rate was poor in 50% with obstructive pneumonia and in 26% with non-obstructive pneumonia . The mortality rate was 11% in the patients with obstructive pneumonia, while it was 61% in the patients with non-obstructive pneumonia . The prognosis was significantly poorer in the patients with non-obstructive pneumonia . We concluded that although the prognosis was not so poor for patients with obstructive pneumonia if the appropriate treatment was given, in the patients with non-obstructive pneumonia, the treatment for underlying diseases and the improvement of their general condition, including the determination of causative microorganisms, was important.

Am J Respir Cell Mol Biol, 2002 May, 26(5), 617 - 26
Early mitochondrial dysfunction, superoxide anion production, and DNA degradation are associated with non-apoptotic death of human airway epithelial cells induced by Pseudomonas aeruginosa exotoxin A; Plotkowski MC et al.; It has been shown that bacterial exoproducts may induce airway epithelium injury . During the epithelial repair process, the respiratory epithelial cells no more establish tight junctional intercellular complexes and may be particularly susceptible to bacterial virulence factors . In this study, we analyzed the effect of Pseudomonas aeruginosa exotoxin A (ETA) at different periods of time and concentrations on 16 HBE 14o(-) human bronchial epithelial cells in culture conditions inducing a phenotype of repairing cells . ETA treatment for 24 and 48 h led to the killing of 40.0 +/- 5.7% and 79.0 +/- 1.4% of the cells, respectively, as determined by the dimethylthiazole 2,5 diphenyl tetrazolium bromide assay . At 1,000 ng/ml, ETA led to the killing of 25.2 +/- 6.6, 59.4 +/- 5.9, and 82.3 +/- 3.7% of the cells, after treatment periods of 7, 24, and 48 h, respectively . Cell death could not be inhibited by z-VAD-fmk, a broad spectrum caspase inhibitor . By transmission electron microscopy, ultrastructural characteristics described in apoptosis were not detected in ETA-treated cells . Instead, the mitochondria of cells treated for 24 and 48 h with ETA at 100 and 1,000 ng/ml were highly condensed . Human nasal polyp epithelial cells in primary culture exposed to ETA at 1,000 ng/ml did not exhibit characteristic features of apoptotic cells either . Cytofluorometric analysis of ETA-treated 16 HBE 14o(-) cells labeled with DiOC(6)(3) and hydroethidine showed a time- and dose-dependent reduction of the mitochondrial transmembrane potential, detected 7 h after ETA treatment, and an increase in superoxide production, detected at 24 h, respectively . By a photometric assay, DNA degradation was also detected 7 h after cell treatment with ETA at 100 and 1,000 ng/ml . Taken together, our results show that ETA-induced death of epithelial respiratory cells was preceded by early mitochondrial dysfunction and superoxide anion production, but was not followed by the classically described apoptotic pathways.

J Appl Microbiol, 2002, 92(4), 729 - 36
Antimicrobial susceptibility changes and T-OMP shifts in pyrithione-passaged planktonic cultures of Pseudomonas aeruginosa PAO1; Abdel-Malek SM et al.; AIMS: The aim of this study was to determine whether passaging Pseudomonas aeruginosa PAO1 with sub-MICs of the pyrithione biocides results in both the induction of decreased susceptibility towards these antimicrobials and associated outer membrane profile changes . METHODS AND RESULTS: Previous work by this group has shown that it is possible to induce susceptibility changes towards the isothiazolone biocides in Ps . aeruginosa PAO1 by successive passages in the presence of increasing sub-MICs of biocide . This procedure was accompanied by the loss of a 35 kDa outer membrane protein, T-OMP . In this experiment, this process was repeated with the biocides sodium pyrithione (NaPT), zinc pyrithione (ZnPT) and cetrimide . The pattern of susceptibility was similar to that observed with the isothiazolone biocides . Upon removal of biocide, the observed MIC did not return to the original pre-exposure value . The onset and development of resistance was accompanied by the loss of T-OMP from outer membrane profiles, which suggests that this is a non-specific membrane channel whose production within the cell is sensitive to biocide presence . The T-OMP reappeared when the cells were passaged in the absence of pyrithione . Cross-resistance studies indicated that induced resistance to one biocide yields partial resistance towards other members of the group and the positive control . CONCLUSIONS: These results indicate that the pyrithione biocides have similar susceptibility profiles in Ps . aeruginosa to those exhibited by the isothiazolones, but that the acquired changes in susceptibility to the pyrithiones is largely irreversible . SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that acquired susceptibility changes towards sub-MICs of selected biocides are multifactorial in nature.

J Appl Microbiol, 2002, 92(4), 618 - 23
Factors influencing the susceptibility of Gram-negative bacteria to toluidine blue O-mediated lethal photosensitization; Komerik N et al.; AIMS: Bacteria can be killed by red light in the presence of a photosensitizer . The purpose of this study was to evaluate the effect of physiological and environmental factors on the susceptibility of some bacteria associated with oral infections in immunocompromised patients to killing by the photosensitizer toluidine blue O (TBO) . METHODS AND RESULTS: Suspensions of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae in human saliva, horse serum or saline were exposed to light from a helium/ neon laser in the presence of TBO . Additional suspensions at various growth phases and pHs were treated in an identical manner . Survivors were enumerated by viable counting . All three species were susceptible to lethal photosensitization under all of the conditions tested . The presence of serum and, to a lesser extent, saliva decreased the level of kill attained . The bactericidal effect was reduced at acid pHs but was unaffected by the growth phase of the organism . CONCLUSIONS: The composition and pH of the fluid in which bacteria are suspended influenced the effectiveness of TBO-mediated lethal photosensitization, whereas killing was unaffected by the growth phase of the organism . SIGNIFICANCE AND IMPACT OF THE STUDY: Environmental factors operating in the mouths of patients with mucositis could reduce the effectiveness of TBO-mediated lethal photosensitization of bacteria associated with this condition.

Plast Reconstr Surg, 2002 Apr 1, 109(4), 1338 - 43
Improvement in burn wound infection and survival with antimicrobial peptide D2A21 (Demegel); Chalekson CP et al.; Naturally occurring antimicrobial peptides have been discovered in both plants and animals . Many of these peptides demonstrate impaired activity or cytotoxicity when applied exogenously . Synthetically engineered antimicrobial peptides have been designed to increase potency and activity against bacteria and fungus yet remain noncytotoxic . The antimicrobial peptide D2A21 (Demegel) has already demonstrated significant activity in vitro against many common hospital pathogens . The purpose of this study was to evaluate the effects of D2A21 in an in vivo infected burn-wound model, examining both quantitative cultures of the wound and survival of the animal . Forty-four Wistar rats were subjected to a 23 percent total body surface area scald burn . Pseudomonas aeruginosa was administered topically with 108 organisms and wounds were then evaluated at day 1, 2, or 3 for eschar and subeschar muscle quantitative culture . The experimental group was treated daily with 1.5% topical D2A21 . The control group was treated with control gel . A second group of Wistar rats (n = 14) were burned and given a 107 inoculum of the same Pseudomonas and evaluated to 14 days for survival and weight changes . This group was subdivided into rats receiving either topical D2A21 or control base daily . The quantitative biopsy results demonstrated that D2A21-treated wounds had no bacterial growth in burn eschar at day 2 or 3, whereas control animals demonstrated growth at greater than 105 organisms by day 2 . Subeschar muscle cultures also demonstrated significantly less bacterial invasion compared with controls on each day tested . D2A21-treated animals had an 85.7 percent survival compared with 0 percent survival in controls . Furthermore, the D2A21-treated groups demonstrated maintenance of body weights, whereas controls had significant weight loss with time . In conclusion, D2A21 demonstrates significant antibacterial activity against Pseudomonas, sterilizing burn eschar and decreasing subeschar bacterial load, allowing for a markedly significant improvement in survival in this infected burn-wound model.

Curr Opin Infect Dis, 2002 Apr, 15(2), 175 - 82
Cystic fibrosis; Brennan AL et al.; Cystic fibrosis is the most common lethal inherited disorder with autosomal recessive inheritance . Major progress has been made in understanding the molecular mechanisms leading to increased susceptibility to Pseudomonas aeruginosa colonization . Persistent respiratory infection with P . aeruginosa leads to progressive pulmonary inflammation and is the major cause of morbidity and mortality . Treatment and prophylaxis of respiratory infection has improved the median survival and quality of life of cystic fibrosis patients . In the future, treatment of the underlying genetic defect may be possible.

Curr Opin Infect Dis, 2001 Aug, 14(4), 403 - 7
Pseudomonas aeruginosa infections in cancer patients: have they gone away?
Bodey GP.
Pseudomonas aeruginosa infection continues to be a threat to cancer patients, especially if they are neutropenic . As many as 50% of infections are community acquired . Prompt, effective therapy results in cures in about 80% of patients, although the presence of shock or pneumonia indicates a poor prognosis . Antibiotic resistance is an increasing problem.

Biophys J, 2002 May, 82(5), 2758 - 66
Role of the coordinating histidine in altering the mixed valency of Cu(A): an electron nuclear double resonance-electron paramagnetic resonance investigation; Lukoyanov D et al.; The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46 . The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core . For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change . Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120 . The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation . On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper . The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation . To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.

Biophys J, 2002 May, 82(5), 2645 - 51
Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed; Pozdnyakova I et al.; Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold . Removal of copper produces an apoprotein with the same structure as holoazurin . To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins . The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins . The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect . Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin . In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin . We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.

Appl Biochem Biotechnol, 2001 Spring, 91-93, 459 - 67
Production of biosurfactant from a new and promising strain of Pseudomonas aeruginosa PA1; Santa Anna LM et al.; The Pseudomonas aeruginosa PA1 strain, isolated from the water of oil production in Sergipe, Northeast Brazil, was evaluated as a potential rhamnolipid type of biosurfactant producer . The production of biosurfactants was investigated using different carbon sources (n-hexadecane, paraffin oil, glycerol, and babassu oil) and inoculum concentrations (0.0016-0.008 g/L) . The best results were obtained with glycerol as the substrate and an initial cell concentration of 0.004 g/L . A C:N ratio of 22.8 led to the greatest production of rhamnolipids (1700 mg/L) and efficiency (1.18 g of rhamnolipid/g of dry wt).

Presse Med, 2002 Mar 23, 31(11), 498 - 502
{Risk factors for the acquisition of Pseudomonas aeruginosa in a surgical intensive care unit}; Duchamp CB et al.; OBJECTIVES: To investigate the risk factors for the acquisition (infection and/or colonization) of Pseudomonas aeruginosa, which is frequently associated with nosocomial infections, in a surgical intensive care unit in Dijon . METHOD: A retrospective case-control study was performed on 57 cases matching with 114 controls, between December 1996 and February 1999 . The statistical method used was a conditional multiple logistic regression model . RESULTS: Three groups of variables were studied (patient characteristics--invasive procedures--previous administration of antibiotics) . The multiple logistic regression analysis confirmed 3 risk factors: duration of sedation, infection with another bacteria and cranio-encephalic trauma . These factors are commonly involved in nosocomial infections . CONCLUSION: This study confirms the interest of infection control measures and the prevention of nosocomial infections, especially in cranio-encephalic trauma . The hypothetical relationship between acquisition of Pseudomonas aeruginosa and previous administration of antibiotics was not confirmed . A more powerful study would perhaps specify this relationship.

J Biol Chem, 2002 Jul 5, 277(27), 24490 - 8 Epub 2002 Apr 16.
Pseudomonas aeruginosa aspartate transcarbamoylase . Characterization of its catalytic and regulatory properties; Vickrey JF et al.; Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function . The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity . Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli . The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized . Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex . As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates . In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates . The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme . Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound . The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.

Antimicrob Agents Chemother, 2002 May, 46(5), 1288 - 94
Characterization and movement of the class 1 integron known as Tn2521 and Tn1405; Partridge SR et al.; Two putative transposons, Tn2521 and Tn1405, carrying determinants for the PSE-4 beta-lactamase and for resistance to streptomycin, spectinomycin, and sulfonamides were previously isolated from the chromosome of Pseudomonas aeruginosa Dalgleish . Detailed mapping and determination of the complete sequence of Tn2521 revealed that it is a class 1 integron, here renamed In33, with a backbone structure identical to that of In4 from Tn1696 . In33 contains two gene cassettes, blaP1 and aadA1, replacing the aacC1-orfE-aadA2-cmlA1 cassette array in In4 . Although In33 does not include any transposition genes, movement of In33 (Tn2521) targeted to a single location in the IncP-1 plasmid R18-18 has been reported previously (M . I . Sinclair and B . W . Holloway, J . Bacteriol . 151:569-579, 1982) . A 5-bp duplication of the target, which lies within the res site recognized by the ParA resolvase of R18-18, was present, indicating that the mechanism of movement was transposition . Together, these data indicate that class 1 integrons that are defective in self-transposition can move under appropriate circumstances . The Tn1405 isolate studied was found to represent only the cassette array of In33, which had replaced the cassette array in the recipient plasmid R388, probably by homologous recombination.

Neurochem Res, 2002 Apr, 27(4), 305 - 12
A new hypothalamic polypeptide is a regulator of myelopoiesis; Galoyan AA et al.; The effect of hypothalamic proline-rich polypeptide (PRP) against Pseudomonas aeruginosa infection in BALB/c mice with leukopenia was investigated . Mice were treated with cyclophosphamide (CPA) and were then injected with PRP 24 h after CPA treatment . The lethal doses of P . aeruginosa were injected to mice when the number of peripheral blood leukocytes reached a nadir on day 5 after CPA administration . The administration of PRP significantly increased the survival of infected mice, and had a pronounced protective effect during the period of development of the infection . The number of bacteria in internal organs of PRP-treated mice was significantly lower than that in control mice . In PRP-treated mice, the neutrophil levels in peripheral blood started to increase 7 days after CPA administration and were consistently higher, and they were more mature than those in controls . Our results may indicate the ability of PRP to stimulate recovery of myelopoiesis and enhance mature neutrophil function.

J Infect Chemother, 2002 Mar, 8(1), 99 - 102
Destructive pulmonary embolism in a patient with community-acquired staphylococcal bacteremia; Miyashita T et al.; We report a 17-year-old man with destructive pulmonary embolism caused by Staphylococcus aureus bacteremia . The patient was not immunocompromised and had neither underlying diseases nor risk factors, such as concomitant influenza viral infection, which exacerbate staphylococcal infections . The rapid and extensive progression of pulmonary involvement in all lung fields make this a rare case; there have been few reports in the literature describing a similar radiographic appearance in patients with community-acquired staphylococcal bacteremia . In-vitro studies did not demonstrate the production of enterotoxins or toxic shock syndrome toxin 1 (TSST-1) by the isolated strain, but genetic analysis detected Panton-Valentine leukocidine gene from the strain . Subsequent empyema with bilateral pneumothorax was prolonged because of superinfection with both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa . Optional surgical treatments, including thoracostomy and thoracopneumoplasty, finally improved his condition.

J Infect Chemother, 2002 Mar, 8(1), 37 - 42
Effectiveness of fosfomycin combined with other antimicrobial agents against multidrug-resistant Pseudomonas aeruginosa isolates using the efficacy time index assay; Okazaki M et al.; We investigated the effectiveness of fosfomycin combined with other antibiotics, such as piperacillin, cefepime, ceftazidime, imipenem, meropenem, aztreonam, gentamicin, or levofloxacin, against 30 Pseudomonas aeruginosa strains, including multidrug-resistant strains, isolated from clinical specimens, using the efficacy time index (ETI) assay . The assay refers to the result of pharmacokinetics obtained from adult men volunteers, and yields an ETI to evaluate the effect of a combination of antimicrobial agents . With the ETI, based on serum concentration 3 h after the administration of two antimicrobial agents, the effectiveness of antimicrobial combinations was evaluated as follows: poor, ETI < 0.5; fair, 0.5 < or = ETI < 1; good, 1 < or = ETI < 8; and excellent, ETI > or = 8 . The combination of fosfomycin and cefepime (efficacy rate {excellent plus good}, 76.7%) and fosfomycin/aztreonam (efficacy rate, 76.7%) appeared to be the most effective, followed by fosfomycin/meropenem (efficacy rate, 76.6%), fosfomycin/imipenem (efficacy rate, 73.3%), fosfomycin/ceftazidime (efficacy rate, 70%), fosfomycin/gentamicin (efficacy rate, 70%), fosfomycin/piperacillin (efficacy rate, 66.7%), and fosfomycin/levofloxacin (efficacy rate, 66.7%) . Fosfomycin/cefepime, fosfomycin/aztreonam, and fosfomycin/meropenem may be clinically useful in selected patients, particularly for P . aeruginosa . The ETI assay provided information on the minimum inhibitory concentration (MIC) of many pairs of combined antimicrobial agents simultaneously . The ETI assay may be a useful technique with which to investigate the effect of combinations of antimicrobial agents against P . aeruginosa, including multidrug-resistant strains.

Am J Respir Crit Care Med, 2002 Apr 15, 165(8), 1176 - 81
Prolonged inflammatory response to acute Pseudomonas challenge in interleukin-10 knockout mice; Chmiel JF et al.; Cystic fibrosis (CF) lung disease is characterized by a neutrophilic infiltrate that is excessive relative to the burden of infection . Decreased interleukin-10 in CF airways may impair proper termination of inflammation, leading to persistence of neutrophils after acute infections have been cleared . This could explain reports of lung inflammation in the absence of bacteria in infants with CF . We evaluated the kinetics of inflammation after transient Pseudomonas aeruginosa challenge in IL-10 knockout (KO) and wild-type (WT) mice . Both types of mice cleared the infection by Day 6 (p > or = 0.29) . However, IL-10 KO mice had more neutrophils in bronchoalveolar lavage fluid than did WT mice on Days 4 (p < 0.0001), 6 (p < 0.0001), and 8 (p = 0.042) . IL-10 KO mice had high concentrations of proinflammatory cytokines in BAL on Days 2 and 4, with some cytokines detectable on Days 6 and 8, whereas cytokines in BAL from WT mice were greatest on Day 2 and undetectable by Day 4 . Moreover, IL-10 KO mice failed to regenerate IkappaBalpha once degraded and subsequently had prolonged activation of NF-kappaB . These data suggest that IL-10 deficiency contributes to prolonged inflammatory responses early in CF, when infection may be transient.

Zhonghua Yan Ke Za Zhi, 2002 Jan, 38(1), 8 - 12
{The variance of pathogenic organisms of purulent ulcerative keratitis}; Zhang W et al.; OBJECTIVE: To identify the variance of pathogens of purulent keratitis and their resistance patterns to antibiotics . METHODS: From January 1995 to October 2000, all patients with suspected infectious corneal ulcer were examined by bacterial, fungal and Acanthameoba culture . The results of microorganism-culture were compared with those in literature . RESULTS: Of the specimens in 1 430 cases, there were 790 cases (55.2%) with positive culture including bacterial isolates in 258 cases (18.0%), fungal isolates in 498 cases (34.8%) and parasites in 34 cases (2.4%) . The Staphylococcus aureous and Pseudomonas aeruginosa that had markedly declined since 1980s represented 6.2% and 21.7% of bacterial isolates respectively . However, the opportunity organism such as Staphylococcus epidermidis represented 27.5%, a tendency of gradual increase . The most common fungal pathogen was fusarium spp that represented 64.5% of all positive fungal cultures, and Aspergillus spp (13.6%) occupied the second place . The resistance of the ocular bacteria to fluoroquinolones gradually increased . Although the Pseudomonas aeruginosa was resistant to gentamycin in 36.2%, it was almost no resistance to tobramycin (3.4%) . The fusarium spp was shown to be resistant to a number of the anti-fungal agents, but it is sensitive to natamycin . CONCLUSION: The survey of pathogens in purulent ulcerative keratitis and their resistance patterns to antibiotics helps in clinical treatment.

Biochem J, 2002 Aug 1, 365(Pt 3), 731 - 8
Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity; Novo C et al.; The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism . A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template . The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit . Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads . Support for the structure for the P . aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

Vet Radiol Ultrasound, 2002 Mar-Apr, 43(2), 178 - 82
An infected hip prosthesis in a dog diagnosed with a 99mTc-ciprofloxacin (infecton) scan; Peremans K et al.; This case report describes the use of the 99mTc-labeled radiopharmaceutical ciprofloxacin (Infecton) in a case of hip prosthesis loosening in a dog . Serial planar radiographs were not conclusive, and culture of the synovial fluid was negative . Antibiotic treatment did not result in improvement of the lameness . Scintigraphy was performed with 99-Tc-Infecton, a tracer claimed to be specific for infection . Antibiotic treatment was interrupted 6 weeks prior to the examination . Planar and tomographic images at 3 h and at 24 h postinjection showed increased activity along the acetabulum and the proximal femoral bone surrounding the femoral prosthesis, indicating focal infection . Bacteriology performed after removal of the implant revealed Pseudomonas aeruginosa.

Saudi Med J, 2002 Apr, 23(4), 396 - 9
An in-vitro study of the effects of various disinfectants on prosthetic and surface materials; Bahannan SA et al.; OBJECTIVE: This study assessed the effect of various disinfectants on several contaminated prosthetic and surface-covering materials . METHODS: The efficacy of 6 disinfectants used at King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, on prosthetic and surface-covering materials, irreversible hydrocolloid and elastomer impression materials, wax, acrylic resin, metal, bench-covering material, and floor carpet . These materials were contaminated with Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus . Counts of viable bacteria on the materials was determined by incubated replica plating on blood agar plates at 5 minute intervals . A 3 way non parametric analysis of variance was used to evaluate the main effects and interactions of the disinfectants, bacteria, and materials . RESULTS: Statistical analysis showed that material, type of disinfectant, and interactions between material and bacteria were significant . Carpet has a significantly higher bacterial count than many other items (P < 0.0001) such as acrylic resin, irreversible hydrocolloid, chrome-cobalt casting, and laminated bench surfaces . CONCLUSION: Quaternary ammonia compound and the tertiary ammonia phenol were the most effective disinfectants . Efficacy of the disinfectant depends partly on the bacteria used for contamination . Carpets in dental clinics showed high potential to retain microorganisms.

J Pediatr, 2002 Mar, 140(3), 299 - 305
Antibiotic prophylaxis in infants and young children with cystic fibrosis: a randomized controlled trial; Stutman HR et al.; OBJECTIVES: To evaluate whether antistaphylococcal prophylaxis in infants and young children with cystic fibrosis (CF) would suppress the acquisition of Staphylococcus aureus and delay the onset of the manifestations of bronchopulmonary disease . STUDY DESIGN: A 7-year, multicenter, double-blind, placebo-controlled study of continuous antistaphylococcal therapy . Otherwise healthy children <2 years of age with CF were randomly assigned to be treated with daily cephalexin (80-100 mg/kg/day) or placebo . Clinical, microbiologic, laboratory, radiographic, and anthropometric outcomes were evaluated . RESULTS: Of 209 children enrolled, 119 completed a 5- to 7-year course of therapy . Mean age at enrollment was 15.6 and 14.1 months in the cephalexin and placebo groups, respectively . Respiratory cultures from children treated with cephalexin were significantly less likely to be positive for S aureus (6.0% vs 30.4%; P <.001) . They were, however, much more likely to be positive for Pseudomonas aeruginosa (25.6% vs 13.5%; P <.009) . These differences became apparent in the first year after enrollment and persisted over the duration of the study . In contrast to these microbiologic differences, there were no differences in clinical outcome measures, including radiographic (Brasfield score, 23.4 vs 23.2) or anthropometric scores or pulmonary function . CONCLUSIONS: Although long-term prophylaxis with cephalexin successfully delayed the acquisition of S aureus, it enhanced colonization with P aeruginosa and did not lead to clinically significant improvement in major health outcomes . These data do not support routine antistaphylococcal prophylaxisin otherwise healthy infants and young children with CF.

Mol Microbiol, 2002 Mar, 43(6), 1641 - 50
The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants; Oliver A et al.; We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients . In four out of 11 independent P . aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P . aeruginosa PAO1 . Here, we report the cloning and sequencing of two additional P . aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P . aeruginosa mismatch repair genes (mutL and uvrD) . We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P . aeruginosa strains isolated from CF patients . Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD) . In four cases (three mutS and one mutL), the genes contained frameshift mutations . The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats . This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation . The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity . Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity . Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair . The results shown here indicate that the putative P . aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.

Chest, 2002 Apr, 121(4), 1149 - 54
Noninvasive positive-pressure ventilation vs . conventional oxygen supplementation in hypoxemic patients undergoing diagnostic bronchoscopy; Antonelli M et al.; OBJECTIVE: We have reported previously on the use of noninvasive positive-pressure ventilation (NPPV) to assist spontaneous breathing in high-risk hypoxemic patients (i.e., PaO(2)/fraction of inspired oxygen {FIO(2)} ratio, < or = 100) who are undergoing diagnostic fiberoptic bronchoscopy (FOB) . The efficacy of this intervention in patients with less severe forms of hypoxemia (i.e., PaO(2)/FIO(2) ratio, < 200) is unknown . PATIENTS AND METHODS: Twenty-six patients with PaO(2)/FIO(2) ratios < or = 200 who required bronchoscopic BAL for suspected nosocomial pneumonia were entered into the study . Thirteen patients were randomized during FOB to receive NPPV, and 13 patients were randomized to receive conventional oxygen supplementation by Venturi mask . The primary end points were changes in the PaO(2)/FIO(2) ratio during FOB and within 60 min of terminating the procedure . RESULTS AND OUTCOME: At study entry, the two groups were similar in terms of age, simplified acute physiologic score II values, and cardiorespiratory parameters . During FOB, the mean (+/- SD) PaO(2)/FIO(2) ratio increased by 82% in the NPPV group (261 +/- 100 vs 139 +/- 38; p < 0.001) and decreased by 10% in the conventional oxygen supplementation group (155 +/- 24 to 139 +/- 38; p = 0.23) . Sixty minutes after undergoing FOB, the NPPV group had a higher mean PaO(2)/FIO(2) ratio (176 +/- 62 vs 140 +/- 38; p = 0.09), a lower mean heart rate (91 +/- 18 vs . 108 +/- 15 beats/min; p = 0.02), and no reduction in mean arterial pressure in comparison to a 15% decrease from the baseline in the control group . One patient in the NPPV group and two patients in the control group required nonemergent intubation . Major bacterial isolates included Staphylococcus aureus (7 of 30 isolates; 23%) and Pseudomonas aeruginosa (12 of 30 isolates; 40%) . CONCLUSION: In patients with severe hypoxemia, NPPV is superior to conventional oxygen supplementation in preventing gas-exchange deterioration during FOB with better hemodynamic tolerance.

Curr Pharm Des, 2002, 8(9), 695 - 702
Tailoring an antibacterial peptide of human lysosomal cathepsin G to enhance its broad-spectrum action against antibiotic-resistant bacterial pathogens; Shafer WM et al.; Neutrophils contain several cationic antimicrobial proteins or peptides (CAPs) that exert antibiotic-like action against bacteria . These host-derived antibiotics kill susceptible bacteria by oxygen-independent mechanisms . Considerable interest in their activity has been generated in recent years due not only to their likely important role in innate host defense against infection, but also their possible use as therapeutic agents in treating infections caused by antibiotic-resistant pathogens . We have studied the antibacterial properties of human lysosomal cathepsin G (cat G) . This highly cationic serine protease contains at least three antibacterial regions that by themselves can exert antibacterial action against Gram-negative bacteria, such as Pseudomonas aeruginosa . Only one of these peptides, defined by residues 117-136 of full-length cat G, has bactericidal action against Gram-positive pathogens, such as Staphylococcus aureus . Due to the broad-spectrum antibacterial action of this peptide, we have sought to define the amino acids within its primary sequence required for this activity and have developed variants with improved activity . This review emphasizes the importance of both cationicity and hydrophobicity as necessary characteristics for the antibacterial action of CAPs . It also proposes the strategy that naturally occurring large human CAPs can be dissected to smaller CAPs and then modified to enhance their activity in vitro . This approach could prove beneficial to those interested in developing antimicrobial peptides as therapeutic agents.

Microbiol Immunol, 2002, 46(2), 75 - 81
Antibacterial properties of antimicrobial-finished textile products; Takai K et al.; The antibacterial properties of five kinds of antimicrobial-finished textile products (AFTPs) were examined against Staphylococcus aureus, including methicillin-resistant (MRSA) strains and Pseudomonas aeruginosa, under wet and dry conditions . Textile products containing Ag . Zn . ammonium Zeolite and chitosan were found to be effective against methicillin-sensitive S . aureus (MSSA) for up to 6 hr of incubation under wet and dry conditions, and effective against MRSA for up to 24 hr of incubation only under wet conditions . Under dry conditions, however, all AFTPs were ineffective against one MRSA strain . When organic matter was added to the incubation mixture, textile products containing Ag . Zn . ammonium Zeolite and chitosan still showed antibacterial activities, but not as strongly . The results of this study suggested the following: (1) There are differences in antibacterial properties among commercially available AFTPs; (2) Determining effectiveness requires several hours of incubation; (3) Water content as an environmental factor can affect effectiveness; and (4) Some bacterial species and strains are not affected by AFTPs . The antibacterial properties of AFTPs in the clinical setting may be of limited value.

Cell Biochem Biophys, 2002, 36(1), 19 - 40
A pH dependence study on the unfolding and refolding of apoazurin: comparison with Zn(II) azurin; Hansen JE et al.; Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a beta-sheet protein arranged into a single domain . Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process . The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains . However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be -27.8 +/- 2.4 kJ mol(-1) . From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1 +/- 6.9 kJ/mol and 12.9 +/- 8.1 kJ/mol, respectively . It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity-in particular, from the presence of Zn(II) azurin . We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5 . The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH . We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains . When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion . Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.

J Biol Chem, 2002 Jun 14, 277(24), 21768 - 75 Epub 2002 Apr 05.
Salicylate biosynthesis in Pseudomonas aeruginosa . Purification and characterization of PchB, a novel bifunctional enzyme displaying isochorismate pyruvate-lyase and chorismate mutase activities; Gaille C et al.; Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of the pchB gene, was purified to homogeneity from Pseudomonas aeruginosa . In the reaction catalyzed by this enzyme, isochorismate --> salicylate + pyruvate, no cofactors appear to be required . At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent K(m) of 12.5 microm for isochorismate and a kcat of 106 min(-1), calculated per monomer . The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments . PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs) . Chorismate was shown to be converted to prephenate by purified PchB in vitro, with an apparent K(m) of 150 microm and a kcat of 7.8 min(-1) . An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB . Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P . aeruginosa pchB gene for growth in minimal medium . A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity . In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity.

Chem Phys Lipids, 2002 Feb, 114(2), 181 - 91
Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01; Nieuwenhuizen WF et al.; Ceramidase (CDase) hydrolyses the N-acyl linkage of the sphingolipid ceramide . We synthesized the non-fluorescent ceramide analogue (4E,2S,3R)-2-N-(10-pyrenedecanoyl)-1,3,17-trihydroxy-17-(3,5-dinitrobenzoyl)-4-heptadecene (10) that becomes fluorescent upon hydrolysis of its N-acyl bond . This novel substrate was used to study several kinetic aspects of the recombinant CDase from the pathogenic bacterium Pseudomonas aeruginosa PA01 . Maximum CDase activity was observed above 1.5 microM substrate, with an apparent K(m) of 0.5+/-0.1 microM and a turnover of 5.5 min(-1) . CDase activity depends on divalent cations without a strong specificity . CDase is inhibited by sphingosine and by several sphingosine analogues . The lack of inhibition by several mammalian CDase inhibitors such as D-erythro-MAPP, L-erythro-MAPP or N-oleoylethanolamine points to a novel active site and/or substrate binding region . The CDase assay described here offers the opportunity to develop and screen for specific bacterial CDase inhibitors of pharmaceutical interest.

Presse Med, 2002 Mar 9, 31(9), 393 - 9
{Are the principles of treatment of chronic osteitis applicable to the diabetic foot?}; Senneville E et al.; OBJECTIVE: The interest of the management of bone infections in the diabetic foot, inspired by the recommendations for the treatment of chronic osteitis, was assessed in this study . METHODS: Twenty bone infections in 17 diabetic patients with moderate to mild infections of the feet were confirmed by the results of X-ray and/or scintigraphic studies and bone surgery biopsy cultures revealing one or more bacteria sensitive to standard osteitis treatment (rifampicine + fluoroquinolone) . The patients had received this treatment per os for a median duration of 6 months (3 to 10 months) . Clinical follow-up was carried out during a consultation at 1, 3 and 6 months during treatment and then by telephone every six months after the end of treatment . Clinical success was defined as the disappearance of any local sign of infection and by the absence of relapse during the post-treatment follow-up period . The evolution of the bone infection was also assessed by the results of a control conducted 3 to 6 months after initiation of the antibiotic treatment . RESULTS: At the end of the treatment, all signs of infection had disappeared in 15/17 patients (88.2%) and no relapse had occurred in 14 (82.3%) patients at the end of a median post-treatment period of 22 months (12 to 41 months) . Resection of necrotic bone was performed at the same time as the bone biopsy in 2 patients . The median duration of hospitalisation was of 14 days (3 to 53 days) . During the study, a multi-resistant germ was isolated in 4 patients (1 Pseudomonas aeruginosa, 3 Staphylococcus aureus) . During the post-treatment follow-up, 3 patients dies from causes unrelated to the infection treated . No serious adverse event was reported during the study . DISCUSSION: The results of this pilot study support the rationale of applying the treatment regimens of chronic osteitis to diabetic lesions of the feet, but are only applicable to comparable patients presenting with non-severe lesions of the feet . Moreover, the use of antibiotics with potent selection of resistance such as rifampicine and fluoroquinolone, requires that bone biopsies be taken, which is not easy in all the diabetic foot care centres . We are presently conducting a study to identify the sub-populations of diabetic patients who could benefit from such treatment.

Microbiology, 2002 Apr, 148(Pt 4), 1161 - 9
Influence of extracellular polymeric substances on deposition and redeposition of Pseudomonas aeruginosa to surfaces; Gomez-Suarez C et al.; In this study, the role of extracellular polymeric substances (EPS) in the initial adhesion of EPS-producing Pseudomonas aeruginosa SG81 and SG81R1, a non-EPS-producing strain, to substrata with different hydrophobicity was investigated . The release of EPS by SG81 was concurrent with a decrease in surface tension of a bacterial suspension from 70 to 45 mJ m(-2) that was absent for SG81R1 . Both strains adhered faster and in higher numbers to a hydrophilic than to a hydrophobic substratum, but the initial deposition rates and numbers of adhering bacteria in a stationary-end point were highest for the non-EPS-producing strain SG81R1, regardless of substratum hydrophobicity . Both strains adhered less to substrata pre-coated with isolated EPS of strain SG81 . Furthermore, it was investigated whether bacteria, detached by passing air-bubbles, had left behind 'footprints' with an influence on adhesion of newly redepositing bacteria . Redeposition on glass was highest for non-EPS-producing SG81R1 and decreased linearly with the number of times these cycles of detachment and deposition were repeated to become similar to the redeposition of SG81 after six cycles . This indicates that P . aeruginosa SG81 leaves the substratum surface nearly completely covered with EPS after detachment, while SG81R1 releases only minor amounts of surface active EPS, completely covering the substratum after repeated cycles of detachment and adhesion . Atomic force microscopy showed a thick and irregular EPS layer (up to 32 nm) after the first detachment cycle of EPS-producing strain SG81, whereas the putatively non-EPS-producing strain SG81R1 left a 9 nm thin layer after one cycle . X-ray photoelectron spectroscopy indicated that the bacterial footprints consisted of uronic acids, the prevalence of which increased with the number of detachment and deposition cycles.

Microbiology, 2002 Apr, 148(Pt 4), 923 - 32
Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1; Reimmann C et al.; Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules ('autoinducers') of the N-acylhomoserine lactone (AHL) type . Two Bacillus sp . isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria . From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain . Expression of the aiiA homologue from isolate A24 in P . aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone . This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming . However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface . In conclusion, introduction of an AHL degradation gene into P . aeruginosa could block cell-cell communication and exoproduct formation, but failed to interfere with surface colonization.

Acta Microbiol Pol, 2001, 50(3-4), 311 - 4
Adherence of Pseudomonas aeruginosa strains to solid surfaces; Wolska K et al.; The aim of this study was to evaluate adherence of 59 strains of Pseudomonas aeruginosa to nitric-acid cleansed glass surfaces . There were differences in adherence between the investigated strains . The highest adherence was noticed among human strains (the average percentage was 13.3 +/- 7.51%) and the lowest adherence was determined among swine strains (the average percentage amounted 6 +/- .37%) . We conclude that strains of Pseudomonas aeruginosa isolated from humans colonise glass surfaces better than strains isolated from animals.

Perit Dial Int, 2002 Jan-Feb, 22(1), 27 - 31
Colonization-resistant antimicrobial-coated peritoneal dialysis catheters: evaluation in a newly developed rat model of persistent Pseudomonas aeruginosa peritonitis; Finelli A et al.; OBJECTIVE: Development of a rat model of persistent peritonitis and evaluation of the ability of liposomal ciprofloxacin hydrogel-coated silicone to resist colonization . DESIGN: A newly developed model of persistent Pseudomonas aeruginosa peritonitis to compare the ability of liposomal ciprofloxacin hydrogel (LCH)-coated silicone versus plain silicone for resistance to bacterial colonization . ANIMALS: Male Sprague-Dawley rats . RESULTS: Inoculating the peritoneum of rats with 1 mL 0.5% agar containing 10(6) colony-forming units (cfu)/mL P . aeruginosa in the presence of a plain silicone coupon resulted in persistent peritonitis for at least 7 days . Plain silicone coupons in all 40 rats were colonized (median 2.54 x 10(3) cfu/cm2; range 5.0 x 10(1)-1.0 x 10(6) cfu/cm2) and peritoneal washings were consistently culture-positive . In contrast, the LCH coupons removed after 7 days from the 40 test rats were sterile, as were the peritoneal washings, and there was no evidence of peritonitis . Blood cultures were negative in both groups . CONCLUSIONS: Liposomal ciprofloxacin hydrogel-coated silicone resists colonization in this rat model of persistent P . aeruginosa peritonitis.

JAMA, 2002 Apr 3, 287(13), 1716 - 21
Inhibition of intestinal epithelial apoptosis and survival in a murine model of pneumonia-induced sepsis; Coopersmith CM et al.; CONTEXT: Increased intestinal epithelial apoptosis is present in both human autopsy studies and animal models of sepsis . Whether altering gut apoptosis decreases mortality in sepsis induced by pathogenic bacteria outside the gut is unknown . OBJECTIVE: To determine if decreasing levels of intestinal cell death improves survival in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis . DESIGN AND MATERIALS: Prospective study in which transgenic mice that overexpress the antiapoptotic protein Bcl-2 in their intestinal epithelium (n = 25) and control littermates (n = 26) were subjected to intratracheal injection of P aeruginosa . MAIN OUTCOME MEASURES: Survival at 7 postoperative days, compared between the 2 groups . Secondary outcomes included quantification of gut epithelial apoptosis . RESULTS: Survival in transgenic mice that overexpress Bcl-2 in the intestinal epithelium was 40% (10/25) compared with 4% (1/26) in control littermates 7 days after intratracheal injection of P aeruginosa (P =.001), with differences in survival noted within 24 hours of surgery . Overexpression of Bcl-2 was associated with a decrease in gut epithelial apoptosis demonstrated by active caspase 3 staining, the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay, and hematoxylin-eosin staining . CONCLUSIONS: In this murine model, inhibiting gut epithelial apoptosis by overexpression of Bcl-2 was associated with a survival advantage in P aeruginosa pneumonia-induced sepsis . These results suggest that intestinal epithelial apoptosis may play a role in sepsis-related mortality.

Comp Med, 2001 Feb, 51(1), 75 - 9
Therapeutic effect of a pig-derived peptide antibiotic on porcine wound infections; Ceccarelli AV et al.; PURPOSE: We investigated the therapeutic potential of the pig-derived antimicrobial peptide protegrin-1 (PG-1) against porcine skin wounds infected with Pseudomonas aeruginosa . MATERIALS AND METHODS: Using a porcine skin wound model, PG-1 was added to the wound fluid either at the time of P . aeruginosa inoculation, four hours after inoculation or 24 hours after inoculation . Wound fluids were analyzed 20-24 hours later by use of colony-forming unit (CFU) assays, semiquantitative immunoblot analysis for PG-1, and radial diffusion assays (RDA) for residual in vitro activity . RESULTS: Results of the CFU assays indicated a 10,000-fold decrease in the number of bacteria when PG-1 was added at the time of inoculation, a 120-fold decrease when added 4 hours after inoculation and a 10-fold decrease when added 24 hours after inoculation . Results of immunoblot analysis and RDA indicated that PG-1 concentrations for each of the three conditions remained increased in wound fluid 20 to 24 hours after treatment, and correlated with increased residual in vitro antimicrobial activity . CONCLUSIONS: These results document that the endogenous antibiotic PG-1 significantly prevented the colonization of P . aeruginosa in wounds and reduced the in vivo bacterial concentration in established wound infections . Therapeutics used in the same animal species from which they were derived are a promising means for preventing and treating localized infections.

Bull Math Biol, 2002 Mar, 64(2), 239 - 59
A mathematical model of partial-thickness burn-wound infection by Pseudomonas aeruginosa: quorum sensing and the build-up to invasion; Koerber AJ et al.; Pseudomonas aeruginosa remains a significant pathogen in burn-wound infection, its pathogenicity being associated with the production of a cocktail of virulence determinants which is regulated by a population-density-dependent mechanism termed quorum sensing . Quorum sensing is effected through the production and binding of signalling molecules . Here we present a mathematical model for the early stages of the infection process by P . aeruginosa in burn wounds which accounts for the quorum sensing system and for the diffusion of signalling molecules in the burn-wound environment . The results of the model and the effects of important parameters are discussed in detail . For example, the effect of the degradation rate of signalling molecules and its significance for anti-signalling therapies is discussed.

J Org Chem, 2002 Apr 5, 67(7), 2144 - 51
5-{4-(1-Hydroxyethyl)phenyl}-10,15,20-triphenylporphyrin as a probe of the transition-state conformation in hydrolase-catalyzed enantioselective transesterifications; Ema T et al.; 5-{4-(1-Hydroxyethyl)phenyl}-10,15,20-triphenylporphyrin (1a) and zinc porphyrin 1b were designed and synthesized to experimentally examine the validity of the transition-state model previously proposed for the lipase-catalyzed kinetic resolution of secondary alcohols . The lipases from Pseudomonas cepacia (lipase PS), Candida antarctica (CHIRAZYME L-2), Rhizomucor miehei (CHIRAZYME L-9), and Pseudomonas aeruginosa (lipase LIP) exhibited excellent enantioselectivity (E >100 at 30 degrees C) . Subtilisin Carlsberg from Bacillus licheniformis (ChiroCLEC-BL) also showed high enantioselectivity for 1a (E = 140 at 30 degrees C), and the thermodynamic parameters were determined: DeltaDeltaH = -6.8 +/- 0.8 kcal mol(-1), DeltaDeltaS = -13 +/- 3 cal mol(-1) K(-1) . Lipases and subtilisin showed R- and S-preference for 1, respectively . The mechanisms underlying the experimental observations are explained in terms of the transition-state models . The large secondary alcohol 1 is a powerful tool for investigating the conformation of the transition state of the enzyme-catalyzed reactions . The fact that 1 was resolved with high enantioselectivity strongly suggests that the gauche conformation, but not the anti conformation, is taken in the transition state, in agreement with the transition-state models involving the stereoelectronic effect.

Comp Med, 2001 Aug, 51(4), 341 - 8
The pig as a model for excisional skin wound healing: characterization of the molecular and cellular biology, and bacteriology of the healing process; Wang JF et al.; A pig model of wound healing was developed by excision of 2-cm-diameter full thickness skin in young Yorkshire pigs . The results indicated that wound re-epithelialization in this animal model took an average of 20 days . Analysis of cellular change was assessed by use of DNA quantification and determination of apoptotic cells in tissue sections . The results indicate that RNA and DNA contents paralleled each other throughout the healing process, and observed changes in the pattern of RNA and DNA content of the scar tissues were consistent with cell loss due to apoptosis in this model . Expression of mRNA for relevant genes was assessed by use of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, using porcine specific primer sets and RNA isolated from normal skin and specimens obtained at various times after wounding . The mRNA values for tumor necrosis factor-alpha (TNF-alpha), connective tissue growth factor (CTGF), insulin-like growth factor II (IGF-II), and decorin were significantly high at specific times after wounding, but mRNA values for the transcription factors (c-fos and c-jun) were significantly decreased . Quantitative bacteriologic results indicated that the total bacterial count in this animal model reached 10(9) colony-forming units (CFU)/g, with the highest value at post-wounding day 7, and Pseudomonas aeruginosa and Staphylocococci aureus were the most common bacteria detected in this model . Further definition of this model should identify unique points in the healing process, and such information could lead to development of therapeutic interventions to improve skin wound healing.

East Afr Med J, 2001 Oct, 78(10), 540 - 3
Septicaemia in high risk neonates at a teaching hospital in Ile-Ife, Nigeria; Adejuyigbe EA et al.; OBJECTIVES: To determine the incidence, predisposing factors, clinical features, bacteriological pattern and antibiotic sensitivity in septicaemia in high-risk newborns . DESIGN: A prospective study . SETTING: Neonatal unit, Ife State Hospital, a unit of the Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife, Nigeria . SUBJECTS: All newborns admitted with clinical features and/or risk factors suggestive of neonatal septicaemia from February 1994 to March 1995 . MAIN OUTCOME MEASURES: Culture results and mortality rates . RESULTS: The incidence of neonatal septicaemia among new born was 22.9 per 1000 livebirths . The predisposing perinatal factors were low socio-economic status, lack of antenatal care, maternal peripartum pyrexia and congenital malformations . Gram-positive bacteria were found to be the most prevalent causative organisms (59.4%) . Staphylococcus aureus (36.2%), Pseudomonas aeruginosa (18.8%) and Coagulase negative Staphylococcus (15.9%) were the commonest causes of septicaemia . Meningitis and UTI were associated diagnoses in 16.7% and 18.2% of the septicaemic babies, respectively . The bacterial isolates showed a high degree of in-vitro antimicrobial resistance . However, all the isolates were sensitive to ofloxacin . Amongst the commonly used antibiotics, gentamicin had the lowest resistance . The overall mortality rate was 33.3% . CONCLUSION: Improvement in the socio-economic status of the populace and availability of affordable antenatal care would reduce the incidence of neonatal septicaemia in Nigeria . Continuous surveillance in every unit, as well as close attention to preventive strategies would be necessary to reduce morbidity and mortality from neonatal septicaemia . We recommend the inclusion of gentamicin in the initial treatment of septicaemia in the neonatal unit of OAUTHC, Ile-Ife, Nigeria.

Pediatr Pulmonol, 2002 Apr, 33(4), 311 - 4
Hypersensitivity to inhaled TOBI following reaction to gentamicin; Spigarelli MG et al.; Cystic fibrosis (CF) is the most common autosomal-recessive disease in Caucasians . Colonization with Pseudomonas aeruginosa (P . aeruginosa) of the CF airways causes deterioration of pulmonary status . TOBI (Tobramycin solution for inhalation) is an inhaled antibiotic that can improve the pulmonary disease . We report on a 9-year old boy with CF who developed a rash following a course of IV gentamicin . The rash resolved after its discontinuation . However, the rash returned all over his body, with the start of inhalation of TOBI therapy . We desensitized the patient using escalating doses of inhaled TOBI . He tolerated the procedure well, and continues to be on TOBI 9 months after desensitization on a once-a-day regimen .

Pediatr Pulmonol, 2002 Apr, 33(4), 269 - 76
Effects of tobramycin solution for inhalation on global ratings of quality of life in patients with cystic fibrosis and Pseudomonas aeruginosa infection; Quittner AL et al.; In a previously published placebo-controlled trial, tobramycin solution for inhalation (TSI) was shown to improve lung function and other outcomes in patients with cystic fibrosis (CF) . The objectives of the current study were to examine the effects of TSI on global ratings of health-related quality of life (HRQOL) by patients (or their parents) and physicians blind to group assignment, and to determine whether any perceived benefits persisted over time . The global ratings of HRQOL in 520 patients with CF and chronic Pseudomonas aeruginosa infection were analyzed retrospectively . Patients were randomly assigned to receive 24 weeks of placebo or treatment with TSI 300 mg b.i.d., both administered in cycles of 28 days on drug (or placebo) followed by 28 days off, for a total of three cycles . After each on-drug cycle, patients or parents, and physicians, were asked to rate whether the patient's condition was better, unchanged, or worse . There was strong agreement between the paired patient/parent and physician global HRQOL ratings across the three cycles . Regression analyses demonstrated that patients in the TSI group were significantly more likely to report improvements in HRQOL than were patients in the placebo group . This effect was found to be both immediate (end of on-drug cycle 1) and delayed (end of subsequent on-drug cycles 2 and 3) (P < 0.05) . In addition, change in forced expired volume in 1 sec (FEV(1)) % predicted values was a significant predictor of improvement in HRQOL ratings by patients and parents . After controlling for change in FEV(1) % predicted, physician ratings showed significant improvement only at the end of cycle 1 . Finally, controlling for initial lung disease severity, longitudinal growth models revealed that patients on TSI and their physicians reported higher HRQOL ratings than did placebo patients and their physicians across the three cycles; however, the magnitude of this effect decreased over time . Results of this study provided consistent evidence that TSI was associated with improved global ratings of HRQOL completed by both patients or parents, and physicians . Although these results are promising, they are limited by the use of a single-item rating of health . Future studies of the effects of TSI should utilize a well-validated, disease-specific measure of HRQOL .

Mol Genet Genomics, 2002 Mar, 267(1), 38 - 44 Epub 2002 Feb 19.
Organization and activation of the late promoters of phiCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa; Alber J et al.; The late genes of the temperate phage of Pseudomonas aeruginosa are organized in an analogous fashion to the corresponding transcription units of the Escherichia coli P2 and P2-like phages . Sequence analysis of four putative late promoter regions, PP(phiCTX), PO(phiCTX), PV(phiCTX) and PF(phiCTX), reveals no similarity to sigma(70)-type promoters or promoter consensus sequences found in Pseudomonas, indicating the apparent need for a phage-encoded protein to control the expression of phiCTX late genes . To elucidate the mode of expression of the late genes, we fused the putative late promoter regions to the promoterless lacZalpha gene, which encodes the N-terminal part of beta-galactosidase as a reporter enzyme, in the promoter-probe vector pME4510 . The candidate transactivator gene orf34 was cloned into expression vector pHA10, to generate the plasmid pHA34 . The two recombinant plasmids were introduced together into E . coli XL1-Blue and P . aeruginosa PAO1S-Lac . Our results demonstrate that in phiCTX three late promoters (PP(phiCTX), PO(phiCTX), and PF(phiCTX)) are activated upon induction by IPTG in PAO1S-Lac carrying the cloned promoters and pHA34 . Deletions and base-pair substitutions obtained by PCR-mediated mutagenesis demonstrated that two conserved sequences, TTGTAG-N(9)-cTACAa and GcCGCGCGCGCGgC, are essential for effective late gene expression . Whereas the late promoters were active in P . aeruginosa, only weak beta-galactosidase activity was obtained in E . coli.

Clin Infect Dis, 2002 Apr 15, 34(8), 1047 - 54 Epub 2002 Mar 15.
Pseudomonas aeruginosa ventilator-associated pneumonia: comparison of episodes due to piperacillin-resistant versus piperacillin-susceptible organisms; Trouillet JL et al.; We sought to determine the epidemiological characteristics of patients in an intensive care unit (ICU) who developed ventilator-associated pneumonia (VAP) caused by piperacillin-resistant Pseudomonas aeruginosa (PRPA; n=34) or piperacillin-susceptible P . aeruginosa (PSPA; n=101) . According to univariate analysis, the factors associated with the development of PRPA VAP were presence of an underlying fatal medical condition, immunocompromised status, longer previous hospital stay, less-severe illness at the time of ICU admission, duration of mechanical ventilation before onset of VAP, number of classes of antibiotic received, and previous exposure to imipenem or fluoroquinolone . Multivariate logistic regression analysis identified the following significant independent factors: presence of an underlying fatal medical condition (odds ratio {OR}, 5.6), previous fluoroquinolone use (OR, 4.6), and initial disease severity (OR, 0.8) . We concluded that the clinical characteristics of patients who develop PRPA VAP differ from those of patients who develop PSPA VAP . Restricted fluoroquinolone use is the sole independent risk factor for PRPA VAP that is open to medical intervention.

Nat Immunol, 2002 Apr, 3(4), 354 - 9 Epub 2002 Mar 25.
Human Toll-like receptor 4 recognizes host-specific LPS modifications; Hajjar AM et al.; Lipopolysaccharide (LPS) is the principal proinflammatory component of the Gram-negative bacterial envelope and is recognized by the Toll-like receptor 4 (TLR4)-MD-2 receptor complex . Bacteria can alter the acylation state of their LPS in response to environmental changes . One opportunistic bacterium, Pseudomonas aeruginosa, synthesizes more highly acylated (hexa-acylated) LPS structures during adaptation to the cystic fibrosis airway . Here we show that human, but not murine, TLR4-MD-2 recognizes this adaptation and transmits robust proinflammatory signals in response to hexa-acylated but not penta-acylated LPS from P . aeruginosa . Whereas responses to lipidIVA and taxol are dependent on murine MD-2, discrimination of P . aeruginosa LPS structures is mediated by an 82-amino-acid region of human TLR4 that is hypervariable across species . Thus, in contrast to mice, humans use TLR4 to recognize a molecular signature of bacterial-host adaptation to modulate the innate immune response.

Int J Biol Macromol, 2002 Apr 8, 30(2), 105 - 11
Monomer composition and sequence of alginates from Pseudomonas aeruginosa; Schurks N et al.; Alginates from four strains of Pseudomonas aeruginosa, one mucoid strain isolated from a technical water system, one strain isolated from a patient with cystic fibrosis and two mutants of this strain with a defect which affects the O-acetylation of the extracellular alginate, have been isolated and analysed for monomer composition and sequence by 13C-nuclear magnetic resonance (NMR) spectroscopy . The detected contributions of different monomer triplets (triads) were compared with values expected from a statistical chain constitution based on the given monomer ratio . While a typical algal alginate presents a nearly statistical distribution of uronic acids in the polymer chain, a strong deviation from the statistical arrangement of mannuronate (M) and guluronate (G) was found in the alginate of the mucoid strains of P . aeruginosa, being most expressed for the triad MMM . This feature is partially lost in the alginate from the mutant strains, indicating that the O-acetylation is linked to a mechanism which takes influence on the chain sequence . The strong preference for MG-pairs in the parent strain of P . aeruginosa may be connected to a stronger binding of cations in the MG-vicinity.

Int J Biol Macromol, 2002 Apr 8, 30(2), 67 - 74
Rheological characteristics of microbial suspensions of Pseudomonas aeruginosa and Bacillus cereus; Al-Asheh S et al.; The rheological properties of the bacteria Pseudomonas aeruginosa and Bacillus cereus have been investigated . The apparent viscosity of the bacterial suspensions has been measured at different conditions . The results showed that the bacterial suspensions' apparent viscosity increased with increasing biomass concentration of each of these strains . The P . aeruginosa suspension followed shear thinning behavior while B . cereus suspension followed shear thickening behavior . The shear stress versus shear rate experimental data were best represented by the Herschel-Bulkley model . The apparent viscosity of the P . aeruginosa and B . cereus suspensions decreased with increasing temperature . The relationship between the apparent viscosity and the shearing time highlighted the rheopectic behavior of the suspensions used in this work.

J Biol Chem, 2002 May 31, 277(22), 19792 - 9 Epub 2002 Mar 21.
Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid; Jaffe EK et al.; Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis . 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS . We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA . Some variants of human PBGS (engineered to resemble E . coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity . The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA . Comparison of the crystal structures for E . coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA) . Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction . A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.

J Antimicrob Chemother, 2002 Apr, 49(4), 631 - 9
Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics; Loughlin MF et al.; Our objective was to determine whether strains of Pseudomonas aeruginosa can adapt to growth in increasing concentrations of the disinfectant benzalkonium chloride (BKC), and whether co-resistance to clinically relevant antimicrobial agents occurs . Attempts were made to determine what phenotypic alterations accompanied resistance and whether these explained the mechanism of resistance . Strains were serially passaged in increasing concentrations of BKC in static nutrient broth cultures . Serotyping and genotyping were used to determine purity of the cultures . Two strains were examined for cross-resistance to other disinfectants and antibiotics by broth dilution MIC determination . Alterations in outer membrane proteins and lipopolysaccharide (LPS) expressed were examined by SDS-PAGE . Cell surface hydrophobicity and charge, uptake of disinfectant and proportion of specific fatty acid content of outer and cytoplasmic membranes were determined . Two P . aeruginosa strains showed a stable increase in resistance to BKC . Co-resistance to other quaternary ammonium compounds was observed in both strains; chloramphenicol and polymyxin B resistance were observed in one and a reduction in resistance to tobramycin observed in the other . However, no increased resistance to other biocides (chlorhexidine, triclosan, thymol) or antibiotics (ceftazidime, imipenem, ciprofloxacin, tobramycin) was detected . Characteristics accompanying resistance included alterations in outer membrane proteins, uptake of BKC, cell surface charge and hydrophobicity, and fatty acid content of the cytoplasmic membrane, although no evidence was found for alterations in LPS . Each of the two strains had different alterations in phenotype, indicating that such adaptation is unique to each strain of P . aeruginosa and does not result from a single mechanism shared by the whole species.

Biochem Biophys Res Commun, 2002 Mar 29, 292(2), 513 - 8
Secondary-site mutation restores the transport defect caused by the transmembrane domain mutation of the xenobiotic transporter MexB in Pseudomonas aeruginosa; Yoneyama H et al.; It has been suggested that the MexB subunit of the MexAB-OprM efflux transporter of Pseudomonas aeruginosa exports xenobiotics in an energy-dependent manner . To investigate the role of the transmembrane segments (TMS) of MexB in the transporter activity, we isolated 24 spontaneous mutants showing hypersusceptibility to antibiotics . Among them, three mutations were located at TMS-3, TMS-4, and TMS-10 having amino acid substitution Leu376vPro, Gly397vVal, and Val928vGly, respectively . A secondary mutation, which suppressed the defect caused by the Val928vGly mutation in TMS-10, was found at the 403rd amino acid residue in TMS-4 with a change of glycine to serine, suggesting that TMS-4 and TMS-10 may be in close proximity . This result provided strong support for the recent notion that negatively charged residues in TMS-4 might form a salt-bridge with a positive charge in TMS-10 (Guan, L., and Nakae, T . (2001) J . Bacteriol . 183, 1734-1739) . The transporter function impaired by the Gly397vVal mutation in TMS-4 was recovered by the secondary mutation, Gln998vHis, in the loop between TMS-11 and TMS-12, thereby suggesting that TMS-4 and TMS-11 or TMS-12 might also be in close proximity . Thus, it is most likely that TMS-4, TMS-10, and TMS-11 or TMS-12 are packed close three dimensionally . (c)2002 Elsevier Science (USA).

Chemotherapy, 2002 Mar, 48(1), 31 - 5
Investigation of various antibiotic combinations using the E-Test method in multiresistant Pseudomonas aeruginosa strains; Kocazeybek B et al.; BACKGROUND: Antibiotic combinations are frequently used in order to obtain wide-spectrum effects in the treatment of serious infections such as septicemia and endocarditis, and also to produce an in vivo effect against strains which are defined as resistant to the known inhibiting or fatal dose of one antibiotic . The synergistic effects of combinations such as aminoglycoside + beta-lactam, aminoglycoside + quinolone and quinolone + beta-lactam on Pseudomonas aeruginosa have been revealed in different studies . The multiple resistance rate of nosocomial P . aeruginosa strains isolated from intensive care units (ICUs) has been reported as high in many studies . METHODS: In this study, the effects of various combinations of antibiotics (aminoglycoside + beta-lactam and aminoglycoside + quinolone) against 101 multiresistant P . aeruginosa strains which were isolated from the ICUs of three different hospitals in Istanbul were investigated using the E-test method . The combinations for which the highest synergistic effects were determined by the E-test method were also tested with the checkerboard method, i.e . in addition to the E-test method, in 19 of a total of 23 strains . RESULTS: When the synergistic results which were obtained with the combinations of aminoglycoside + beta-lactam were compared with those of the aminoglycoside + quinolone combinations, they were determined to be higher for the two aminoglycosides gentamicin (GM) and tobramycin (TM) . We determined the synergistic rates to be 23, 21, 19, 18, 16, 14, 10 and 10% for GM + ceftriaxone (TX), GM + piperacillin (PP), GM + ceftazidime (TZ), TM + PP, TM + TX, TM + TZ, GM + ciprofloxacin (CI) and TM + CI, respectively . The GM + TX combination - for which the highest synergistic effects were determined with the E-test stripes - was also determined as synergistic with the checkerboard method in 19 of a total of 23 strains (23%), and the agreement rate between the two methods was 100% (kappa > 0.7) . The highest synergistic effects against strains which were sensitive to both of the antibiotics which constitute the combinations were found for the GM + TX combination, as 50%, whereas for strains which were resistant to both of the antibiotics, this was found for the TM + PP combination, also as 50% . CONCLUSIONS: We consider that the minimal inhibitory concentration values of antibiotics are not sufficient alone in order to constitute a combination for multiresistant strains and it would be advisable to begin a treatment by applying a combination study . The E-test method has been evaluated as a good alternative for combination investigations because of its ease both of application and evaluation and also for its good agreement with the standard checkerboard method .

Infect Immun, 2002 Apr, 70(4), 2230 - 2
Membrane localization contributes to the in vivo ADP-ribosylation of Ras by Pseudomonas aeruginosa ExoS; Riese MJ et al.; Type III-delivered exoenzyme S (ExoS) preferentially ADP-ribosylated membrane-associated His(6)HRas, relative to its cytosolic derivative His(6)HRas Delta CAAX . This indicates that the subcellular protein distribution contributes to in vivo ADP-ribosylation by ExoS.

Infect Immun, 2002 Apr, 70(4), 2198 - 205
Pseudomonas aeruginosa ExoT acts in vivo as a GTPase-activating protein for RhoA, Rac1, and Cdc42; Kazmierczak BI et al.; The Pseudomonas aeruginosa protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases . Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells . We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases . GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other P . aeruginosa type III secreted proteins . We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.

Infect Immun, 2002 Apr, 70(4), 2187 - 97
Balance of pro- and anti-inflammatory cytokines correlates with outcome of acute experimental Pseudomonas aeruginosa keratitis; Thakur A et al.; The purpose of this study was to elucidate the expression of pro- and anti-inflammatory cytokines in mouse corneas infected with Pseudomonas aeruginosa . Three bacterial strains (invasive, cytotoxic, or CLARE {contact lens-induced acute red eye}) which have recently been shown to produce distinct patterns of corneal disease in the mouse were used . The left mouse (BALB/c) corneas were scarified and infected with 2 x 10(6) CFU of one of the three P . aeruginosa strains, while right eyes served as controls . Animals were examined at 1, 4, 8, 16, and 24 h with a slit lamp biomicroscope to grade the severity of infection . Following examination, eyes were collected and processed for histopathology, multiprobe RNase protection assay for cytokine mRNA, enzyme-linked immunosorbent assay to quantitate cytokine proteins, and myeloperoxidase activity to quantitate polymorphonuclear leukocytes . The kinetics of appearance and magnitude of expression of key cytokines varied significantly in the three different phenotypes of P . aeruginosa infection . The predominant cytokines expressed in response to all three phenotypes were interleukin-1 beta (IL-1 beta), IL-1Ra, and IL-6 . In response to the invasive strain, which induced severe corneal inflammation, significantly lower ratios of IL-1Ra to IL-1 beta were present at all time points, whereas corneas challenged with the CLARE strain, which induced very mild inflammation, showed a high ratio of IL-1Ra to IL-1 beta . The outcome of infection in bacterial keratitis correlated with the relative induction of these pro- and anti-inflammatory cytokines, and exogenous administration of recombinant rIL-1Ra (rIL-1Ra) was able to reduce the disease severity significantly . These findings point to the therapeutic potential of rIL-1Ra protein in possible treatment strategies for bacterial keratitis.

Infect Immun, 2002 Apr, 70(4), 1783 - 90
Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis; Erickson DL et al.; Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa . The chronic infections caused by P . aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality . As regulators of virulence determinants, P . aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF . We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF . We detected biologically active levels of N-(3-oxododecanoyl)-L-homoserine (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) in sputum from CF patients . Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF . We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF . We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system . Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.

Blood, 2002 Apr 1, 99(7), 2483 - 9
Ex vivo development of functional human lymph node and bronchus-associated lymphoid tissue; Tirouvanziam R et al.; We introduce a novel in vivo model of human mucosal immunity, based on the implantation of human fetal bronchial mucosa and autologous peribronchial lymph node (PLN) in the severe combined immunodeficiency (SCID) mouse . In the SCID host, human fetal bronchi implanted alone retain macrophages and mast cells but lose T cells . In contrast, fetal bronchi co-implanted with PLN contain, in addition to macrophages and mast cells, numerous T cells and B cells, often clustered in intramucosal bronchus-associated lymphoid tissue (BALT) . Functionally, bronchus-PLN cografts are able to mount robust alphabeta and gammadelta T-cell-mediated immune responses to Pseudomonas aeruginosa and 3,4-epoxy-3-methyl-1-butyl-diphosphate challenges . No other autologous lymphoid organ (bone marrow, thymus, liver) allows for BALT development in co-implanted bronchi, which suggests special ontogenetic and functional relations between extramucosal PLN and intramucosal BALT . Overall, the bronchus-PLN cograft appears as a promising model for human bronchial immune development and function . Our study is the first to document long-term ex vivo maintenance of functional human lymph nodes as native appendices to mucosal tissue . Our results, therefore, suggest a simple strategy for developing similar experimental models of human immune function in other mucosae.

Curr Top Med Chem, 2001 May, 1(1), 73 - 82
Siderophore-antibiotic conjugates used as trojan horses against Pseudomonas aeruginosa; Budzikiewicz H; Pseudomonas aeruginosa is a dangerous opportunistic bacterium responsible for frequently lethal hospital (nosocomial) infections . It endangers especially severely injured patients suffering from large wounds or severe burns, as well as persons whose immune system is weakened . An extremely critical situation exists for patients suffering from mucoviscidosis (cystic fibrosis), when P . aeruginosa infects the bronchial tubes . P . aeruginosa is resistant against many disinfecting agents and, more important, an increasing number of strains especially from hospital isolates have become highly resistant against most antibiotics . The low permeability of the outer membrane and an active export mechanism for low molecular weight substances are the main reasons for the resistance . In addition, beta-lactamase activity affects treatment with beta-lactam antibiotics . An approach to overcome the problem of resistance lies in the synthesis of antibiotics conjugated with compounds active as siderophores . In this way the transport ways for iron complexes into the cell can be used ("Trojan Horse strategy"), and the presence of large substituents reduces the export and the beta-lactamase activity . The results obtained with natural (pyoverdins) and synthetic (mainly catecholate) siderophores will be reviewed.

Curr Top Med Chem, 2001 May, 1(1), 59 - 71
Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance; Poole K et al.; Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy . Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date . These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains . Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown . These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor {OMF}) and a periplasmic membrane fusion protein (MFP) that links the other two . In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing . While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P . aeruginosa makes them reasonable targets for therapeutic intervention.

Curr Top Med Chem, 2001 May, 1(1), 1 - 6
Siderophores of the human pathogenic fluorescent pseudomonads; Budzikiewicz H; Bacteria need a sufficient supply of iron in ionic form for their metabolism . When living in an environment where this is not possible (as in the soil due to the presence of highly unsoluble ferric oxide hydrates, or in living organisms where iron is bound to peptidic chelators) Fe3+ complexing compounds, called siderophores, are produced . The siderophores of Pseudomonas aeruginosa, a dangerous opportunistic human pathogen, and of related potentially pathogenic species will be presented.

Allergy Asthma Proc, 2002 Jan-Feb, 23(1), 19 - 25
Inflammatory mediators in cystic fibrosis lung disease; Berger M; Cystic fibrosis (CF) is a complex multisystem disorder caused by mutations in a membrane glycoprotein called the CF transmembrane regulator (CFTR), which has as its major function serving as a Cl- channel . The relationship between defects in CFTR and development of lung disease remains incompletely understood . Chronic lung disease, characterized by persistent infection with a peculiar type of Pseudomonas aeruginosa, bronchiectasis, and airway obstruction is the major cause of morbidity and mortality in CF patients . The inflammatory response to the chronic infection resembles that induced by lipopolysaccharide (LPS) and is mediated primarily by cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and IL-8, whose synthesis is activated by the transcription factor nuclear factor kappa B (NF-kappa B) . Large numbers of neutrophils dominate the inflammatory response and excessive concentrations of their products create a vicious cycle that becomes injurious rather than protective and eventually claims the life of the patient.

Eur J Med Res, 2002 Feb 21, 7(2), 79 - 80
Eradication of initial Pseudomonas aeruginosa colonization in patients with cystic fibrosis; Griese M et al.; The optimal treatment for the eradication of initial P . aeruginosa infection in CF is still unclear . Recently long-term inhaled tobramycin has been proposed . Here we report the results with brief inhaled and/or systemic anti-pseudomonal treatments . Initial P . aeruginosa colonization was successfully eradicated as demonstrated by negative repetitive throat cultures or sputa and serum antipseudomonal antibodies in 15 of 17 patients for at least two years . Randomized, controlled trials are urgently needed to define the optimal protocol for the eradication of P . aeruginosa . Pseudomonas aeruginosa, infection, treatment, antibodies; inhaled tobramycin

Crit Care Med, 2002 Feb, 30(2), 442 - 7
Beneficial effects of nitric oxide inhalation on pulmonary bacterial clearance; Jean D et al.; OBJECTIVE: The beneficial effects of nitric oxide inhalation on oxygenation during acute respiratory distress syndrome are well described . In contrast, the effects of nitric oxide on pulmonary inflammatory response are much less known in vivo . The objectives of this study were to evaluate the effects of nitric oxide inhalation on bacterial clearance during bacterial pneumonia and on alveolar neutrophil functions . DESIGN: Controlled animal study . SETTING: Research laboratory of an academic institution . SUBJECTS: Male Sprague-Dawley rats . INTERVENTIONS: Severe pneumonia was induced by alveolar instillation of live Pseudomonas aeruginosa (1.5 x 10(8) colony-forming units/kg) in rats . After instillation, rats were exposed to oxygen alone (FIO(2) 100%) or to oxygen (FIO(2) approximately 100%) plus nitric oxide (10 ppm) during 24 hrs . MEASUREMENTS AND MAIN RESULTS: Oxygen plus nitric oxide inhalation compared with oxygen alone increased recruitment of alveolar neutrophils (32.5 +/- 4.6 x 10(6) cells/mL vs . 23.4 +/- 1.9 x 10(6) cells/mL, p <.05) and improved bacterial clearance in the bronchoalveolar lavage fluid (8.1 +/- 4.2 x 10(2) vs . 1.6 +/- 1.0 x 10(5) colony-forming units/mL, p <.05) and in the pulmonary parenchyma (1.7 +/- 1.14 x 10(7) vs . 3.4 +/- 1.5 x 10(8) colony-forming units/mL, p <.05) . However, neither protein concentration in the bronchoalveolar lavage fluid nor mortality rates were modified by nitric oxide inhalation . The ex vivo alveolar neutrophil functions were similar regardless of whether rats previously inhaled nitric oxide . In vitro experiments demonstrated that nitric oxide donor had a direct bactericidal effect against P . aeruginosa and did not modify alveolar neutrophil functions . CONCLUSIONS: These results suggest a beneficial effect of nitric oxide inhalation on bacterial clearance of P . aeruginosa attributable to both a direct bactericidal effect and an influx of alveolar neutrophils with preserved functions.

Crit Care Med, 2002 Feb, 30(2), 315 - 22
Impairment of polymorphonuclear neutrophil functions precedes nosocomial infections in critically ill patients; Stephan F et al.; OBJECTIVE: A postinjury immunodepression involving neutrophil functions has been described in critically ill patients . The aim of this prospective study was to search for a relationship between an impairment of neutrophil functions and the subsequent development of nosocomial infection . DESIGN: Twenty-one severely ill (simplified acute physiology score II >20 on admission), nonimmunosuppressed patients who were receiving no antibiotics active against methicillin-resistant Staphylococcus aureus and highly resistant Pseudomonas aeruginosa were included . Twelve healthy subjects constituted a control group . MEASUREMENTS: Neutrophil functions (phagocytosis and bactericidal activity toward S . aureus and P . aeruginosa in homologous plasma, reactive oxygen species secretion) were studied at day 4 +/- 1 after admission, and occurrence of nosocomial infection was prospectively recorded over the following 5 days . Interleukin-10 concentration was assessed by enzyme-linked immunosorbent assay . Results are expressed as median (25th-75th percentiles) . MAIN RESULTS: Six out of the 21 patients acquired a nosocomial infection during the 5 days after blood sampling (infected group) . Compared with the patients who did not acquire nosocomial infection (noninfected group, n = 15), the neutrophils of the infected group demonstrated a higher percentage of intracellular bacterial survival (17% {2% to 67%} vs . infected: 62% {22% to 100%}, p <.05), leading to an impairment of S . aureus killing in homologous plasma (killed bacteria: 4.93 log(10) colony forming units/mL {4.24-5.29} vs . infected: 3.62 log(10) colony forming units/mL {0.00-4.58}, p <.05) . Interleukin-10 plasma concentration was higher in infected patients (78 pg/mL {60-83}) compared with noninfected patients (22 pg/mL {14-58}, p <.05) . By contrast, P . aeruginosa killing was similar in patients whether or not they acquired a nosocomial infection . CONCLUSION: A decrease in S . aureus killing capabilities of neutrophils can be evidenced within the days before occurrence of a nosocomial infection.

J Bacteriol, 2002 Apr, 184(7), 2045 - 9
Differential effects of mutations in tonB1 on intrinsic multidrug resistance and iron acquisition in Pseudomonas aeruginosa; Zhao Q et al.; Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa . Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-a-vis their contribution to drug resistance only.

Chest, 2002 Mar, 121(3), 863 - 70
Silver-coated endotracheal tubes associated with reduced bacterial burden in the lungs of mechanically ventilated dogs; Olson ME et al.; STUDY OBJECTIVES: To evaluate the influence of silver-coated endotracheal tubes on the lung bacterial burden of mechanically ventilated dogs . DESIGN: Randomized, double-blinded, controlled experiment . SETTING: Animal research facility of a regional medical university . PATIENTS OR PARTICIPANTS: Eleven healthy adult dogs . INTERVENTIONS: The dogs were intubated either with cuffed, noncoated endotracheal tubes or with endotracheal tubes having a novel antimicrobial silver hydrogel coating and were challenged with buccal administration of Pseudomonas aeruginosa . MEASUREMENTS AND RESULTS: The silver coating delayed the appearance of bacteria on the inner surface of the endotracheal tubes ({mean +/- SD} duration of mechanical ventilation before appearance of bacteria, 3.2 +/- 0.8 days; mean duration of mechanical ventilation, 1.8 +/- 0.4 days; p = 0.016) . The mean total aerobic bacterial burden in the lung parenchyma was statistically lower among the dogs receiving the silver-coated endotracheal tubes compared to those not receiving them (4.8 +/- 0.8 vs 5.4 +/- 9 log cfu/g lung tissue, respectively; p = 0.010) . Pronounced differences were seen in the gross and histologic assessments of inflammation in the lung . Using an increasing severity scale of 0 to 12 to assess four components of histology (ie, hyperemia, edema, cellular infiltration, and bacterial presence), dogs receiving noncoated endotracheal tubes had statistically greater histology scores compared to dogs receiving silver-coated endotracheal tubes (7.1 plus minus 1.6 vs 2.8 plus minus 1.2, respectively; p < 0.001) . CONCLUSION: These results suggest that the silver coating of endotracheal tubes may delay the onset of and decrease the severity of lung colonization by aerobic bacteria . Based on these results, clinical studies are planned to determine the safety and clinical efficacy of silver-coated endotracheal tubes in patients requiring mechanical ventilation in the ICU setting.

Med Clin (Barc), 2002 Mar 9, 118(8), 299 - 301
{Life-threatening hemoptysis in cystic fibrosis: clinical characteristics and management in 36 episodes}; Maiz L et al.; BACKGROUND: Our goal was to evaluate the clinical characteristics and management of life-threatening hemoptysis in patients with cystic fibrosis (CF) . PATIENTS AND METHOD: We included adult CF patients followed up at the Cystic Fibrosis Units of the Autonomous Community of Madrid who had life-threatening hemoptysis from June 1990 to December 1999 . RESULTS: Twelve CF patients (4 females) developed 36 episodes of life-threatening hemoptysis (30 massive and 6 recurrent) . Lung disease was moderate to severe . Sputum cultures revealed Pseudomonas aeruginosa in 10 patients . Thirteen episodes (36%) resolved upon antibiotic treatment and 3 (8%) after antibiotic therapy and bronchoscopy . Bronchial artery embolization (BAE) was performed in 20 of 36 events . Immediate technique success was achieved in 80% episodes (16 of 20) after one session, 85% (17/20) after two sessions, and 95% (19/20) after three sessions . No major complications associated with the procedure were seen . The overall recurrence rate per episode was 69% (24 of 35 episodes in 6 patients) with a mean time of recurrence of 13 months . There were no massive hemoptysis-associated deaths during the follow-up . CONCLUSIONS: Life-threatening hemoptysis is a frequent complication in CF patients who have moderate or severe lung disease . When conservative therapeutic measures (including antibiotics) fail to control it, BAE should be performed . When performed by expert professionals, BAE is effective and safe to immediate control of life-threatening hemoptysis in patients with CF.

Rev Mal Respir, 2001 Oct, 18(5), 549 - 51
{Allergic bronchopulmonary aspergillosis disclosing mucoviscidosis}; Coltey B et al.; In predisposed patients, allergic bronchopulmonary aspergillosis (ABPA) can arise from aspergillus bronchial colonization . We report the case of a young woman who presented with a right basal pneumonia, ground glass opacities and mediastinal adenopathies on CT scan . Biological, radiological and clinical criteria, as well as an history of childhood asthma, allowed the initial diagnosis of ABPA . However, the unusual coexistence of an additional infection with Pseudomonas Aeruginosa evoked the diagnosis of cystic fibrosis, confirmed by a sweat test and genetic analysis . Under corticosteroid and antifungal therapy and antibiotics, the clinical and radiological evolution was favourable but immuno-allergic sensitisation persisted . The ABPA-cystic fibrosis association is not rare with an estimated prevalence of 2% to 11% according to previous studies . This variability is partly explained by the difficulty of the diagnosis due to confounding clinical, radiological, and biological signs between ABPA and cystic fibrosis . Many predictive development factors of ABPA in the context of cystic fibrosis have been reported, including respiratory function, personal or familial atopy, colonization with Pseudomonas Aeruginosa and age . As in non cystic fibrosis patients, the treatment requires systemic corticotherapy and itraconazole . ABPA is still often under diagnosed and should be evoked in the context of cystic fibrosis.

Recenti Prog Med, 2002 Feb, 93(2), 104 - 7
{External otitis}; Olina M et al.; Otitis externa is one of the most common diseases in ORL practice, during summer; the treatment of otitis externa may be simple and easy or protracted and frustrating, also with fatal outcome . Many local factors may interfere with the normal defences against infections in the external auditory canal . Removing or dissolving the cerumen by water or other instruments eliminates an important barrier to infections: its acids inhibit the growth of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and fungi (Aspergillus) . Also skin abrasions or irritation, allergic diseases and many systemic condition like anaemia, vitamin deficiency, endocrine disorders (diabetes) and various forms of dermatitis cause a lower resistance to infections in external auditory canal . Even if the prognosis remains benign in the majority of cases, important complications could appear like: malignant otitis externa, facial nerve paralysis, tympanic bone osteomyelitis, pericondrytis . Successful treatment depends on a proper diagnosis and therapy: the most important factor in the treatment is repeated debridement of the external auditory canal by the physician . The use of Castellani' Tintura rubra, hydroalcoholic solution of phenic fuchsin, can be very effective for bacteria and mycotic eradication . Culturing of ear canal infection could be performed on the second or third visit if the otitis externa is not responding to therapy . Complication are not frequent, but malignant otitis externa can be mortal . Dermatological consultation is often necessary for correct diagnosis.

Bioorg Med Chem, 2002 May, 10(5), 1595 - 610
Structure--activity relationships of 1beta-methyl-2-(5-phenylpyrrolidin-3-ylthio)carbapenems; Sato H et al.; Structure--activity relationship studies of 1beta-methyl-2-{(3S,5R)-5-(4-aminomethylphenyl)pyrrolidin-3-ylthio}carbapenems, especially those pertaining to the relationship between antibacterial activity and side-chain structure were conducted . These studies suggested that the trans-(3S,5R)-5-phenylpyrrolidin-3-ylthio side-chain and the aminomethyl group at the 4-position of the phenyl ring play a key role in enhancing the antibacterial activity against the MRSA and Pseudomonas aeruginosa strains . In particular, the basicity of a substituent at the 4-position of the phenyl ring were shown to greatly contribute to the antibacterial activity against MRSA and methicillin-resistant Staphyloccocus epidermidis strains . In contrast, the amidine group was shown to lead to potent antibacterial activity against P . aeruginosa strains comparable to that of imipenem, however, a good correlation between the basicity of the 4-substituent and antipseudomonal activity was not observed . In conclusion, the 4-aminomethyl or methylaminomethyl group on the phenyl ring was the best substituent for antipseudomonal activity.

Insect Biochem Mol Biol, 2002 Apr, 32(4), 389 - 95
Molecular identification of an endosymbiotic bacterium associated with pederin biosynthesis in Paederus sabaeus (Coleoptera: Staphylinidae); Kellner RL; Biosynthesis of the structurally complex hemolymph toxin pederin is an eminent character of Paederus females . For that capability, however, they rely on endosymbiotic bacteria that are lacking in aposymbiotic females . The bacterial inhabitants of the two phenotypes in Paederus sabaeus are evaluated in a PCR-based analysis of 16S rDNA . A certain fragment, which is not found in aposymbiotic females, is highly dominant in the other, biosynthesizing females and thus identifies the endosymbiont . Its DNA sequence reveals a member of the gamma subdivision of the Proteobacteria that is clustered within the genus Pseudomonas (sensu stricto) as it is most closely related to Pseudomonas aeruginosa . These bacteria appear as the hypothesized common producers of pederin and the pederin family of analogs from marine sponges.

FEMS Microbiol Lett, 2002 Jan 22, 207(1), 63 - 8
Transcriptional regulation of mexR, the repressor of Pseudomonas aeruginosa mexAB-oprM multidrug efflux pump; Sanchez P et al.; The transcription start site of mexR, encoding the repressor of the Pseudomonas aeruginosa mexAB-oprM multidrug efflux pump, has been determined by S1 mapping . One signal corresponding to a single promoter has been found, whereas three major signals were observed for the mexA messenger . Further analysis demonstrated that mexA has just one promoter that overlaps with the mexR promoter, with the other two signals observed by S1 probably being the consequence of RNA processing . Transcription of mexR and mexA from the aforementioned promoters is regulated by MexR . We show that bacterial growth phase affects expression of these promoters as well . mexR expression was higher at the exponential growth phase and declined afterwards, whereas mexA expression was triggered at the onset of the stationary growth phase . A model for the regulation of mexR and mexA expression, which includes an analysis of the interplay between both promoters, is proposed.

Presse Med, 2002 Feb 16, 31(6), 263 - 70
{Mucoviscidosis in adults}; Stern M et al.; FROM CHILDREN TO ADULTS: Mucoviscidosis, a genetic autosomal recessive disease, is not only a paediatric disease, but with the progress in therapy, has become a disease of adults . Today, median survival of patients is of 30 years and, in France, more than one third of patients are adults . CLINICAL SYMPTOMS IN ADULTS: Are predominantly respiratory, with dilatation of the bronchi and characteristic colonization flora . Chronic bronchial Pseudomonas aeruginosa colonisation is frequently encountered in adults . It develops in successive episodes towards chronic respiratory failure . Other than this typical form, diagnosed in the first years of life, the discovery of the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene and its mutations permits diagnosis of mucoviscidosis in patients presenting with mild, or even monosymptomatic forms of the disease . Today, diagnosis of mucoviscidosis relies on the association of characteristic organ damage and an abnormality in CFTR (sweat test and/or difference in nasal potential) or the revelation of gene mutations on each allele . REGARDING TREATMENT: Respiratory failure is the core of daily therapeutic efforts . Respiratory physical therapy, effort re-education and muscle exercising are essential . Antibiotherapy is aimed at treating, spacing out or preventing the infectious exacerbations, in order to stall the functional degradation . Repeated, sequential cycles of intravenous infusions of antibiotics are required in P . aeruginosa chronic bronchial colonization . Treatment of the primary pyocyanic colonisation and inhaled antiobiotherapy appear promising . Pulmonary transplantation is a recognized and efficient therapeutic in advanced stages of respiratory failure . The discomfort and time the patient has to spend on daily treatments requires regular monitoring, to improve compliance and to improve the quality of life of these patients.

Am J Physiol Lung Cell Mol Physiol, 2002 Apr, 282(4), L751 - 6
Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin; Lillehoj EP et al.; We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC . Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001) . The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches . Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P . aeruginosa strain PAK . Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P . aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin . We conclude that flagellin is an adhesin of P . aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.

Biochem J, 2002 Mar 15, 362(Pt 3), 619 - 26
Activation of bacterial ceramidase by anionic glycerophospholipids: possible involvement in ceramide hydrolysis on atopic skin by Pseudomonas ceramidase; Kita K et al.; We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) {Okino, Tani, Imayama and Ito (1998) J . Biol . Chem . 273, 14368--14373} . In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents . Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective . Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific omega-hydroxyacyl Cer, by the enzyme in the absence of detergents . It was confirmed that several strains of P . aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S . aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol . Since both P . aeruginosa and S . aureus are suspected of being present in microflora of atopic skin, we speculate that S . aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase.

Zhonghua Shao Shang Za Zhi, 2001 Apr, 17(2), 91 - 2
{The change in resistance of Pseudomonas aeruginosa to gentamicin during in vitro subculturing}; Dai L et al.; OBJECTIVE: To observe the change in resistance of Pseudomonas aeruginosa (PA) to gentamicin (GM) during in vitro subculturing . METHODS: Twenty -- six strains of PA with resistance to GM isolated from burn patients were subcultured in vitro for 30 generations . The strains were also divided into high level (20 strains) and medium level (6 strains) of resistant groups according to their MIC (minimum inhibition concentration) values . The MIC and MBC (minimal bactericidal concentration) levels were examined in every strains of the 1st, 10th, 20th, and 30th filial generations of PA strain . RESULTS: There was no significant general difference of MIC and MBC values among the filial generations in each group (P < 0.05) when multiple comparisons of MIC and MBC mean values were made among every filial generations in high and medium levels of GM resistant groups . CONCLUSION: There was no evident difference in GM resistance among all the filial generations of PA . The result indicated that, the PA with different levels of GM resistance clinically found might come from different PA strains.

Zhonghua Shao Shang Za Zhi, 2001 Apr, 17(2), 80 - 2
{Micro -- ecological investigation of burn wound infection}; Wang W et al.; OBJECTIVE: To analyze the epidemic tendency of the bacteria causing burn wound infection, and to explore the influencing factors . METHODS: Bacterial ecology on burn wound was investigated in 210 cases of burn patients admitted to our hospital from January 1999 to December 2000, and the results were compared to those done in the 1970s, 1980s and 1990s . RESULTS: Since 1999, G(+)cocci increased obviously (52.17%) when compared with those of the past three decades (P < 0.01), while G(minus sign) bacilli decreased significantly (46.48%, P < 0.01) . The ratio of Pseudomonas aeruginosa in recent two years was evidently lower than those in the early 1970s and 1990s (P < 0.01) while the ratio of staphylococcus aureus was much higher than those in the middle of 1980s and 1990s (P < 0.01), in which methacillin -- resistant Staphylococcus aureus (MRSA) accounted for 85% or so, which increased significantly than those in the middle of 1980s and the beginning of 1990s (P < 0.01) with an increasing tendency . CONCLUSION: The increasing tendency of Staphylococcus aureus in our unit for the recent two years might closely be related to the preferential application of Imipenem in burn infection control . Furthermore, the ratio of MRSA in Staphylococcus aureus could not be lowered by vancomycin.

Zhonghua Shao Shang Za Zhi, 2001 Apr, 17(2), 75 - 9
{An experimental study on the release of endotoxin from gram negative bacteria induced by antibiotics}; Xu N et al.; OBJECTIVE: To explore the characteristics and possible mechanism of LPS released from Gram negative bacteria induced by antibiotics, so as to improve clinical management of endotoxemia and sepsis . METHODS: Cultures containing PA103 subtype of Pseudomonas aeruginosa (PA) and E coli 25922 subtype of E coli were treated with four kinds of antibiotics as Imipenam (IMP), ceftazidime (CTZ), amikacin (AMN) and pefloxacine (PFX) in four concentrations of 0.5, 1, 5 and 10 MIC for 8 hours . The changes in the bacterial quantity and morphology and the supernatant levels of free LPS of the culture media were observed at different time points . RESULTS: All the four kinds of antibiotics could kill the tested bacteria in similar degree, but lead to the different types of morphological changes of the bacteria . In detail, IMP could convert the bacteria into spherical shape, while CTZ and PFX made the bacteria to filamentous shape . But AMN could induce lysis of bacterial thallus . Under same condition, the ability of different kinds and concentrations of antibiotics to induce LPS release ranked as CTZ > PFX > IMP > AMN, 0.5MIC > 1MIC > 5MIC > 10MIC . Along with the prolongation of the action time, the LPS release increased . Furthermore, PA103 released less endotoxin than E . coli after the action of antibiotics . CONCLUSION: All of the four antibiotics, i,e, IMP, CTZ, AMN and PFX could induce PA103 and E coli 25922 to release different levels of LPS, which was related to bacterial morphological changes . The LPS release from the bacteria was correlated to the antibiotics applied, concentrations, action time and the bacterial features . Antibiotics with less ability of inducing LPS release were recommended for clinical management of the sepsis and/or septic shock caused by Gram negative bacteria.

Zhonghua Shao Shang Za Zhi, 2000 Apr, 16(2), 93 - 5
{An experimental study of antibiotic -- induced endotoxin release in Pseudomonas aeruginosa bacteremia inburned rats}; Wang S et al.; OBJECTIVE: To assess the intensity of endotoxin release in Pseudomonas aeruginosa challenged burned rats after the administration of various antibiotics . METHODS: Rats were inflicted with 30% TBSA burn, and they were challenged with Pseudomonas aeruginosa (PA103) . After the administration of different beta lactam antibiotics, serum endotoxin level and blood bacterial count were determined . RESULTS: All the antibiotics used were effective in eradicating the bacteria with concomitant release of endotoxin in varying amounts . It was found that Imipenem released the least amount of endotoxin, followed in order by Cefoperazone, Ceftazidime, and Cefotxime . CONCLUSIONS: Different antibiotics possessed varying capacity of inducing endotoxin release from PA, and the amount released did not bear any relation with the bactericidal capability of the antibiotics.

Clin Exp Immunol, 2002 Feb, 127(2), 206 - 13
Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response; Moser C et al.; Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases . Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection . To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re-infected with P . aeruginosa 14 days after the initial infection . Singly-infected BALB/c mice, as well as non-infected mice, were used as controls . Decreased mortality and milder lung inflammation in re-infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly-infected mice . The improved outcome in re-infected mice correlated with changes in CD4 cell numbers . Surface expression of LFA-1 on pulmonary CD4 cells was increased in re-infected compared with singly-infected mice . Moreover, resistance to re-infection was paralleled by a shift towards a Th1-dominated response and increased IL-12 production . No significant increase in serum IgG was observed in the re-infected mice . In conclusion, these results indicate a protective role for a Th1-dominated response, independent of antibody production, in chronic P . aeruginosa lung infection in CF.

Infect Control Hosp Epidemiol, 2001 Dec, 22(12), 771 - 5
Nosocomial infections in pediatric cardiac surgery, Italy; Valera M et al.; OBJECTIVE: To evaluate the incidence of nosocomial infection (NI) in pediatric patients who received cardiothoracic surgery and to identify possible associated risk factors . DESIGN: Prospective observational study . SETTING: The cardiac surgery and cardiac intensive care units at the Regina Margherita Children's Hospital, Turin, Italy . PATIENTS: All patients who underwent surgery from July 20, 1998, to July 19, 1999, were enrolled, except patients with operative catheterization only . METHODS: Clinical data were collected daily from July 20, 1998, to July 19, 1999 . NIs were diagnosed according to US Centers for Disease Control and Prevention criteria . RESULTS: 104 patients were included in the present study, 80 (76.9%) of whom underwent extracorporeal circulation . The NI ratio was 48.1% (50/104); the percentage of patients with NI was 30.8% (32/104): 23.1% developed one infection, 7.7% two or more . The rate of NI was 2.17 per 100 days of hospitalization (50/2,304) . The most common pathogen was Pseudomonas aeruginosa . Important risk factors were length of preoperative admission >5 days, total length of admission >10 days, open chest during postoperative phase, and cyanotic heart disease . There was a significant association between sepsis and central venous catheterization for 3 days or more . Rate of sepsis was 19 per 1,000 catheter days (16/852) . CONCLUSION: NIs represent a frequent complication for children who undergo heart surgery . Based on our data, we suggest decreasing the preoperative stay as much as possible . The higher NI incidence in patients with an open chest postoperatively suggests that an alternative antibiotic strategy should be considered for these patients.

Pathol Biol (Paris), 2002 Feb, 50(1), 12 - 7
{Susceptibility of Pseudomonas aeruginosa to antibiotics isolated from patients of intensive care units in France in 1998 . Resistant phenotypes to beta-lactams}; Rio Y et al.; Pseudomonas aeruginosa is responsible for nosocomial infections and demonstrates many types of resistance mechanisms to antibiotics . Thus, in vitro susceptibility survey are frequently required . In this study, susceptibility has been assessed on 105 non redundant consecutive strains isolated from ICU's in 18 general hospitals, from 01.02.98 to 30.06.98 . Only clinically significant samples have been considered . MICs have been measured for nine beta-lactams, three aminoglycosides, one fluoroquinolone and colistine . For ticarcilline resistant strains, phenotype has been assessed on Mueller-Hinton medium supplemented with beta-lactamases inhibitor . Transferable beta-lactamases has been identified using pl and PCR . MIC 50 and MIC 90 (mg/L) for beta-lactams are the following (MIC 50-->90): ticarcilline (16-->512), ticarcilline + clavulanic acid (16-->512), piperacilline (4-->512), piperacilline + tazobactam (4-->64), aztreonam (4-->16), cefsulodine (4-->32), ceftazidime (2-->16), cefepime (4-->16), imipeneme (1-->8) . For aminoglycosides: gentamicine (2-->32), tobramycine (1-->32), amikacine (4-->16) . For ciprofloxacine (0.25-->32) and colistine (0.5-->2) . According to CA-SFM break points recommendations, 50% of isolated strains are resistant to gentamicine, one out of three for ticarcilline + clavulanic acid (29%), one out of four for tobramycine (25%) and ciprofloxacine (25%), one out of ten for amikacine (9%), tazocilline (8%) and imipeneme (9%) . Resistance to ceftazidime and aztreonam is uncommon (respectively 2%-1%) and never observed for cefepim . For ticarcilline resistant strains, (38% of total isolates) the following phenotypes have been detected: 6.7% non enzymatic resistance, 15.2% transferable beta-lactamase (TEM 4.8%, CARB 4.8%, TEM + CARB 4.8% and OXA-10 and derivated 0.9%) and 16.2% high level cephalosporinase . Extended-spectrum beta-lactamase has never been detected . TEM beta-lactamase is associated with resistance to amikacine and ciprofloxacine.

J Bacteriol, 2002 Mar, 184(6), 1779 - 82
Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa; Robert V et al.; Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system . This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton . We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton . In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis . We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton . Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.

J Bacteriol, 2002 Mar, 184(6), 1503 - 13
Mutational analysis of the TonB1 energy coupler of Pseudomonas aeruginosa; Zhao Q et al.; Siderophore-mediated iron transport in Pseudomonas aeruginosa is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity . To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the tonB1 gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined . The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both P . aeruginosa and Escherichia coli . Appending it to the N terminus of the nonfunctional (in P . aeruginosa) Escherichia coli TonB protein (TonB(Ec)) rendered TonB(Ec) weakly active in P . aeruginosa and did not compromise the activity of this protein in E . coli . Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in P . aeruginosa and E . coli . Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB(Ec) function in E . coli, was shown here to be essential for TonB1 activity in E . coli but not in P . aeruginosa . Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both P . aeruginosa and E . coli, including substitutions at Tyr264, Glu274, Lys278, and Asp304 . Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in E . coli but not in P . aeruginosa . The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in P . aeruginosa . Several TonB1-TonB(Ec) chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in P . aeruginosa, although chimeras possessing an E . coli C terminus were active in E . coli.

J Microbiol Methods, 2002 May, 49(3), 285 - 93
Bactericidal activity of electrolyzed acid water from solution containing sodium chloride at low concentration, in comparison with that at high concentration; Kiura H et al.; Electrolyzed strong acid water (ESW) containing free chlorine at various concentrations is becoming to be available in clinical settings as a disinfectant . ESW is prepared by electrolysis of a NaCl solution, and has a corrosive activity against medical instruments . Although lower concentrations of NaCl and free chlorine are desired to eliminate corrosion, the germicidal effect of ESW with low NaCl and free-chlorine concentrations (ESW-L) has not been fully clarified . In this study, we demonstrated that ESW-L possesses bactericidal activity against Mycobacteria and spores of Bacillus subtilis . The effect was slightly weaker than that of ESW containing higher NaCl and free-chlorine concentrations (ESW-H), but acceptable as a disinfectant . To clarify the mechanism of the bactericidal activity, we investigated ESW-L-treated Pseudomonas aeruginosa by transmission electron microscopy, a bacterial enzyme assay and restriction fragment length polymorphism pattern (RFLP) assay . Since the bacterium, whose growth was completely inhibited by ESW-L, revealed the inactivation of cytoplasmic enzyme, blebs and breaks in its outer membrane and remained complete RFLP of DNA, damage of the outer membrane and inactivation of cytoplasmic enzyme are the important determinants of the bactericidal activity.

Infect Control Hosp Epidemiol, 2002 Jan, 23(1), 44 - 6
Sensor-operated faucets: a possible source of nosocomial infection?
Assadian O, El-Madani N, Seper E, Mustafa S, Aspock C, Koller W, Rotter ML.
Recently, contamination of sensor-operated faucets (SOFs) with Pseudomonas aeruginosa was observed . To evaluate odds ratios, we conducted a case-control study in which handle-operated faucets served as controls . No statistically significant difference in P . aeruginosa counts was observed between SOFs and regular faucets in our study (odds ratio, 0.0; 95% confidence interval, 0.0 to 39.0; two-sided P exact = .99).

Acta Chir Belg, 2001 Nov-Dec, 101(6), 304 - 7
Chronic osteomyelitis after sternotomy; Thijssens K et al.; Two patients with chronic sternal osteomyelitis after an initially uncomplicated coronary artery bypass grafting (CABG) operation are described . Chronic osteomyelitis, caused in both cases by Pseudomonas aeruginosa, occurred six and four months after CABG respectively . Because chronic infection failed to respond to local wound care and medical therapy, more radical treatment was needed . Steel wires were removed and surgical debridement was performed . In one patient, an additional omental transposition was performed . In both cases radical debridement in combination with antibiotics successfully eradicated the infection.

Eur Surg Res, 2002 Jan-Apr, 34(1-2), 61 - 7
Chronic porcine two-hit model with hemorrhagic shock and Pseudomonas aeruginosa sepsis; Eissner B et al.; BACKGROUND: Sepsis is still a major cause of death despite well-developed therapeutical strategies such as antibiotics and supportive medication . The aim of this study was to characterize the long-term effects of a two-hit porcine sepsis model with a hemorrhagic shock as 'first hit' followed by a Pseudomonas aeruginosa infusion as 'second hit' . MATERIALS AND METHODS: Twelve juvenile healthy pigs were anesthetized and hemodynamically monitored . The two-hit group (n = 6) underwent a hemorrhagic shock with a 50% reduction of the mean arterial pressure and/or cardiac index for 45 min, followed by resuscitation, while the control group (n = 6) received no pretreatment . All chronically catheterized conscious pigs were challenged with a P . aeruginosa infusion (1.6 x 10(7) CFU/kg/h for the first 24 h followed by 1.6 x 10(6) CFU/kg/h for the next 24 h) and observed for another 48 h . RESULTS: The two-hit group showed the following significant differences to the control group: higher APACHE II scores prior to sepsis induction, increased persisting mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) during bacterial challenge . In contrast, systemic vascular resistance (SVRI) was reduced at the end of the study . Throughout the observation period, the mean arterial pressure (MAP) was significantly reduced . CONCLUSIONS: The present study shows that the clinical course and hemodynamic effects of a P . aeruginosa sepsis will be aggravated by a preceding hemorrhagic shock during an observation period of 96 h . This two-hit model represents a valid, clinically relevant experimental protocol in sepsis research .

Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 3159 - 64 Epub 2002 Feb 26.
The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum; Pukatzki S et al.; Genetically accessible host models are useful for studying microbial pathogenesis because they offer the means to identify novel strategies that pathogens use to evade immune mechanisms, cause cellular injury, and induce disease . We have developed conditions under which the human pathogen Pseudomonas aeruginosa infects Dictyostelium discoideum, a genetically tractable eukaryotic organism . When D . discoideum is plated on nutrient agar plates with different P . aeruginosa strains, the bacteria form lawns on these plates with amoebae embedded in them . Virulent P . aeruginosa strains kill these amoebae and leave an intact bacterial lawn . A number of P . aeruginosa mutants have been identified that are avirulent in this assay . Amoebae feed on these bacteria and form plaques in their bacterial lawns . One avirulent mutant strain carries an insertional mutation in the lasR gene . LasR is a transcription factor that controls a number of virulence genes in a density-dependent fashion . Another class of avirulent P . aeruginosa mutants is defective in type III secretion . One mutant lacks the PscJ protein, a structural component of the secretion apparatus, suggesting that cytotoxins are injected into the D . discoideum cell . One of these cytotoxins is ExoU, and exoU mutants are avirulent toward D . discoideum . Complementation of the lasR and exoU mutations restores virulence . Therefore, P . aeruginosa uses conserved virulence pathways to kill D . discoideum.

J Leukoc Biol, 2002 Mar, 71(3), 458 - 68
Distinct fates of monocytes and T cells directly activated by Pseudomonas aeruginosa exoenzyme S; Epelman S et al.; Gram-negative infections can cause overwhelming inflammatory responses . Although factors other than LPS are clearly involved, these factors and their mechanisms of action have been poorly defined . During studies of LPS-independent inflammatory responses of the gram-negative pathogen Pseudomonas aeruginosa, an important virulence factor (exoenzyme S) was shown to be a potent mitogen for T cells . The current work demonstrates that exoenzyme S selectively induced transcription and secretion of biologically active cytokines and chemokines (chemotactic for neutrophils and T cells) from monocytes . Exoenzyme S stimulated highly purified monocytes independent of T cells . In addition, exoenzyme S stimulated T cells directly; neither T-cell activation (CD69) nor apoptosis (hypodiploidy) required the presence of monocytes . However, T-cell activation was enhanced via a noncontact-dependent mechanism as a result of the secretion of TNF-alpha and IL-6 . This study identifies a unique property of a gram-negative-derived microbial product capable of activating multiple cell types and suggests a mechanism by which exoenzyme S contributes to the immunopathogenesis of cystic fibrosis and sepsis in patients infected with P . aeruginosa.

Clin Microbiol Infect, 1996 Jun, 1(4), 261 - 265
Typing of Pseudomonas aeruginosa strains from patients with cystic fibrosis: phenotyping versus genotyping; Renders N et al.; OBJECTIVE: To assess the discriminatory power of two genotypic and two phenotypic techniques by analysis of Pseudomonas aeruginosa sputum isolates obtained with long term intervals from 29 independent cystic fibrosis (CF) patients . METHODS: Fifty-eight strains of P . aeruginosa were subjected to serotyping and pyocin production was assessed . Arbitrarily primed polymerase chain reaction (AP PCR) and pulsed-field gel electrophoresis (PFGE) were applied in order to detect genetic polymorphisms . RESULTS: From the results of different typing techniques, it appeared that the numbers of separate types varied between 11 and 43, and the percentage of identical P . aeruginosa pairs from individual patients varied between 51% and 72%, depending on the test system used . AP PCR and PFGE displayed enhanced resolution when compared to serotyping and pyocin typing; both DNA typing techniques generated concordant results, although differences in resolution are apparent . This results in 15% discordance, which may be the result of differences in the definitions of (sub)clonal relatedness as applied for AP PCR and PFGE, respectively . CONCLUSIONS: Molecular typing techniques are superior to phenotyping where P . aeruginosa is concerned . AP PCR is a fast and useful technique for determining clonality among P . aeruginosa strains from chronically colonized CF patients . It is clear, however, that the interpretation of data and comparative analysis of PFGE and AP PCR results necessitates additional (international) standardization and the development of practical guidelines.

J Am Chem Soc, 2002 Mar 6, 124(9), 2035 - 41
An ab initio quantum-chemical study of the blue-copper site of azurin; van Gastel M et al.; The electronic structure of the blue-copper site of Pseudomonas aeruginosa azurin has been investigated by ab initio multireference determinantal configuration interaction (MRD-CI) calculations . A truncated site consisting of copper and its three equatorial ligands has been studied with emphasis on the g tensor and the nitrogen hyperfine tensors of the coordinating histidines . In the ground state the singly occupied molecular orbital (SOMO) involves a copper 3d orbital pi antibonded to the cysteine sulfur and sigma antibonded to the histidine nitrogens . A proper description of the electron-paramagnetic-resonance parameters has been achieved through the use of an effective core potential for copper up to and including the 3s electrons . Both the complete g tensor and the anisotropic hyperfine tensors at the nitrogens are essentially reproduced . Mulliken spin densities of 35 and 59% on copper and sulfur, respectively, and 2.1 and 1.7% on the respective coordinating nitrogens reflect the delocalized character of the SOMO and the inequivalence of the histidines.

J Antimicrob Chemother, 2002 Mar, 49(3), 561 - 5
A nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum beta-lactamase GES-2 in South Africa; Poirel L et al.; Eight Pseudomonas aeruginosa clinical strains that produce the clavulanic-acid-inhibited beta-lactamase GES-2 were isolated from patients of a South African hospital from March to July 2000 . They were clonally related and each harboured a 150 kb conjugative plasmid carrying a class 1 integron containing a gene cassette encoding GES-2, followed by those for beta-lactamase OXA-5 and an aminoglycoside modifying AAC(3)I-like enzyme . Hence, incidences of infection, several fatal, due to bacteria displaying clavulanate-inhibited resistance to extended-spectrum cephalosporins and reduced susceptibility to imipenem in Pretoria Academic Hospital, South Africa, can be explained, at least in part, by the spread of P . aeruginosa expressing the GES-2 beta-lactamase.

Clin Microbiol Infect, 1998 Apr, 4(4), 199 - 204
Resistance to amikacin and gentamicin among Gram-negative bloodstream isolates in a university hospital between 1989 and 1994; Harbarth S et al.; OBJECTIVE: To characterize antimicrobial resistance patterns to amikacin (AN) and gentamicin (GM) among Gram-negative bloodstream isolates and to determine the possible relationship between use of AN and GM and the occurrence of antibiotic resistance during a 6-year period . METHODS: Standard media and techniques of isolation and identification were used . Antimicrobial susceptibility testing was performed with the disk diffusion method and API rapid ATB E strips . Data on consumption of aminoglycosides were collected by the central hospital pharmacy and were expressed as daily defined doses . RESULTS: One thousand nine hundred and four bloodstream isolates were tested for AN and GM susceptibility between 1989 and 1994 . Activities of AN and GM remained high during the study period against most isolates of Gram-negative bacteria . No relationship could be observed between the use of AN/GM and the rate of AN/GM resistance . Nosocomial Gram-negative bloodstream isolates showed a higher degree of resistance towards both AN (3.9% of all nosocomial isolates) and GM (7.9%) than community-acquired isolates (1.8% toward AN and 3.1% towards GM, respectively) . There was a significant increase (P=0.004) in the risk of GM resistance in patients with nosocomial Gram-negative bacteremia detected more than 14 days after admission . The proportion of GM-susceptible Pseudomonas aeruginosa isolates decreased linearly from 97% for infections acquired between day 3 and day 10 following admission to 80% for bacteremia developing 30 days or more after admission (P=0.008) . CONCLUSIONS: AN and GM remain highly active antimicrobial drugs for treatment of GNB in times of growing resistance to cephalosporins and fluoroquinolones.

Clin Microbiol Infect, 1997 Feb, 3(6), 621 - 628
Multiresistant Pseudomonas aeruginosa serogroup O:11 outbreak in an intensive care unit; Tassios PT et al.; OBJECTIVE: To determine whether 15 multiresistant Pseudomonas aeruginosa isolates from an intensive care unit (ICU) outbreak were related, were endemic, and belonged to the O:12 European clone . METHODS: Forty-six P . aeruginosa isolates from a large hospital were investigated with respect to their antibiotic resistance profiles, serogroups, bacteriocin types and DNA fingerprints obtained by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with Xbal . RESULTS: Fourteen of the ICU outbreak isolates were indeed identical with respect to their serogroup, O:11, pyocin type, 10/a, and PFGE type, A . Clone A was endemic and dominant throughout the hospital, even though, within the ICU, it underwent phenotypic alterations, such as loss of cell wall lipopolysaccharide side-chains, or acquisition of ceftazidime and imipenem resistance . Bacteriocin typing was more discriminatory than serotyping, but PFGE could differentiate further among phenotypically identical strains . It also allowed the tracking of an O:6 strain, as it was becoming gradually more resistant and undergoing a bacteriocin-type conversion while remaining genotypically unaltered . CONCLUSIONS: Using three typing methods, a nosocomial multiresistant strain distinct from the previously described dominant European O:12 clone was characterized, and the ability of PFGE to identify clonal isolates even when these appear phenotypically distinct was demonstrated.

Clin Microbiol Infect, 1997 Apr, 3(2), 222 - 228
Computerized restriction endonuclease analysis compared with O-serotype and phage type in the epidemiologic fingerprinting of Pseudomonas aeruginosa strains; Garaizar J et al.; OBJECTIVE: To assess restriction endonuclease analysis (REA) of chromosomal DNA using SalI enzyme, low-concentration (0.4%) agarose gels and digitalized data management of the REA patterns obtained for the typing of clinical Pseudomonas aeruginosa isolates . METHODS: A group of 67 clinical unrelated isolates from 10 Spanish hospitals was used to study the discriminatory power, reproducibility and typeability of REA typing . RESULTS: A SalI REA pattern consisted of a variety (1--10) of restriction bands in the range between 12.2 and 48.5 kb and an unresolvable smear of low-molecular-weight bands . Forty different SalI REA patterns with an index of discrimination of 0.979 were obtained . Low typeability (91.04%) was the major limitation of REA typing . Analysis of blinded subcultures of eight Pseudomonas aeruginosa strains showed the reproducibility of REA typing to be 87.5% . Combined phenotypic typing (O-serotyping and phage typing) performed on the same group of strains showed comparable discrimination but much lower reproducibility . Isolates selected from five clusters of nosocomial infections in hospitals in the UK were typed by REA typing, and the results show high agreement when compared with conventional phenotypic typing methods in distinguishing between strains . CONCLUSIONS: These data underline the usefulness of REA typing enhanced with digitalized data management for the epidemiologic subtyping of clinical Pseudomonas aeruginosa isolates.

Clin Microbiol Infect, 1997 Feb, 3(1), 73 - 81
Comparative in vitro pharmacodynamics of BO-2727, meropenem and imipenem against Gram-positive and Gram-negative bacteria; Odenholt I et al.; OBJECTIVE: To investigate and compare the in vitro pharmacodynamics of three carbapenems: imipenem, meropenem and BO-2727 . METHODS: The following studies were performed: (1) comparative studies of the rate of killing of the three carbapenems of reference strains of Gram-positive and Gram-negative bacteria at a concentration corresponding to the 1-h serum level following 500 mg intravenously in humans; (2) comparative studies of the rate of killing of BO-2727, meropenem and imipenem at different antibiotic concentrations of reference strains of Gram-positive and Gram-negative bacteria; (3) comparative studies of the rate of killing of BO-2727, meropenem and imipenem of bacteria which are phenotypically tolerant; (4) studies of the postantibiotic effect of BO-2727 using viable counts and optical density; (5) studies of the postantibiotic sub-MIC effect (PA SME) of BO-2727 using optical density . RESULTS: No difference in killing rate was noted between the three carbapenems, and there was no concentration-dependent killing of the Gram-negative strains after 6 h . A pronounced paradoxical effect was seen against Staphylococcus aureus . All three antibiotics were able to kill phenotypically tolerant bacteria . Only very short or no postantibiotic effect of BO-2727 was found against the investigated strains . Very long PA SMEs were noted for the Gram-negative strains, although there was a pronounced variation for the different strains of Pseudomonas aeruginosa . CONCLUSION: There was no significant difference between the studied carbapenems in their pharmacodynamic properties . All three antibiotics acted similarly to other beta-lactam antibiotics.

Am J Ther, 1996 Mar, 3(3), 212 - 218
Comparative Effects of Ceftazidime Two or Three Times Daily in Patients with Skin and Skin Structure Infections; Gentry LO et al.; BACKGROUND: Skin and skin structure infections are among the most common infectious diagnoses in both the hospital and community settings . If untreated, these infections can produce serious complications . Successful antimicrobial therapy for these infections requires coverage against the causative pathogens, particularly Staphylococcus aureus . OBJECTIVE: This randomized, single-blind, multicenter study was designed to compare the efficacy and safety of intravenous ceftazidime 2 g given two times daily (BID) with that of intravenous ceftazidime 1 g given three times daily (TID) for the treatment of skin and skin structure infections caused by ceftazidime-sensitive pathogens . METHODS: Adults (greater-than-or-equal18 years) were eligible for enrollment if they were hospitalized or in home health care settings and they had a skin or skin structure infection caused by a ceftazidime-sensitive pathogen . Patients were randomly assigned to receive ceftazidime 2 g every 12 h or ceftazidime 1 g every 8 h as an intermittent infusion over 15--30 min . Treatment was continued for 2--3 days beyond the time the patient became asymptomatic or evidence of bacterial eradication was obtained; however, total treatment duration had to be at least 5 days . Patients were assessed for their clinical and bacteriological response at the end of treatment and for their clinical response at follow-up . RESULTS: A total of 806 patients were enrolled in the study, 406 of whom received ceftazidime 2 g BID and 400 of whom received ceftazidime 1 g TID . Both treatments were administered for a mean duration of 9 days . At the end of therapy, 248 of 264 (94%) clinically evaluable patients receiving ceftazidime BID and 258 of 275% (94%) clinically evaluable patients receiving ceftazidime TID achieved clinical cure or improvement (p = 0.953) . Pathogens were eradicated or presumed to be eradicated from 217 of 256 (85%) bacteriologically evaluable patients receiving ceftazidime BID and from 228 of 266 (86%) bacteriologically evaluable patients receiving ceftazidime TID (p = 0.760) . Of 1131 isolates, the most common pathogens were S . aureus (30%) and Pseudomonas aeruginosa (10%) . Both regimens were well tolerated with only 13 patients (3%) in the BID group and 16 patients (4%) in the TID group withdrawing because of an adverse event . CONCLUSIONS: These data indicate that ceftazidime 2 g given twice daily is as effective as ceftazidime 1 g given three times daily for the treatment of skin and skin structure infections . In addition, the twice-daily regimen has the advantage of convenience.

Acta Derm Venereol, 2001 Nov-Dec, 81(6), 406 - 9
Differential proteinase expression by Pseudomonas aeruginosa derived from chronic leg ulcers; Schmidtchen A et al.; Pseudomonas aeruginosa colonizes 20-30% of all venous leg ulcers . Hypothetically, P . aeraginosa could release proteases and cytotoxic substances in the environment of chronic ulcers, thus negatively affecting the wound-healing activity in this patient group . Here we show that P . aeruginosa isolates from leg ulcers exhibit a highly variable expression of the proteinases elastase and alkaline proteinase . We propose that bacterial phenotype should be taken into account in future studies on the clinical outcome of leg ulcers colonized by P . aeruginosa.

Bioorg Med Chem Lett, 2002 Mar 11, 12(5), 763 - 6
Peptidomimetics of efflux pump inhibitors potentiate the activity of levofloxacin in Pseudomonas aeruginosa; Renau TE et al.; Several classes of peptidomimetics of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595) have been prepared and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin in Pseudomonas aeruginosa . A number of the new analogues were as active or more active than the lead, demonstrating that a peptide backbone is not essential for activity.

Eur J Biochem, 2002 Jan, 269(2), 593 - 601
Functional expression of Pseudomonas aeruginosa GDP-4-keto-6-deoxy-D-mannose reductase which synthesizes GDP-rhamnose; Maki M et al.; Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals . A-band lipopolysaccharide of P . aeruginosa contains d-rhamnosylated O-antigen . The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose . It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD) . Here, we describe the enzymatic characterization of P . aeruginosa RMD expressed in Saccharomyces cerevisiae . Previous success in functional expression of bacterial gmd genes in S . cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose . Thus, coexpression of the Helicobacter pylori gmd and P . aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose . This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy . The functional expression of P . aeruginosa RMD in S . cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.

Eur J Epidemiol, 2001, 17(5), 491 - 6
Hansenula anomala outbreak at a surgical intensive care unit: a search for risk factors; Kalenic S et al.; During a 5-month period, Hansenula anomala (H . anomala), an opportunistic fungus, caused an outbreak of infections in eight adult patients treated at a surgical intensive care unit (ICU) . The source of the infections and route of transmission could not be identified . A case-control study included 32 patients treated simultaneously at the surgical ICU . Univariate analysis pointed to the following significant risk factors: blood alkalosis, reduced urea, duration of hospitalization, bacteremia and colonization with Pseudomonas aeruginosa, and an APACHE II score >17 (during bacteremia or fungemia) . The stepwise logistic regression multivariate analysis showed only the duration of blood alkalosis to be significant in case patients.

Nihon Kokyuki Gakkai Zasshi, 2001 Nov, 39(11), 877 - 81
{A case of multiple emphysematous bullae treated with living-donor lung transplantation from identical twin brothers}; Miwa T et al.; A 30-year-old man with multiple emphysematous bullae and bronchiectasis was admitted to Toyama Medical and Pharmaceutical University Hospital because of exertional dyspnea and fever . His chest radiograph and CT scan revealed multiple large bullae in the right lung and infiltrative shadows in the left middle lung field . Pseudomonas aeruginosa was isolated from his sputum culture . Although standard therapies including various antibiotics were administered, his respiratory condition was exacerbated, accompanied with the enlargement of bullae in the right lung and the consequent shift of the mediastinum to the left . The patient and his family proposed lung transplantation, and we concluded that lung transplantation would be an appropriate treatment for his disease . We transported the patient to Osaka University Hospital . Living-donor lung transplantation from the patient's identical twin brothers was successfully performed.

Infect Immun, 2002 Mar, 70(3), 1507 - 17
Construction and characterization of a live, attenuated aroA deletion mutant of Pseudomonas aeruginosa as a candidate intranasal vaccine; Priebe GP et al.; Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses . With the goal of developing a broadly protective P . aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P . aeruginosa serogroup O2/O5 strain PAO1 . The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA . The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids . When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects . No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal . Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P . aeruginosa serogroup O2/O5 strains . The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains . This live, attenuated P . aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P . aeruginosa O2/O5 serogroup.

Infect Immun, 2002 Mar, 70(3), 1352 - 8
Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1; Wieland CW et al.; Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients . P . aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation . Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta-6, gamma interferon (IFN-gamma), MIP-1 alpha MIP-2 in the lungs but did not affect IL-18 levels . ETA did not induce TNF-alpha and was a weak inducer of IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-2 . Remarkably, ETA reduced constitutive lung IL-18 levels . LPS was the only factor inducing IFN-gamma . LPS, PLC, and ETA all induced cell infiltration in the lungs . The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated . When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-alpha, IL-1 beta, and IFN-gamma than did wild-type (WT) mice . Similarly, a milder effect of ETA on IL-1 beta and IL-18 was observed for IRF-1 KO than for WT mice . In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice . Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA . IRF-1 deficiency had no effect on MIP-1 alpha and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA . Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8(+) and NK cells in IRF-1 KO mice compared to percentages observed for WT mice . These data indicate that different virulence factors from P . aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.

Zhonghua Yan Ke Za Zhi, 2000 Jan, 36(1), 43 - 5
{Effect on keratocyte-mediated collagen degradation by Pseudomonas aeruginosa}; Hao J et al.; OBJECTIVE: To study the pathogenesis of cornea melting (ulceration) by pseudomona (P) aeruginosa for instruction of clinical treatment . METHODS: Type I collagen gels with or without suspended keratocytes were incubated for 24 hours under medium containing sterile P . aeruginosa culture broth . Native collagen fibrils were removed from the media by ultrafiltration . The ultrafiltrates were then hydrolyzed, and the amount of hydroxyproline was measured spectrophotometrically . The effect of a synthetic matrix metalloproteinase (MMP) inhibitor, Galardin, on collagen degradation was also examined . RESULTS: P . aeruginosa broth induced type I collagen gel degradation directly . In the presence of keratocytes, degradation by P . aeruginosa broth was enhanced . Galardin significantly reduced the amount of collagen degraded by P . aeruginosa culture broth, no matter keratocytes were present or not . CONCLUSION: P . aeruginosa culture broth directly degrades type I collagen and also increases keratocyte-mediated collagen degradation . The result is helpful to the clinical treatment of cornea melting caused by P . aeruginosa, and the mechanism should be further studied.

J Appl Microbiol, 2001 Dec, 91(6), 972 - 84
The relationships and susceptibilities of some industrial, laboratory and clinical isolates of Pseudomonas aeruginosa to some antibiotics and biocides; Lambert RJ et al.; AIMS: To provide evidence to support or refute the hypothesis that cross-resistance between antibiotics and biocides can occur . METHODS AND RESULTS: Fifty-five strains of Pseudomonas aeruginosa were tested for their resistance to anti-pseudomonal antibacterials . Twenty clinical, 19 industrial and 16 culture collection isolates were used . The MIC was found for the antibiotics amikacin, ceftazidime, ciprofloxacin, gentamycin, ticarcillin, tobramycin, imipenem and polymyxin B . The MIC was also found for the biocides benzalkonium chloride and chlorhexidine . The analysis of the data was based on the production of a normal distribution of the log (MIC) plots for each antimicrobial . Strains were then labelled as resistant, intermediate or sensitive based on the mean and standard deviation of the distributions . CONCLUSIONS: In general the clinical isolates were the most recalcitrant organisms, with the industrial isolates being the most sensitive . SIGNIFICANCE AND IMPACT OF THE STUDY: The work shows that antibiotic/biocide correlations do occur, especially with clinical strains . That such correlations were not found with industrial isolates suggests that the clinical environment is responsible for the correlation . We could infer that it is the selective pressure of antibiotic usage that differentiates the clinical environment from the industrial.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 854 - 8
Risk factors for piperacillin-tazobactam-resistant Pseudomonas aeruginosa among hospitalized patients; Harris AD et al.; Antimicrobial resistance is an emerging problem with Pseudomonas aeruginosa . This study determined risk factors for the recovery of piperacillin-tazobactam-resistant P . aeruginosa from clinical cultures from hospitalized patients . A case-control study design was used to compare two groups of case patients with control patients . The first group of case patients was defined by nosocomial isolation of piperacillin-tazobactam-resistant P . aeruginosa, and the second group of cases yielded piperacillin-tazobactam-susceptible P . aeruginosa . Controls were selected in a 6:1 ratio from the same medical or surgical services among which piperacillin-tazobactam-resistant P . aeruginosa arose in patients . Risk factors analyzed included antimicrobial drug exposure, comorbid conditions, and demographics . Bivariate and multivariable analyses were performed . Piperacillin-tazobactam-resistant P . aeruginosa was isolated from 179 patients, and piperacillin-tazobactam-susceptible P . aeruginosa was isolated from 624 patients over a 2.5-year period . Piperacillin-tazobactam (odds ratio {OR} = 6.82; 95% confidence interval {CI}, 4.56 to 10.21), imipenem (OR = 2.42; 95% CI, 1.19 to 4.94), aminoglycosides (OR = 2.18; 95% CI, 1.44 to 3.28), vancomycin (OR = 1.87; 95% CI, 1.21 to 2.89), and broad-spectrum cephalosporins (OR = 2.38; 95% CI, 1.45 to 3.88) were the antibiotics associated with the isolation of piperacillin-tazobactam-resistant P . aeruginosa . Exposure to vancomycin (OR = 1.53; 95% CI, 1.13 to 2.06) or ampicillin-sulbactam (OR = 2.28; 95% CI, 1.62 to 3.21) was associated with recovery of piperacillin-tazobactam-susceptible P . aeruginosa . In this study, antibiotics associated with piperacillin-tazobactam-susceptible P . aeruginosa were different from antibiotics associated with piperacillin-tazobactam-resistant P . aeruginosa . Piperacillin-tazobactam was a strong risk factor for piperacillin-tazobactam-resistant P . aeruginosa . Our results suggest that the nosocomial isolation of piperacillin-tazobactam-resistant P . aeruginosa may be affected by multiple antibiotics.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 689 - 94
Antibacterial properties of dermaseptin S4 derivatives with in vivo activity; Navon-Venezia S et al.; Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents . Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K4K20-S4 and two shorter versions, K4-S4(1-16) and K4-S4(1-13) . The range of MICs of K4K20-S4 against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 microg/ml . MICs of the short derivatives were rather similar or two to fourfold higher . Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either E . coli or S . aureus by 6 log units in 30 min or less . Compared with MSI-78 or PG-1, K4-S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes . Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the L- or D isomer of either of the dermaseptin derivatives . The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with P . aeruginosa . Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K4-S4(1-16) or K4-S4(1-13), respectively . In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K4-S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h . Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 665 - 71
Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility; Valdezate S et al.; The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S . maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml . GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5% . Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E . coli (73.0 to 88.6%) were observed . Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs . The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected . The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg) . In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE . The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs . An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains . These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S . maltophilia.

Antimicrob Agents Chemother, 2002 Mar, 46(3), 638 - 45
Molecular characterization of a novel class 1 integron containing bla(GES-1) and a fused product of aac3-Ib/aac6'-Ib' gene cassettes in Pseudomonas aeruginosa; Dubois V et al.; As seen by the disk diffusion method, the clinical strain of Pseudomonas aeruginosa Pa695, resistant to all extended-spectrum cephalosporins and aminoglycosides, exhibited an unusual synergistic effect between ceftazidime and imipenem . This isolate produced an extended-spectrum beta-lactamase (ESBL) with a pI of 5.8 that appeared to be chromosomally encoded . Cloning experiments revealed that this ESBL was encoded by bla(GES-1), previously described in an integron from Klebsiella pneumoniae . In P . aeruginosa Pa695, a higher level of resistance to ceftazidime than to ticarcillin was observed, and no synergy between the beta-lactamase inhibitors and extended-spectrum cephalosporins was detected, in contrast to the resistance pattern observed in K . pneumoniae . Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located in a class 1 integron, which contained another sequence corresponding to the fused aac3-Ib and aac6'-Ib' gene cassettes . The fusion product was functional, as was the product of each gene cloned separately: AAC3-I, despite the deletion of the four last amino acids, and AAC6', which carried three amino acid changes compared with the most homologous sequence . The AAC3-I protein conferred an expected gentamicin and fortimicin resistance, and the AAC6', despite the Leu-119-->Ser substitution, yielded resistance to kanamycin, tobramycin, and dibekacin, but slightly affected netilmicin and amikacin, and had no apparent effect on gentamicin . The fusion product conveyed a large profile of resistance, combining the AAC6' activity with a higher level of gentamicin resistance without accompanying fortimicin resistance.

Int J Antimicrob Agents, 2002 Feb, 19(2), 159 - 62
Use of Etests with carbapenems for Gram-negative rods producing beta-lactamases; Kocazeybek BS et al.; The in vitro activity of imipenem and meropenem on strains of Gram-negative rods producing extended-spectrum beta-lactamase (ESBL) and inducible beta-lactamase (IBL) was studied using the Etest . In all, 185 strains from the surgical intensive care units of four different hospitals were looked at over 2 years . Of these, 94 were ESBL producers and 91 were IBL positive . The in vitro sensitivities of imipenem and meropenem were 89.7 and 95.1%, respectively, against all strains . The imipenem and meropenem sensitivities of Klebsiella spp . were 98.4 and 100%, respectively, but imipenem resistance (21.6%) in Pseudomonas aeruginosa strains was higher than that of meropenem (10.8%).

Bioorg Chem, 2001 Dec, 29(6), 387 - 97
4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane; Royt PW et al.; The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC . Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline . This compound has been previously isolated and named pseudan IX, a name which we use here . We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical . Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII . Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract . The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron .

Roum Arch Microbiol Immunol, 1999 Apr-Jul, 58(2), 101 - 9
Experimental studies on the bacterial product CANTASTIM derived from Pseudomonas aeruginosa . V . Protective effect in endotoxin shock; Salageanu A et al.; In this paper, we investigated the effect of the treatment with the bacterial immunomodulator CANTASTIM in a model of endotoxin shock in mice . Among the different mouse models described for septic shock, we have chosen the low-dose endotoxin model using D-galactosamine sensitized mice . We noticed a significant increase in the survival rate of the mice treated with CANTASTIM before the endotoxin challenge . This protective effect was correlated with a strong reduction in the level of TNF alpha in the sera of treated mice . Prior exposure to CANTASTIM also attenuated subsequent ex vivo nitric oxide production by peritoneal macrophages of the mice . In this model of endotoxin shock, the major role has been attributed to TNF alpha acting through its receptor TNFRI (p55) . A downregulation of this receptor as a consequence of the treatment with CANTASTIM may be hypothesized . However, the intervention of CANTASTIM in other points in the cytokine network involved in endotoxin shock cannot be excluded.

Pflugers Arch, 2001, 443 Suppl 1, S55 - 61 Epub 2001 Sep 28.
Altered sialyl- and fucosyl-linkage on mucins in cystic fibrosis patients promotes formation of the sialyl-Lewis X determinant on salivary MUC-5B and MUC-7; Shori DK et al.; Destruction of the lungs as a consequence of recurrent infections with microorganisms such as Pseudomonas aeruginosa remains the underlying cause of most morbidity and mortality in cystic fibrosis (CF) . We have hypothesized that changes in the glycosylation of key tracheal mucins such as MUC5B and MUC7 might increase the risk of pulmonary disease in CF patients . However, in preference to sputum we have examined the sugar-chains on these mucins in saliva because in the latter not only can the glycoproteins be collected from controls, but they are essentially free from modifications made following bacterial infection in disease . Proteins in ductal or whole-mouth saliva from 20 CF patients with the Delta F-508 CFTR mutation and age-and sex-matched controls were separated by SDS-PAGE and blotted onto nitrocellulose and then probed with labelled lectins of known specificity . Linkage of terminal sialic acid on the blotted mucins was determined using Sambucus nigra agglutinin (detects the 2-->6 linkage) and Maackia amurensis agglutinin (the 2-->3 linkage) . Fucose was detected by Ulex europaeus agglutinin-1 (1-->2 linkage) and Aleuria aurantia agglutinin (1-->3 linkage) . We found that each mucin shows a characteristic glycosylation pattern and in controls most of the sialic acid is 2-->6 linked on MG1 (MUC 5B) and 2-->3 linked on MG2 (MUC 7) . CF is associated with a shift from a 2-->6 linkage to a 2-->3 linkage on MG1 with some patients showing almost no 2-->6 linkage; 2-->3 linkage on MG2 is similarly increased in disease in some individuals . The expression of fucose on these mucins is also raised in CF patients . These shift to a 2-->3 linkage of sialic acid, and with increased fucosylation this promotes the formation of sialyl-Lewis X antigen detected on CF mucins in our study . These changes will be tested for their correlation with the severity of lung disease . We gratefully acknowledge support from the European Union Biomed-II Programme.

Pflugers Arch, 2001, 443 Suppl 1, S40 - 4 Epub 2001 Aug 31.
Relationship between IkappaBalpha deficiency, NFkappaB activity and interleukin-8 production in CF human airway epithelial cells; Tabary O et al.; Several recent reports have suggested that airway inflammation may precede infection and relate to an endogenous dysregulation of pro-inflammatory cytokines in cystic fibrosis (CF) airways . Evidence suggests that activation of the nuclear factor kappa B (NFkappaB), which regulates the inflammatory gene transcription, depends on the degradation of the inhibitory factor IkappaBalpha . We show that, in in situ human DeltaF508 CF bronchial tissues, inhibitor factor IkappaBalpha is not present in gland cells, although endogenous levels of chemokine IL-8 are high . These data are confirmed by studying cultured CF human bronchial gland cells, in which a lack of cytosolic IkappaBalpha and high levels of activated NFkappaB, concomitant with IL-8 overproduction (a 13-fold increase) are found when compared to non-CF bronchial gland cells . Interestingly, treatment of CF gland cells with the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, results in a significant decrease ( P < 0.001) in IL-8 production down to levels released by non-CF gland cells . The addition of genistein also reverses the effects of lipopolysaccharide (LPS) Pseudomonas-aeruginosa-induced nuclear translocation of NFkappaB by increasing IkappaBalpha protein level (65%) in CF gland cells . Our data indicate that the induction of IkappaBalpha protein in CF airway glandular epithelial cells may be a novel mechanism by which IL-8-mediated lung inflammatory events are markedly reduced in CF patients, at least at the airway glandular level.

Pflugers Arch, 2001, 443 Suppl 1, S36 - 9 Epub 2001 Jul 07.
CFTR may play a role in regulated secretion by lymphocytes: a new hypothesis for the pathophysiology of cystic fibrosis; Bubien JK; Human lymphocytes and pancreatic acinar cells have a common function: both cell types secrete specific proteins in response to extracellular signals . Acinar cells secrete digestive enzymes, while lymphocytes secrete antibodies and cytokines . Both cell types utilize similar receptor-mediated activation systems, similar signal transduction pathways (i.e., alpha adrenergic receptors, and cAMP), and express the cystic fibrosis transmembrane conductance regulator (CFTR) . Preliminary tests of the hypothesis that B lymphocytes are capable of regulated secretion were carried out using transformed lymphocytes . lambda light chain secretion rates were measured in response to treatment with 8-CPT-cAMP . A rapid transient increase in secretion was observed in non-CF lymphocytes . This effect was absent in CF lymphocytes . A failure of regulated secretion could cause a reduced response to antigen presentation, and an inability to completely clear pathogens such as Pseudomonas aeruginosa . Another piece of circumstantial evidence is that lung-transplanted CF patients remain chronically ill . While immunosuppressive therapy may contribute to the chronic illness, the phenomenon is more acute in CF lung-transplant patients than non-CF lung-transplant recipients receiving the same immunosuppressive therapy . A defect in regulated secretion of antibodies and cytokines in response to antigens may be the source of a long suspected, but as yet unproved CFTR-mediated immunological defect underlying the pulmonary morbidity and mortality in cystic fibrosis (CF).

N Engl J Med, 2002 Feb 14, 346(7), 491 - 6
Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis; Shinefield H et al.; BACKGROUND: In patients with decreased resistance to infection, Staphylococcus aureus is a major cause of bacteremia and its complications . The capsular polysaccharides are essential for the pathogenesis of and immunity to S . aureus infection and are targets for vaccines . METHODS: In a double-blind trial involving patients with end-stage renal disease who were receiving hemodialysis, we evaluated the safety, immunogenicity, and efficacy of a vaccine with S . aureus type 5 and 8 capsular polysaccharides conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A . Between April 1998 and August 1999, 1804 adult patients at 73 hemodialysis centers were randomly assigned to receive a single intramuscular injection of either vaccine or saline . IgG antibodies to S . aureus type 5 and 8 capsular polysaccharides were measured for up to two years, and episodes of S . aureus bacteremia were recorded . Efficacy was estimated by comparing the incidence of S . aureus bacteremia in the patients who received the vaccine with the incidence in the control patients . RESULTS: Reactions to the vaccine were generally mild to moderate, and most resolved within two days . The capsular polysaccharides elicited an antibody response of at least 80 microg per milliliter (the estimated minimal level conferring protection) in 80 percent of patients for type 5 and in 75 percent of patients for type 8 . The efficacy during weeks 3 to 54 was only 26 percent (P=0.23) . However, between weeks 3 and 40 after vaccination, S . aureus bacteremia developed in 11 of 892 patients in the vaccine group who could be evaluated for bacteremia, as compared with 26 of 906 patients in the control group (estimate of efficacy, 57 percent; 95 percent confidence interval, 10 to 81 percent; nominal P=0.02) . CONCLUSIONS: In patients receiving hemodialysis, a conjugate vaccine can confer partial immunity against S . aureus bacteremia for approximately 40 weeks, after which protection wanes as antibody levels decrease.

Clin Microbiol Infect, 2001 Dec, 7(12), 706 - 8
Pseudomonas aeruginosa: antibiotic susceptibility and genotypic characterization of strains isolated in the intensive care unit; Bertrand X et al.; A prospective study was carried out to assess the incidence and the local antibiotic susceptibility of Pseudomonas aeruginosa in intensive care units (ICUs) and to characterize cross-transmission by using pulsed-field gel electrophoresis as an epidemiologic tool . For this purpose, we screened surveillance cultures and routine clinical cultures from patients admitted to two adult ICUs during a 2-year period . Antibiotic susceptibility was determined by a disk diffusion method . The overall incidence of P . aeruginosa was 19.1 cases per 100 patients . Our findings concerning the antibiotic resistance of clinical isolates were concordant with those of other studies . Genotyping revealed that approximately 53.5% of P . aeruginosa colonization was acquired via cross-transmission; the other cases probably originated from endogenous sources . Cross-colonization seems to make a large contribution to the spread of P . aeruginosa in ICUs.

Artif Organs, 2001 Dec, 25(12), 951 - 60
Pyrogen retention by highly permeable synthetic membranes during in vitro dialysis; Lonnemann G et al.; Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment . After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively) . Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C . Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs . Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood . High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min . After 60 min, membrane-dependent differences were no longer detectable . Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane . Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments . DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood . Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate . High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone . In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.

Infect Control Hosp Epidemiol, 2001 Nov, 22(11), 720 - 3
Presence of polyclonal Pseudomonas aeruginosa in an intensive care unit: a 27-month prospective study on molecular epidemiology; Cortes P et al.; Pseudomonas aeruginosa was isolated in 22.2% of 305 intensive care unit environmental cultures . A high genetic heterogeneity (18 pulsotypes) was evident . Taps and related surfaces were a stable reservoir for certain pulsotypes . The 15.4% of the P . aeruginosa-positive cultures were polyclonal . Different colony morphotypes should be assayed in surveillance studies.

J Invest Dermatol, 2002 Feb, 118(2), 275 - 81
Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation; Liu AY et al.; Intact human epidermis resists invasion by pathogenic microbes but the biochemical basis of its resistance is not well understood . Recently, an antimicrobial peptide, human beta-defensin-2, was discovered in inflamed epidermis . We used a recombinant baculovirus/insect cell system to produce human beta-defensin-2 and confirmed that at micromolar concentrations it has a broad spectrum of antimicrobial activity, with the striking exception of Staphylococcus aureus . Immunostaining with a polyclonal antibody to human beta-defensin-2 showed that the expression of human beta-defensin-2 peptide by human keratinocytes required differentiation of the cells (either by increased calcium concentration or by growth and maturation in epidermal organotypic culture) as well as a cytokine or bacterial stimulus . Interleukin-1alpha, interleukin-1beta, or live Pseudomonas aeruginosa proved to be the most effective stimuli whereas other bacteria and cytokines had little or no ability to induce human beta-defensin-2 synthesis . In interleukin-1alpha-stimulated epidermal cultures, human beta-defensin-2 first appeared in the cytoplasm in differentiated suprabasal layers of skin, next in a more peripheral web-like distribution in the upper layers of the epidermis, and then over a few days migrated to the stratum corneum . By semiquantitative Western blot analysis of epidermal lysates, the average concentration of human beta-defensin-2 in stimulated organotypic epidermal culture reached 15--70 microg per gram of tissue, i.e., 3.5-16 microM, well within the range required for antimicrobial activity . Because of the restricted pattern of human beta-defensin-2 distribution in the epidermis, its local concentration must be much higher . Defensins and other antimicrobial peptides of inflamed epidermis are likely to play an important antimicrobial role in host defense against cutaneous pathogens.

J Biotechnol, 2002 Mar 28, 94(2), 195 - 212
The enhancement by surfactants of hexadecane degradation by Pseudomonas aeruginosa varies with substrate availability; Noordman WH et al.; The rhamnolipid biosurfactant produced by Pseudomonas aeruginosa influences various processes related to hydrocarbon degradation . However, degradation can only be enhanced by the surfactant when it stimulates a process that is rate limiting under the applied conditions . Therefore we determined how rhamnolipid influences hexadecane degradation by P . aeruginosa UG2 under conditions differing in hexadecane availability . The rate of hexadecane degradation in shake flask cultures was lower for hexadecane entrapped in a matrix with 6 nm pores (silica 60) or in quartz sand than for hexadecane immobilized in matrices with pore sizes larger than 300 nm or for hexadecane present as a separate liquid phase . This indicates that the availability of hexadecane decreased with decreasing pore size under these conditions . The rate-limiting step for hexadecane entrapped in silica 60 was the mass transfer of substrate from the matrix to the bulk liquid phase, whereas for hexadecane present as a second liquid phase it was the uptake of the substrate by the cells . Hexadecane degradation in batch incubations was accelerated by the addition of rhamnolipid or other surfactants in all experiments except in those where hexadecane was entrapped in silica 60, indicating that the surfactants stimulated uptake of hexadecane by the cells . Since rhamnolipid stimulated the degradation rate in batch experiments to a greater extent than any of the other 14 surfactants tested, hexadecane uptake was apparently more enhanced by rhamnolipid than by the other surfactants . Although rhamnolipid did not stimulate the release of hexadecane from silica 60 under conditions of intense agitation, it significantly enhanced this rate during column experiments in the absence of strain UG2 . The results demonstrate that rhamnolipid enhances degradation by stimulating release of entrapped substrate in column studies under conditions of low agitation and by stimulating uptake of substrate by the cells, especially when degradation is not limited by release of substrate from the matrices.

Drug Discov Today, 2002 Feb 15, 7(4), 247 - 58
Bacterial toxins as tools for mucosal vaccination; Mrsny RJ et al.; Several studies have demonstrated that the biological properties of secreted bacterial toxins could be harnessed for the induction of mucosal and systemic immunity following application at epithelial surfaces . Although the properties and potential application of several of these toxins will be discussed in this review, special focus will be placed on Pseudomonas aeruginosa exotoxin A (PE) . A non-toxic form of PE (ntPE) into which antigenic epitopes can be integrated appears to be a particularly promising vaccination tool, which is able to cross the polarized epithelia of the gastrointestinal, respiratory and reproductive tracts and selectively target macrophages and dendritic cells.

Z Naturforsch {C}, 2001 Nov-Dec, 56(11-12), 933 - 8
A new pyoverdin from Pseudomonas aeruginosa R'; Ruangviriyachai C et al.; From a Pseudomonas aeruginosa hospital isolate a new pyoverdin was isolated . It is identical with that of Pseudomonas aeruginosa strain R except that in the peptide chain L-Gln is missing.

Pediatr Pulmonol, 2002 Mar, 33(3), 201 - 7
Mannose-binding lectin (MBL) therapy in an MBL-deficient patient with severe cystic fibrosis lung disease; Garred P et al.; Deficiency of mannose-binding lectin has been shown to be a risk factor for cystic fibrosis (CF) patients . We, therefore, decided to treat a patient with CF, mannose-binding lectin deficiency, severe bronchopulmonary Pseudomonas aeruginosa infection, and rapid deterioration of lung function with purified mannose-binding lectin in an attempt to ameliorate the course of the lung disease . The mannose-binding lectin used originated from pooled human donor plasma and was given as an intravenous infusion twice a week for a period of 3 months . The patients's clinical condition was stabilized during the treatment period, but was not improved . No adverse events were observed . However, the lung function assessed as percent forced expiratory volume in 1 sec (FEV1%) and percent forced vital capacirt (FVC%) correlated significantly with the mannose-binding serum lectin levels (rho=+0.68, P=0.008, and rho=+0.73, P=0.004) . Additionally, an inverse correlation with the acute phase-reactant C-reactive protein and the proinflammatory cytokine IL-6 was observed (rho=-0.49, P=0.007 and rho=-0.41, P=0.04, respectively) . The results emphasize the importance of mannose-binding lectin as a secondary disease modifier in CF . Moreover, purified mannose-binding lectin can safely be administered to chronically ill patients, and may be a potential treatment in CF and other diseases in which mannose-binding lectin deficiency plays a pathophysiological role .

Bioorg Med Chem, 2002 Apr, 10(4), 869 - 74
Chloropyrimidines as a new class of antimicrobial agents; Agarwal N et al.; In the course of our investigations of pyrimidines as antimycotic agents, we have identified a sub-class, with significant in vitro activity against mycobacteria . The salient feature of these pyrimidine derivatives (3a-o and 7a,b) is their appended aryl, heteroaryl and alkylthio substituent at position 6 and also alkylthio substituent at position 2 . The rational design, synthesis, and evaluation of the in vitro antibacterial activity against six pathogenic bacteria including virulent and non-virulent strains of Mycobacterium tuberculosis is described . Some of the synthesized compounds (3c, 3h, 3i, 3o) have displayed only potent in vitro antimycobacterial activity with MIC of 0.75 microg/mL except 3i which also demonstrated activity against Escherichia coli at 12.5 microg/mL concentration . Only two compounds, 3a and 3b, demonstrated antibacterial activity against Pseudomonas aeruginosa and E . coli with MIC 12.5 microg/mL . All the synthesized compounds were also evaluated for their antimycotic activity against five pathogenic fungi but only some of them 3j-n and 7a,b were found most potent against Aspergillus fumigatus and Trichophyton mentagrophytes.

J Biomed Mater Res, 2002 Apr, 60(1), 206 - 15
Prophylactic treatment of gram-positive and gram-negative abdominal implant infections using locally delivered polyclonal antibodies; Poelstra KA et al.; The increasing clinical incidence and host risk of biomaterial-centered infections, as well as the reduced effectiveness of clinically relevant antibiotics to treat such infections, provide compelling reasons to develop new approaches for treating implanted biomaterials in a surgical context . We describe the direct local delivery of polyclonal human antibodies to abdominal surgical implant sites to reduce infection severity and mortality in a lethal murine model of surgical implant-centered peritoneal infection . Surgical implant-centered peritonitis was produced in 180 female CF-1 mice by the direct inoculation of surgical-grade polypropylene mesh disks placed in the peritoneal cavity with lethal doses of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa . Mice randomly received a resorbable antibody delivery vehicle at the implant site: either a blank carboxymethylcellulose (CMC) aqueous gel or the same CMC gel containing 10 mg of pooled polyclonal human immunoglobulin G locally on the implant after infection, either alone or in combination with systemic doses of cefazolin or vancomycin antibiotics . Human antibodies were rapidly released (first-order kinetics) from the gel carrier to both peritoneal fluids and serum in both infection scenarios . Inocula required for lethal infection were substantially reduced by surgery and the presence of the implant versus a closed lethal peritonitis model . Survival to 10 days with two different gram-negative P . aeruginosa strains was significantly enhanced (p < 0.01) by the direct application of CMC gel containing antibodies alone to the surgical implant site . Human-equivalent doses of systemic vancomycin provided a significantly improved benefit (p < 0.01) against lethal, implant-centered, gram-positive MRSA infection . However, locally delivered polyclonal human antibodies in combination with a range of systemic vancomycin doses against MRSA failed to improve host survival . Successful antibody therapy against gram-negative, implant-centered infections complements the clinically routine use of systemic antibiotics, providing a mechanism of protection independent of antibiotic resistance .

Dermatology, 2002, 204(1), 50 - 5
Treatment of refractory atopic dermatitis using 'wet-wrap' dressings and diluted corticosteroids: results of standardized treatment in both children and adults; Devillers AC et al.; BACKGROUND: 'Wet-wrap' dressings with diluted corticosteroids form an alternative treatment in patients with refractory atopic dermatitis (AD) . OBJECTIVE: To evaluate a standardized treatment, using wet-wrap dressings with diluted corticosteroids, in patients with refractory AD . METHODS: Results of treatment, complications and possible side effects were retrospectively evaluated in 14 children and 12 adults . RESULTS: Skin lesions improved dramatically during 1 week of inpatient treatment . A significant decrease in early-morning serum cortisol levels was measured . Levels below the normal range were only observed after 1 week in 2 adults and on day 4 in 3 children . Suppression of the hypothalamus-pituitary-adrenal-cortex axis in 1 adult and a new exacerbation of AD in 2 children and 3 adults complicated long-term treatment at home . Additional complications included folliculitis, a Pseudomonas aeruginosa infection, a secondary bacterial infection and refractory skin lesions between bandages . CONCLUSION: Wet-wrap dressings and diluted corticosteroids form an effective treatment in patients with refractory AD .

Curr Opin Microbiol, 2002 Feb, 5(1), 81 - 6
CFTR mutations and host susceptibility to Pseudomonas aeruginosa lung infection; Pier GB; The susceptibility of cystic fibrosis patients to bacterial pathogens is associated with deficient airway antimicrobial peptide activity, and airway-surface-liquid dehydration with decreased transport velocity and hypersecretion of mucus . Susceptibility to Pseudomonas aeruginosa infection has been linked to the role of the cystic fibrosis transmembrane conductance regulator protein as a receptor for P . aeruginosa . Binding of P . aeruginosa coordinates lung clearance as part of innate immunity . The function of CFTR in innate immunity to P . aeruginosa infection is multifactorial, with one key component being a specific ligand-receptor interaction between the protein and the microbe.

J Pept Sci, 2002 Jan, 8(1), 36 - 43
The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin; Strom MB et al.; We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin . We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins . All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9 . Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa . The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively . The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM) . The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity . All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).

Biochem Biophys Res Commun, 2002 Feb 15, 291(1), 1 - 7
Cyanide binding to cd(1) nitrite reductase from Pseudomonas aeruginosa: role of the active-site His369 in ligand stabilization; Sun W et al.; Cyanide binding to fully reduced Pseudomonas aeruginosa cd(1) nitrite reductase (Pa cd(1) NiR) has been investigated for the wild-type enzyme and a site-directed mutant in which the active-site His369 was replaced by Ala . This mutation reduces the affinity toward cyanide (by approximately 13-fold) and especially decreases the rate of binding of cyanide to the reduced d(1) heme (by approximately 100-fold) . The crystal structure of wild-type reduced Pa cd(1) NiR saturated with cyanide was determined to a resolution of 2.7 A . Cyanide binds to the iron of the d(1) heme, with an Fe-C-N angle of 168 degrees for both subunits of the dimer and only His369 is within hydrogen bonding distance of the nitrogen atom of the ligand . These results suggest that in Pa cd(1) NiR the invariant distal residue His369 plays a dominant role in controlling the binding of anionic ligands and allow the discussion of the mechanism of cyanide binding to the wild-type enzyme . (c)2002 Elsevier Science (USA).

Nippon Geka Gakkai Zasshi, 2001 Dec, 102(12), 846 - 50
{Guidelines for antibiotic agents in the field of respiratory tract surgery}; Hirano T et al.; This paper provides information on the use of antibiotic agents during the postoperative period from three aspects . 1) It is important to evaluate the risk factors of postoperative infection based on the patient's preoperative condition . Diseases treated with respiratory tract surgery are frequently caused by heavy smoking . Therefore it must be remembered that the patients may have low respiratory function . 2) In the prevention of postoperative infection, the special circumstances after respiratory surgery (e.g., the disintegration of the clearance system in the airway caused by the dissection of lymph nodes, the suppression of respiration, and cough caused by thoracotomy accompanied by resection of the ribs) must be considered . Therefore we usually administer antibiotic agents for 3 to 4 days . In general, we select second-generation cephalosporins, be cause gram-negative rod infection frequently occurs . 3) Postoperative infection is diagnosed based on fluctuations in fever, laboratory data (number of white blood cells and C-reactive protein), chest X-ray findings, and properties of drainage fluid . When bacteria are not identified, we must consider MRSA as a gram-positive bacterium and Pseudomonas aeruginosa as a gram-negative bacterium . After the identification of bacteria causing the infection, the antibiotic agents may be changed based on the results of sensitivity tests . The appropriate usage of antibiotic agents in the field of respiratory tract surgery is discussed based on actual clinical experience in our department.

J Surg Res, 2002 Feb, 102(2), 193 - 7
The effect of sepsis on wound healing; Rico RM et al.; BACKGROUND: Normal wound healing is a regulated sequence of events that successfully restore tissue integrity . Previous studies have suggested that wound healing is impaired in a septic host . The current study examines the effect of sepsis on the inflammatory and proliferative phases of wound healing at a remote site of secondary injury . METHODS: Polyvinyl alcohol sponges, either inoculated with a standard dose of Pseudomonas aeruginosa (experimental) or soaked in normal saline (control), were placed subcutaneously in the anterior abdominal region of male B6D2F1 mice . Immediately following sponge placement, full thickness excisional dermal wounds were created on the dorsum . Wound healing was examined at days 3, 5, and 7 postinjury . The infiltration of neutrophils and macrophages into wounds was quantified, and the reepithelialization rate and collagen content were measured . RESULTS: Peripheral neutrophil counts were significantly elevated in infected mice, yet neutrophil content of the remote wound of infected animals was significantly reduced (5% of control, P < 0.05) . Wounds of infected mice also showed a 30% reduction in the macrophage content . Wounds of infected animals exhibited delayed reepithelialization (76 +/- 3 vs 97 +/- 3% at day 5, P < 0.05) and collagen synthesis (55.3 +/- 9.5 vs 105 +/- 13.0 microg/wound, P < 0.05) . CONCLUSION: Systemic infection alters both the inflammatory and the proliferative processes at remote sites of injury . Multiple factors seem likely to contribute to the increased incidence of wound complications in septic patients . (c)2001 Elsevier Science.

J Thorac Imaging, 2002 Jan, 17(1), 53 - 7
Spectrum of CT findings in nosocomial Pseudomonas aeruginosa pneumonia; Shah RM et al.; The purpose of this study was to evaluate CT findings in nosocomial Pseudomonas aeruginosa Pneumonia (PAP) and to compare features of PAP in patients with isolated P . aeruginosa cultures and those with coexistent infections . A retrospective database search revealed 28 patients with nosocomial PAP (12 men, 16 women; mean age, 57 years) in which thoracic CT had been performed within a mean of 1.7 days from the time of respiratory culture . Two chest radiologists blinded to culture data performed a consensus reading noting distribution and pattern of consolidation, ground-glass opacity, nodules, peribronchial infiltration, necrosis, effusions, and pleural enhancement . Coexistent respiratory cultures were recorded . Consolidation was present in all patients, involving multiple lobes in 23 (82%) and demonstrating upper zonal involvement in 23 (82%) . Nodular features were present in 14 (50%), including tree-in-bud patterns with centrilobular distributions in 9 (64%) and larger, randomly distributed nodules in 5 (36%) . Five of five patients with consolidations limited to the lower lung zones had associated upper lung nodules . Ground-glass opacity was seen in nine (31%) and peribronchial infiltration in 16 (57%) . Necrosis was present in eight (29%) . Thirteen (46%) bilateral and five (18%) unilateral pleural effusions were present with enhancement occurring in two (1%) . Coexistent positive respiratory cultures were identified in 13 patients . The distribution of consolidation, frequency and distribution of nodules, and frequency of necrosis did not differ significantly between patients with and without other positive cultures . With CT, PAP most commonly presents with multifocal airspace consolidation . Nodular features were identified in half, with one-third demonstrating tree-in-bud opacities . Unsuspected necrosis occurred in one-third of cases . CT findings in patients with and without other respiratory isolates did not differ in the distribution and frequency of consolidations, nodularity, or necrosis.

J Clin Invest, 2002 Feb, 109(3), 317 - 25
Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients; Worlitzsch D et al.; Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways . Here we show that, in CF patients with established lung disease, Pseudomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens . In vitro studies revealed that CF-specific increases in epithelial O(2) consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection . Motile P . aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production . With P . aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted . These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P . aeruginosa adapted to anaerobic environments.

Biodegradation, 2001, 12(3), 149 - 57
Induction of the halobenzoate catabolic pathway and cometabolism of ortho-chlorobenzoates in Pseudomonas aeruginosa 142 grown on glucose-supplemented media; Corbella ME et al.; The aerobic cometabolism of ortho-substituted chlorobenzoates by Pseudomonas aeruginosa strain 142 growing on glucose-supplemented medium was analyzed . The strain, which can use 2-chlorobenzoate (2-CBA) and 2,4-dichlorobenzoate (2,4-DCBA) as sole carbon and energy sources, showed high rates of 2-CBA metabolism in glucose-fed cells . In contrast, 2,4-DCBA was metabolized only after extended incubation of the full grown culture and depletion of glucose . In addition to the ortho-dehalogenation (ohb142) genes encoding the alpha and beta subunits of the oxygenase component of a 2-halobenzoate dioxygenase, strain 142 harbours a closely related ohbABCDFG gene cluster previously identified in P . aeruginosa JB2 (ohbJB2) . The genes for the chlorocatechol ortho-catabolic pathway were identified and sequenced in this strain, showing a near complete identity with the clcABD operon of the pAC27 plasmid . Relative quantification of mRNA by RT-PCR shows a preferential induction of ohb142 by 2-CBA, which is abolished in glucose-grown cultures . The alternate ohbJB2 and clc genes were expressed preferentially in 2,4-DCBA grown cultures . Only ohbJB2 appears to be expressed in the presence of the carbohydrate . Detection of chlorocatechol-1,2-dioxygenase activity in 2,4-DCBA plus glucose grown cultures suggests the presence of an alternate system for the ortho-cleavage of chlorobenzoates . The recruitment of elements from two halobenzoate dioxygenase systems with different induction patterns, together with a chlorocatechol degradative pathway not repressed by carbon catabolite, may allow P . aeruginosa 142 to cometabolize haloaromatics in carbohydrate grown cultures.

Electrophoresis, 2002 Jan, 23(1), 8 - 14
Differential staining of western blots of avian egg white glycoproteins using diverse lectins; Lerrer B et al.; Avian egg white glycoproteins which differ in structure and carbohydrate composition, vary in their interactions with diverse lectins . Generally, wheat germ agglutinin (WGA) and concanavalin A (Con A) are used for the identification and separation of those of the chicken . In the present study, interactions of a battery of lectins, including: the above two, several galactophilic lectins (from Aplysia gonad (AGL), Erythrina corallodendron (ECorL), peanut (PNA) and Pseudomonas aeruginosa (PA-IL)), and fucose-binding lectins (from Ulex europaeus (UEA-I), Ulva lactuca (ULL) and P . aeruginosa (PA-IlL), which also binds mannose) with chicken, quail and pigeon egg white glycoproteins, were examined using both hemagglutination inhibition and Western blot analyses . The chicken egg white glycoproteins interacted most strongly with WGA, followed by Con A >> AGL = PA-IlL . The quail glycoprotein order of affinities was: Con A >> WGA = AGL = PA-IlL, while that of the pigeon was: AGL > PA-IL > WGA > Con A = PA-IlL . The blocking of the other lectins by the egg whites were insignificant . The results demonstrated the selectivity and efficiency of the five most reactive lectins for differential tagging of avian egg white glycoproteins and unveiled the profound heterogeneity of the latter, as well as the possible potential lectin usage for improving purification and quality control of the desired glycoproteins.

Electrophoresis, 2001 Dec, 22(20), 4368 - 74
Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns; Vilain S et al.; In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry . In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa . This improvement of focusing concerns more particularly basic proteins . This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips . Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

J Immunol, 2002 Feb 15, 168(4), 1861 - 8
Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection; Usher LR et al.; Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear . Phenazine products of P . aeruginosa can induce death in Caenorhabditis elegans . We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing . We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro . Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P . aeruginosa . 1-hydroxyphenazine was without effect . In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h . Comparison of wild-type and phenazine-deleted strains of P . aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain . In clinical isolates of P . aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture . Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients . Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP . Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis . We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P . aeruginosa in human tissue.

Can J Microbiol, 2001 Dec, 47(12), 1095 - 100
Interactions of Pseudomonas aeruginosa PA-IIL lectin with quail egg white glycoproteins; Lerrer B et al.; Pseudomonas aeruginosa produces several lectins, including the galactophilic PA-IL and the fucose- and mannose-binding PA-IIL . The great advantage of these two lectins is their stability in purified preparations . Following observations that pigeon egg white blocks Escherichia coli P-fimbriae and PA-IL, we examined the interactions of diverse avian egg white components with PA-IIL . This lectin may represent both mannose- and fucose-specific microbial adhesins . For comparison, Con A (which also binds mannose) and Ulex europaeus lectin (UEA-I, which binds fucose) were analyzed in parallel . The lectin interactions with chicken, quail, and pigeon egg whites and several purified chicken egg white glycoproteins were examined by a hemagglutination inhibition test and Western blotting . Both analyses showed that like Con A and unlike UEA-I, which was not sensitive to any of these three egg whites, PA-IIL most strongly reacted with the quail egg white . However, in contrast with Con A, its interactions with the chicken egg white components, excluding avidin, were very poor . The results of this study might indicate the possibility that some of the egg white components that interacted with the above two mannose-binding lectins (exhibiting individual heterogeneity) might be associated with the innate immunity against mannose-specific microbial or viral adhesion during the fowl embryonic period.

Syst Appl Microbiol, 2001 Nov, 24(3), 423 - 9
Rapid parallel detection of hygienically relevant microorganisms in water samples by PCR and specific hybridization in microtiter plates; Frahm E et al.; A molecular biological test protocol for the parallel detection of enterococci and Pseudomonas aeruginosa in drinking water was developed . Amplicons labelled with digoxigenin during PCR were hybridized to specific 23S rDNA targeted oligonucleotide probes immobilized in microtiter plates . Detection was performed by addition of anti-digoxigenin-peroxidase-conjugate and chromogenic substrate . Specificity of the probes was evaluated by using pure cultures . First evaluation data with natural water samples in comparison to conventional microbiological analysis according to the German Drinking Water Regulation showed good agreement . Its feasible and rapid performance should be advantageous for use in routine drinking water quality control . Further comparative evaluation studies need to be undertaken to determine the true applicability for routine testing of water samples.

Recenti Prog Med, 2001 Dec, 92(12), 754 - 5
{Hemoptysis in a patient with post-obstructive pneumonia due to a broncholith}; Ravenna F et al.; A case of a 51-year-old man admitted to the hospital for hemoptysis after a three-week history of fever and cough is presented . The chest x-ray film revealed consolidation in the left upper lobe . Because microbiologic studies of the bronchial lavage showed the grew of Pseudomonas aeruginosa, the patient was treated with piperacilline and pefloxacin . Haemoptysis and abnormal temperature was persistent for several days . Revaluation of the chest x-ray permitted to discover a little calcified nodule of 1 mm diameter . CT scan of the thorax confirmed the consolidation of the left upper lobe and the little calcification . A second fiberoptic bronchoscopy was performed in 10th day, using a bronchoscope with a smaller diameter; it was possible to observe the occlusion of the subsegment bronchus LB 1&2 due to a mobile protruding mass . The mass was gentle removed by the use of alligator bioptic forceps; it presented as a grey, hard, 2 mm diameter body with irregular surface like a "floating mine" . On microscopical examination of the mass, aspergillus hyphae appeared as broad septate filaments . Culture of the samples were negative . Haemoptysis and fever stopped after FOB . Chest x-ray in 19th day was negative: consolidation and calcification were absent.

J Biol Chem, 2002 Apr 5, 277(14), 12082 - 8 Epub 2002 Jan 30.
Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS; Riese MJ et al.; Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin . The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain . We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity . Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP) . In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment . Using {(32)P}NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS . Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro . To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of {(32)P}NAD, the substrate for ADP-ribosylation . After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated . The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.

Diagn Microbiol Infect Dis, 2002 Jan, 42(1), 75 - 8
In vitro synergy testing of levofloxacin, ofloxacin, and ciprofloxacin in combination with aztreonam, ceftazidime, or piperacillin against Pseudomonas aeruginosa; Pendland SL et al.; The synergistic potential of levofloxacin, ofloxacin and ciprofloxacin combined with aztreonam, ceftazidime, or piperacillin was compared using 24 strains of Pseudomonas aeruginosa with varying susceptibility profiles . Levofloxacin and ciprofloxacin demonstrated similar in vitro activity, with ofloxacin demonstrating less activity compared to the other agents . Predominantly additive effects were seen with all combinations, with no significant differences detected between the fluoroquinolone agents.

Diagn Microbiol Infect Dis, 2002 Jan, 42(1), 71 - 3
Clinical evaluation of the Vitek automated system with cards GNS 122 and 127 and VTK-R07.01 software for antimicrobial susceptibility testing of Pseudomonas aeruginosa; Chandler LJ et al.; The performance of the Vitek Automated Susceptibility Testing System software version VTKR07.01 (bioMerieux Vitek, Hazelwood, MO), for testing Pseudomonas aeruginosa was evaluated by comparing results for 200 clinical isolates with those of disk diffusion and manual broth microtiter dilution testing . For cefepime, the restricted major error rate was 0.53% and the minor error rate was 12.5% . For piperacillin, the restricted major error rate was 2.15% . For ticarcillin/clavulanic acid, restricted very major and major error rates of 6.5% and 3.2%, respectively, occurred . The results of our study indicate that the Vitek system performs within acceptable limits when testing piperacillin, but remains problematic for testing cefepime and ticarcillin-clavulanic acid.

World J Gastroenterol, 1998 Oct, 4(5), 388 - 391
Gene therapy for human colorectal carcinoma using human CEA promoter contro led bacterial ADP-ribosylating toxin genes human CEA: PEA & DTA gene transfer; Cao GW et al.; AIM:To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes . METHODS:Pseudomonas exotoxin A domain {II+III (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa.PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically . After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA)promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively . In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out . RESULTS:Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro . Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION:The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.

Invest Ophthalmol Vis Sci, 2002 Feb, 43(2), 419 - 24
Role of IL-12 and IFN-gamma in Pseudomonas aeruginosa corneal infection; Hazlett LD et al.; PURPOSE: In Pseudomonas aeruginosa ocular infection, T-helper cell 1-responsive mouse strains are susceptible (the cornea perforates), and neutralization of IFN-gamma before infection has been shown to delay the onset of perforation . IFN-gamma is the predominant cytokine induced by IL-12, and positive regulation of IL-12 by IFN-gamma, if unchecked, leads to excessive cytokine production and toxicity . Despite its potential importance, the role of IL-12 in ocular infection with P . aeruginosa remains unexplored and was the purpose of this study . METHODS: IL-12 knockout mice, histopathology, RT/PCR and ELISA analyses, immunocytochemistry, and quantitation of viable bacteria in cornea were used to examine the role of IL-12 in IFN-gamma production and the susceptibility phenotype . RESULTS: To directly test the effect of IL-12 on IFN-gamma production, IL-12 knockout and wild-type C57BL/6 mice were used . Both groups of mice were susceptible to infection, with corneal perforation seen at 5 to 7 days after infection . RT-PCR and ELISA analyses confirmed that IL-12 message and protein levels were elevated after infection only in the wild-type mouse cornea . Other differences between the two groups were detected . Knockout versus wild-type mice showed a significant decrease in IFN-gamma mRNA levels in the cornea and cervical lymph nodes and decreased TNF-alpha protein levels in cornea . Corneas of knockout mice also had a significant increase in bacterial load at 5 days after infection when compared with wild-type mice . CONCLUSIONS: These data provide evidence that IL-12 is important in IFN-gamma production and in the absence of the cytokine, both IFN-gamma and TNF-alpha levels in cornea are significantly decreased, resulting in unchecked bacterial growth and perforation.

Curr Opin Investig Drugs, 2001 Feb, 2(2), 212 - 5
MC-207110 Daiichi Seiyaku/Microcide Pharmaceuticals; Barrett JF; Microcide and Daiichi are collaborating on the discovery of new bacterial efflux pump inhibitorsfor the treatment of drug-resistant Pseudomonas aeruginosa and other bacterial infections . The bacterial efflux pump technology program focuses on Gram-negative bacteria that have developed antibiotic resistance by means of the efflux pump {192681}, {341408} . MC-207110 was the initial screening hitfrom Microcide's library and is a low molecular weight dipeptide amide, which shows minimal antibacterial activity but potentiated the activity of levofloxacin by 8-fold at 10 microg/ml . This lead is being optimized to improve on its biological and pharmacological profile {341183} . Optimization studies have been conducted on MC-207110, yielding a class of broad-spectrum efflux pump inhibitors for P aeruginosa . A member of this class has demonstrated in vivo activity against P aeruginosa in a murine neutropenic thigh model {351686}.

J Antimicrob Chemother, 2002 Feb, 49(2), 403 - 6
In vitro activity of the aerosolized agents colistin and tobramycin and five intravenous agents against Pseudomonas aeruginosa isolated from cystic fibrosis patients in southwestern Germany; Schulin T; Three hundred and eighty-five mucoid and non-mucoid strains of Pseudomonas aeruginosa isolated from 192 sputa from 57 adult cystic fibrosis patients were studied . Susceptibility testing using an agar dilution technique was performed for ceftazidime, ciprofloxacin, fosfomycin, meropenem and piperacillin, and the aerosolized agents colistin and tobramycin . Meropenem, ceftazidime and piperacillin were the most potent agents (susceptibility 86.2%, 84.2% and 84%, respectively) . Tobramycin and colistin susceptibility rates were lower, but in light of higher intrabronchial concentrations of these drugs a greater percentage of strains might still be clinically susceptible . Only 46.2% and 41.8% of isolates were susceptible to ciprofloxacin and fosfomycin, respectively.

J Antimicrob Chemother, 2002 Feb, 49(2), 391 - 4
Reproducibility of control organism zone diameters for batches of IsoSensitest agar manufactured from 1996 to 2000 using the BSAC disc susceptibility test method; Landrygan J et al.; The BSAC Working Party on Susceptibility Testing has recently suggested that the performance of IsoSensitest agar has changed since 1991 . Twenty batches of IsoSensitest agar that had been manufactured between 1996 and 2000 were tested using the BSAC standardized disc susceptibility testing method . Antibiotic discs containing amoxicillin 10 microg, ceftazidime 30 microg, gentamicin 10 microg, ciprofloxacin 1 microg and colistin sulphate 25 microg were tested on each batch of media 12 times against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 where appropriate . There was a small reduction in zone sizes for most antibiotics on batches of media that were near their expiration date, but otherwise zone sizes were remarkably consistent . We could find no evidence to suggest that a change in the performance of IsoSensitest agar for the disc diffusion method had occurred since 1996.

Bioorg Med Chem Lett, 2002 Feb 11, 12(3), 383 - 6
Synthesis and antibacterial evaluation of novel 2-{N-Imidoylpyrrolidinyl} carbapenems; Hattori K et al.; The synthesis, antibacterial activity and DHP-susceptibility of a series of novel carbapenems, directly linked with heterocyclic moiety are described . Especially, the compounds linked pyrrolidine-carbapenem exhibited to have a good antibacterial activity against Staphylococcus aureus (MRSA) as well as Pseudomonas aeruginosa to maintain a good stability towards DHP-I.

Biochemistry, 2002 Feb 5, 41(5), 1663 - 71
Recycling of pyoverdin on the FpvA receptor after ferric pyoverdin uptake and dissociation in Pseudomonas aeruginosa; Schalk IJ et al.; Under iron-limiting conditions, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin (PaA), which, after complexing iron, is transported back into the cells via its outer membrane receptor FpvA . The recent finding that all FpvA receptors on the bacterial cell surface are loaded with iron-free PaA under iron limiting conditions has raised questions about the mechanism by which P . aeruginosa transports efficiently iron . We used {(3)H}PaA' {(55)Fe}PaA-Fe, and a kinetically stable chromium-PaA complex to show that iron loading of the receptor occurs through a siderophore displacement mechanism in vivo . Moreover, the fluorescence properties of iron-free PaA revealed that, after PaA-Fe uptake and dissociation, the PaA molecule is recycled into the extracellular medium . We used fluorescence resonance energy transfer (FRET) between the PaA chromophore and the FpvA tryptophans in vivo to monitor the kinetics of PaA displacement by PaA-Fe at the cell surface, the dissociation of iron from the siderophore, and the recycling of PaA back to the receptor on the outer membrane of the bacteria in real time . The loading status of FpvA (PaA versus PaA-Fe) was shown to depend on the relative concentration of the two forms of pyoverdin in the growth medium.

J Mass Spectrom, 2002 Jan, 37(1), 41 - 6
Liquid chromatographic/mass spectrometric detection of the 3-(3-hydroxyalkanoyloxy) alkanoic acid precursors of rhamnolipids in Pseudomonas aeruginosa cultures; Lepine F et al.; A series of pseudomolecular and fragment ions attributed to 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) were detected by liquid chromatography/mass spectrometry among the rhamnolipids observed in a Pseudomonas aeruginosa strain 57RP supernatant . The fragmentation mechanism leading to the formation of the fragment ions was determined by a deuterium exchange experiment and by using a standard HAA mixture obtained from the mild acidic hydrolysis of rhamnolipids of known composition . The structure and the response factor of these free HAAs were determined . The HAAs relative composition differs between free HAAs and those present in rhamnolipids, the former being enriched in lower molecular mass congeners and depleted in the heavier ones . Within an isomeric pair, the isomer with the shortest 3-hydroxyalkaloyl residue at the hydroxyl end was more abundant than the one with the heavier 3-hydroxyalkaloyl acid at this position, and the ratios of their relative abundances were similar for free HAAs and those in rhamnolipids . Experiments with deuterium-labeled rhamnolipids demonstrated that free HAAs are part of a pool used for rhamnolipid biosynthesis and are not rhamnolipid degradation products .

J Infect Chemother, 2001 Dec, 7(4), 258 - 62
Survey of antibiotic resistance in Pseudomonas aeruginosa by The Tokyo Johoku Association of Pseudomonas Studies; Kato K et al.; Pseudomonas aeruginosa resistance (minimum inhibitory concentration {MIC}, > or =16 microg/ml defined as resistant) to meropenem, imipenem, panipenem, piperacillin, ceftazidime, cefozopran, cefoperazone, sulbactam/cefoperazone, amikacin, and tobramycin, as well as cross-resistance profiles, were investigated in P . aeruginosa strains isolated at eight hospitals in the Johoku area, Tokyo, during November 1998 . Overall, 8.3% of isolates were imipenem-resistant and 4.6% were ceftazidime-resistant . However, the incidence of antibiotic-resistant P . aeruginosa was distinctly different at each hospital . P . aeruginosa resistance to imipenem ranged from (MIC) 1 to 64 microg/ml (MIC90 32 microg/ml), and its resistance to ceftazidime ranged from 2 to more than 128 microg/ml (MIC90, 64 microg/ml) . Meropenem (MIC range, < or =0.25 to 16 microg/ml) was more active than panipenem (MIC range, 2 to 64 microg/ml) . Cefozopran was more active than piperacillin, cefoperazone, or sulbactam/cefoperazone, but many strains were resistant to cefoperazone (17/57) . Our analysis found cross-resistance to many beta-lactams, but the degree of cross-resistance was very variable.

J Infect Chemother, 2000 Dec, 6(4), 200 - 5
Determination of intracellular and extracellular beta-lactamase activities of Pseudomonas aeruginosa after exposure to beta-lactams in vitro and in vivo; Matsumura N et al.; Beta-lactamase production in Pseudomonas aeruginosa was determined in in-vitro models and in rat pouch infection models after exposure to ceftazidime, imipenem, and piperacillin . Exposure of 28 P . aeruginosa strains to 1/4 minimum inhibitory concentration (MIC) of ceftazidime, imipenem, and piperacillin for 24 h enhanced intracellular beta-lactamase activities in 14, 22, and 6 strains, respectively, of the 28 clinical strains tested, and enhanced extracellular beta-lactamase activities which were not detected without exposure to antibiotics, in 7, 23, and 1 of the 28 strains, respectively . Extracellular beta-lactamase activity from P . aeruginosa S-1278, producing an inducible beta-lactamase, scarcely increased after exposure to ceftazidime and piperacillin 24 h after incubation, while the activity increased after exposure to imipenem over the range of 1/8 to 8 MIC . In the rat granuloma pouch models infected with P . aeruginosa S-1278, ceftazidime and piperacillin, after single administration (20 mg/kg) and serial administration (20 mg/kg per day x 3 days), did not enhance extracellular beta-lactamase activities . However, the activities were enhanced with single and serial administrations of imipenem, and levels over 10 mU/ml were detected until the third day . The beta-lactamase activity, similar to the activity found in rat pouches after serial administration of imipenem, inactivated various cephalosporins . In conclusion, extracellular beta-lactamase activity was detected both in vitro and in vivo after exposure to a good inducer, and extracellular beta-lactamase remained at infection site at levels that could inactivate cephalosporins.

J Infect Chemother, 2000 Sep, 6(3), 184 - 7
Isolation of Pseudomonas aeruginosa producing OXA-4 beta-lactamase in a Japanese hospital in 1996; Marumo K et al.; One-hundred and fifty isolates of Pseudomonas aeruginosa were collected from different patients' specimens at Showa University Fujigaoka Hospital between April and August 1996 . The 23 isolates resistant to piperacillin (minimum inhibitory concentration, >or=100 microg/ml) were widely distributed in the hospital wards . Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and isoelectric focusing (IEF) methods, we detected OXA-4 beta-lactamase hydrolyzing penams in 4 of the resistant isolates (17%) with O-serotype E . However, no other external beta-lactamase was detected by the IEF method . The hybridization results showed that the OXA-4 genes were located in chromosome, but not in plasmid . Furthermore, the isolation rate was significantly lower than that found between 1992 and 1993 (61%; P < 0.01; chi2-test), suggesting a decreased rate of hospital infection caused by P . aeruginosa producing the OXA-4 enzyme.

J Infect Chemother, 2000 Sep, 6(3), 155 - 61
A new model of pulmonary superinfection with Aspergillus fumigatus and Pseudomonas aeruginosa in mice; Yonezawa M et al.; We have produced a new model of pulmonary super-infection with Aspergillus fumigatus and Pseudomonas aeruginosa in immunosuppressed mice . Male ICR mice were given an intratracheal inoculation of 4 x 10(5) conidia of A . fumigatus in agar beads, and were immunosuppressed with 100 mg/kg subcutaneous injections of cortisone acetate on days 7, 9, 12, 14, and 16 after inoculation . Twelve days after inoculation, with the agar beads, the mice were challenged with the intranasal instillation of 2 x 10(6) CFU of P . aeruginosa . The survival rates of superinfected, A . fumigatus-alone, P . aeruginosa-alone, and non-infected mice were 50%, 30%, 90%, and 100% 14 days after pseudomonal infection (26 days after inoculation of A . fumigatus), respectively . In the superinfected mice, both A . fumigatus and P . aeruginosa were detected more than 10 days after pseudomonal infection (22 days after inoculation of A . fumigatus) . Histopathological examination revealed peribronchial necrosis around A . fumigatus hyphae and inflammation by P . aeruginosa . This infection model in mice would be useful for studying the pathogenesis of superinfection.

J Infect Chemother, 2000 Jun, 6(2), 86 - 92
Synergistic bactericidal effects of acrinol and tetracycline against Pseudomonas aeruginosa; Saji M et al.; Combined treatment of acrinol (Ac) and tetracycline hydrochloride (Tc) against Pseudomonas aeruginosa strains isolated from clinical specimens synergistically increased the bactericidal effect . The minimum bactericidal concentration (MBC) of Ac against P . aeruginosa strain no . 985 was 200 microg/ml, while the MBC of Ac against strains no . 47 and no . 783 was above 800 microg/ml for each . The MBC of Tc was above 400 microg/ml against each of the tested strains . However, simultaneous treatment with 25 microg/ml Ac and 200 microg/ml Tc against P . aeruginosa strain no . 985 decreased the viable cell number from 107 cfu/ml to <10 cfu/ml within 24 h, while a higher concentration of Tc (400 microg/ml) with Ac (25 microg/ml) reduced the viable cell number to <10 cfu/ml within 8 h . A similar synergistic bactericidal effect of Ac and Tc was observed in strains no . 47 and no . 783 by treatment with 200 microg/ml Ac and 200 microg/ml or 400 microg/ml Tc . The degree of bactericidal effect against P . aeruginosa was proportional to the concentration of Tc under the condition of a constant concentration of Ac . Furthermore, Ac-treated cells of strain no . 47 were killed by a following Tc treatment, but cells pretreated with Tc did not show such a sensitivity to Ac . To induce the synergistic effect of Ac and Tc, Ac must be applied to P . aeruginosa before or at the same time as Tc.

J Infect Chemother, 2000 Mar, 6(1), 26 - 9
Development of a rapid assay for screening point mutations associated with quinolone resistance in the Pseudomonas aeruginosa parC gene; Deguchi T et al.; To detect quinolone resistance-associated mutations within the Ser-80 and Glu-84 codons of the Pseudomonas aeruginosa parC gene, we developed a rapid and simple assay based on polymerase chain reaction (PCR) amplification of the region of the parC gene containing the mutation sites and digestion of the PCR products with a restriction enzyme . The mutations generating alterations at Ser-80 and Glu-84 were detected as restriction fragment length polymorphisms of the PCR products digested with HinfI . Among 22 clinical isolates tested by this assay, mutations at the Ser-80 and Glu-84 codons were detected in all 10 isolates in which the presence of the mutations had been confirmed previously by DNA sequencing . This rapid and simple assay could be a useful screening device for genetic alterations associated with resistance to quinolones in the P . aeruginosa parC gene.

J Infect Chemother, 2000 Mar, 6(1), 1 - 7
Potential of macrolide antibiotics to inhibit protein synthesis of Pseudomonas aeruginosa: suppression of virulence factors and stress response; Tateda K et al.; Recently we have reported that sub-minimum inhibitory concentrations (MICs) of macrolide antibiotics, such as erythromycin, clarithromycin, and azithromycin, induce loss of viability of Pseudomonas aeruginosa with longer incubation periods . In the present study we examined the effects of sub-MICs of macrolide antibiotics on protein synthesis and the expression of heat shock proteins (Gro-EL) in P . aeruginosa and the association of these factors with the viability of P . aeruginosa . In seven strains of P . aeruginosa clinical isolates, inhibition of protein synthesis was generally observed in bacteria grown on agar with sub-MIC azithromycin (8 microg/ml) at 24 h, and this was followed by loss of viability after an additional 24-h incubation . The inhibition of protein synthesis was shown in bacteria treated with sub-MICs of erythromycin and clarithromycin, but not with sub-MICs of other antibiotics examined (josamycin, tobramycin, ofloxacin, clindamycin, and ceftazidime) even at relatively high sub-MICs . In the heat shock condition (45 degrees C), strong expression of the heat shock protein Gro-EL was induced in bacteria grown on antibiotic-free medium, whereas there was a delay of such a response in bacteria exposed to 4 microg/ml of azithromycin . Reflecting these results, an abrupt reduction of viability in azithromycin-treated bacteria was observed within 3 h in the heat shock condition . Western blot analysis, using specific antibody for Gro-EL, demonstrated that erythromycin, clarithromycin, and azithromycin, at concentrations of 0.5-2 microg/ml, inhibited the expression of lower-molecular weight Gro-EL bands in the constitutive state . These results indicated that macrolides, at concentrations far below the MICs, suppressed protein synthesis in P . aeruginosa, an effect which may be associated with the inhibition of P . aeruginosa virulence and its loss of viability with longer incubation . Moreover, it is likely that the macrolides may sensitize bacteria to stresses, as these antibiotics induced alterations in a major stress protein, Gro-EL, in constitutive and inducible states.

J Infect Chemother, 1999 Dec, 5(4), 201 - 205
Present situation of serotyping of Pseudomonas aeruginosa in Japan and correlation among three kinds of commercially available serotyping kits; Goto S et al.; We carried out the serotyping of 196 freshly isolated strains of Pseudomonas aeruginosa with international antigenic typing system (IATS) antibodies from Rougier Bio-Tech (RBT kit) and other two kinds of antibodies, from Meiji Seika Kaisha (Mei assay kit) and Denka Seiken (Seiken kit) to ascertain the relationship among the serotypes obtained by the different methods . All the test strains belonging to the serogroups A, C, E, F, G, H, I, J, and K were well correlated with each other with the Mei assay and the Seiken kits . However, 21 of 26 strains of serogroup M typed with the Mei assay kit were non-typable with the Seiken kit . It was confirmed that serogroups obtained with both these methods were closely related; 171 of the 196 strains of P . aeruginosa tested (87.2%) were classified into identical serogroups . From the serotyping results for P . aeruginosa strains with the Seiken and the RBT kits and with the Mei assay and the RBT kits, it was found that the serotypes of 172 of the 192 strains tested (89.6%) and those of 164 of the 170 strains tested (96.5%), respectively were well correlated . However, the serotyping of P . aeruginosa with the RBT kit may be problematic, as the kit did not contain the antisera agglutinated with the serogroup M strains of P . aeruginosa classified with the Mei assay and Seiken kits.

J Infect Chemother, 1999 Sep, 5(3), 168 - 170
Estimation of outer membrane permeability of carbapenem antibiotics to Pseudomonas aeruginosa; Iyobe S et al.; The outer membrane permeability of carbapenems (imipenem {IPM}, panipenem {PAPM}, meropenem {MEPM}, and biapenem {BIPM}) and ceftazidime (CAZ) to Pseudomonas aeruginosa was determined by the Zimmermann and Rosselet method . The permeability coefficients of beta-lactams tested at 50 &mgr;M concentration of substrates ranged from (0.40 +/- 0.10) x 10-6 cm/s for CAZ to (2.33 +/- 0.33) x 10-6 cm/s for IPM, indicating that the outer membrane permeability of carbapenems to P . aeruginosa was high in comparison with that of CAZ . In particular, IPM and BIPM showed a higher rate of penetration than MEPM and PAPM.

Clin Infect Dis, 2002 Mar 1, 34(5), 603 - 11 Epub 2002 Jan 23.
Nosocomial spread of the integron-located veb-1-like cassette encoding an extended-pectrum beta-lactamase in Pseudomonas aeruginosa in Thailand; Girlich D et al.; The beta-lactamase gene content and epidemiology of ceftazidime-resistant Pseudomonas aeruginosa isolates (24% of the total number of P . aeruginosa isolates) were investigated at a University Hospital in Thailand during a 4-month period in 1999 . Of 33 nonrepetitive clinical isolates, 31 produced a VEB-1-like clavulanic acid-inhibited extended-spectrum beta-lactamase (ESBL) . These isolates belonged to different pulsed-field gel electrophoresis types and subtypes . In 1 case, the bla(VEB-1)-like gene was plasmid located . The bla(VEB-1)-like genes were present as a gene cassette on class 1 integrons that varied in size and structure . In most cases, the veb-1 cassette was associated with an arr-2 cassette (rifampin resistance), aminoglycoside resistance gene cassettes, and an oxa-10-like cassette encoding a narrow-spectrum oxacillinase-type beta-lactamase . The present study indicates that ESBLs may be endemic in P . aeruginosa and illustrates that integrons are efficient means for their spread.

J Bacteriol, 2002 Feb, 184(4), 1132 - 9
The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine lactone contributes to virulence and induces inflammation in vivo; Smith RS et al.; Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl . These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI . LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone . There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells . In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P . aeruginosa had on colonization of the lung . We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P . aeruginosa to colonize the lung . To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice . In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3 . Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression . The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain . We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment . Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections . Therefore, the quorum-sensing systems of P . aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.

J Bacteriol, 2002 Feb, 184(4), 1057 - 64
Global genomic analysis of AlgU (sigma(E))-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis; Firoved AM et al.; The conversion of Pseudomonas aeruginosa to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF) . A major pathway of conversion to mucoidy in clinical strains of P . aeruginosa is dependent upon activation of the alternative sigma factor AlgU (P . aeruginosa sigma(E)) . Here we initiated studies of AlgU-dependent global expression patterns in P . aeruginosa in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy . Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (sigma(E)) promoter sites on the P . aeruginosa chromosome . Each candidate promoter was individually tested by reverse transcription and mRNA 5'-end mapping using RNA isolated from algU(+) and algU::Tc(r) mutant cells . A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped . Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes . Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes, lptA and lptB, had proinflammatory activity . In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung . Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given sigma factor (collectively termed here sigmulon) using conventional molecular tools . In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity . This link may be of significance for infections and inflammatory processes in CF.

Therapie, 2001 Sep-Oct, 56(5), 519 - 24
Ciprofloxacin pharmacokinetics in young cystic fibrosis patients after repeated oral doses; Odoul F et al.; Until recently, only compassionate use of ciprofloxacin in children with cystic fibrosis was possible despite limited pharmacokinetic data . We studied five subjects with cystic fibrosis and exacerbation of pulmonary infection . A 15 mg/kg b.i.d . regimen of oral ciprofloxacin was administered . On the 15th day, eight blood samples were collected and plasma concentrations were measured by HPLC . The actual dose administered ranged from 10 to 14 mg/kg b.i.d., due to the fixed-dosage formulation . Corresponding AUC0-12 ranged from 8.2 to 11.9 mg.h/L . Plasma concentrations were maintained above individual MICs for a median time of 12.7 h over 24 h . The median area under the inhibitory curve was 52.8, which is about half the proposed target value for ciprofloxacin in pulmonary infections with Gram-negative bacteria such as Pseudomonas aeruginosa . A higher dose, administered from a specific formulation to ensure precise dosing, must be given in order to obtain adequate concentrations in cystic fibrosis children.

Rev Med Interne, 2001 Dec, 22 Suppl 3, 367s - 373s
{Cystic fibrosis in adulthood}; Durieu I; Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the CFTR gene . The number of adult CF patients increased dramatically, since life expectancy is now around thirty years . CF is usually a pediatric disease . In adult patients the disease associate a diffuse bronchectasia with chronic colonisation of sputum with Pseudomonas aeruginosa, and pancreatic insufficiency . Mortality is usually related to respiratory insufficiency . One third of adult patients develop diabetes mellitus . A diagnosis of CF can be made in adult patients particularly when it exists male infertility with congenital absence of vas deferens, chronic sinusitis or diffuse bronchectasia or chronic pancreatitis, acute and recurrent pancreatitis, allergic bronchopulmonary aspergillosis . The diagnosis is established with positive sweat chloride concentration, or double CFTR mutations and/or other suggestive organ involvement.

Am J Respir Cell Mol Biol, 2002 Feb, 26(2), 216 - 23
Pseudomonas aeruginosa and tumor necrosis factor-alpha attenuate Clara cell secretory protein promoter function; Harrod KS et al.; The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals . Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury . CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection . In the present study, the role of decreased promoter function in the regulation of CCSP by P.a . was assessed using an in vitro co-culture system and in vivo studies of transgenic mice . CCSP promoter activity in lung epithelial cells was markedly decreased by P.a . or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner . Regulation of CCSP promoter function by either P.a . or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity . Decreased regulation of the CCSP promoter by P.a . or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a . or TNF-alpha, respectively . A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells . TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a . administration . Using a transgenic mouse model, P.a . administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo . The attenuation of CCSP promoter activity in lung epithelial cells by P.a . involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

Acta Biotheor, 2001, 49(4), 207 - 18
Positive feedback circuits and adaptive regulations in bacteria; Guespin-Michel J et al.; The mechanisms by which bacteria adapt to changes in their environment involve transcriptional regulation in which a transcriptional regulator responds to signal(s) from the environment and regulates (positively or negatively) the expression of several genes or operons . Some of these regulators exert a positive feedback on their own expression . This is a necessary (although not sufficient) condition for the occurrence of multistationarity . One biological consequence of multistationarity may be epigenetic modifications, a hypothesis unusual to microbiologists, in spite of some well-known epigenetic modifications in bacteria . We propose here that the occurrence of mucoidy in the opportunistic pathogen Pseudomonas aeruginosa, which is currently attributed to mutations only, may also be an epigenetic modification . A theoretical approach using a generalised logical analysis lends credit to this hypothesis and suggests experiments to ascertain it.

J Bioenerg Biomembr, 2001 Dec, 33(6), 513 - 21
Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease; Ko YH et al.; The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter-like protein found in the plasma membrane of animal cells . CFTR is believed to function primarily as a Cl- channel, but evidence is mounting that this protein has other roles as well . Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains . These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process . Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1 . The most common mutation causing CF is the deletion (delta) of a single phenylalanine (F) in position 508 within a putative helix located in NBF1 . CF patients bearing this deltaF508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30 . Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the deltaF508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect . In general, this pioneering or "ground roots" research has succeeded according to plan . This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory . For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and "cost effective" strategies to identify agents that correct the structural defect in the deltaF508 CFTR protein causing most cases of CF.

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Apr, 24(4), 204 - 7
{Alterations of pulmonary surfactant during Pseudomonas aeruginosa pneumonia of immunocompromised rats}; Li Z et al.; OBJECTIVE: To analyze the alterations of pulmonary surfactant in immunocompromised host with Pseudomonas aeruginosa (PA) pneumonia . METHODS: 50 rats were randomly divided into two groups, one immunosuppressed with cyclophosphamide and cortisone acetate as ICH group, another as control group (CON), their lung tissue and bronchoalveolar lavage fluid (BALF) were collected before PA challenging and 3 h, 6 h, 9 h, 24 h after PA challenging intratracheally, wet/dry ratio (W/D) of lung tissue were measured, concentrations of total protein (TP), total phospholipids (TPL), disaturated phosphatidylcholine (DSPC) and surfactant protein A (SP-A) in BALF were analyzed . RESULTS: The ratios of DSPC/TPL and DSPC/TP decreased significantly in both ICH and CON groups after PA infection, there were not significant differences between the two groups, no change in the concentrations of TPL and DSPC . Concentration of SP-A and SP-A/TP in ICH group decreased remarkably 6 h after PA pulmonary infection than before {(1.8 +/- 1.1) microgram/ml vs (4.2 +/- 1.5) microgram/ml, (1.4 +/- 0.7) microgram/mg vs (11.7 +/- 8.1) microgram/mg, P < 0.05}, meanwhile there were no significant alterations in CON group . W/D and TP concentrations increased in both groups after PA challenging, however the alterations were much greater in ICH group (P < 0.05) . Alterations of SP-A appeared a negative correlation with alterations of TP and W/D (r = -0.793, P < 0.01, r = -0.769, P < 0.01) . The ratios of SP-A/TPL and SP-A/DSPC decreased significantly 6 h after PA challenging than before, the ratios were much lower in ICH group than in CON group during 6 h-9 h after PA inoculation . CONCLUSION: After PA pulmonary infection, alterations of phospholipids could initially appear a relative decrement of DSPC during acute phase of infection, much remarkable decrease of SP-A in ICH group could be associated with the more severe lung injury, alterations of SP-A were more obvious than surfactant lipids in ICH.

Biochem J, 2002 Feb 1, 361(Pt 3), 577 - 85
Modulation of the reactivity of the essential cysteine residue of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa; Gonzalez-Segura L et al.; Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine . In the human opportunistic pathogen Pseudomonas aeruginosa this reaction is an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors . As with every aldehyde dehydrogenase studied so far, BADH possesses an essential cysteine residue involved in the formation of the intermediate thiohemiacetal with the aldehyde substrate . We report here that the chemical modification of this residue is conveniently measured by the loss in enzyme activity, which allowed us to explore its reactivity in a pH range around neutrality . The pH dependence of the observed second-order rate constant of BADH inactivation by methyl methanethiosulphonate (MMTS) suggests that at low pH values the essential cysteine residue exists as thiolate by the formation of an ion pair with a positively charged residue . The estimated macroscopic pK values are 8.6 and 4.0 for the free and ion-pair-forming thiolate respectively . The reactivity towards MMTS of both thiolate forms is notably lower than that of model compounds of similar pK, suggesting a considerable steric inhibition by the structure of the protein . Binding of the dinucleotides rapidly induced a significant and transitory increment of thiolate reactivity, followed by a relatively slow change to an almost unreactive form . Thus it seems that to gain protection against oxidation without compromising catalytic efficiency, BADH from P . aeruginosa has evolved a complex and previously undescribed mechanism, involving several conformational rearrangements of the active site, to suit the reactivity of the essential thiol to the availability of coenzyme and substrate.

Crit Care Med, 2001 Dec, 29(12), 2239 - 44
Clinical aspiration-related practice patterns in the intensive care unit: a physician survey; Rebuck JA et al.; OBJECTIVE: To characterize physician practice patterns regarding the clinical, microbiological, and antimicrobial-related events of suspected or documented aspiration and aspiration pneumonia within the intensive care unit . DESIGN: National mail survey . SETTING: University medical center . STUDY POPULATION: Critical care physician members of the Society of Critical Care Medicine . INTERVENTIONS: Survey questionnaire . MEASUREMENTS AND MAIN RESULTS: The response rate was 645 (32%) of 2,000 mailed surveys; analysis of data represents completed questionnaires from 605 respondents . Intensivists (42.3%), pulmonologists (22.6%), and surgeons (21.6%) represent the majority of respondents . Altered level of consciousness (67.9%) in the intensive care unit was identified as the predominant predisposing factor for aspiration pneumonia . Sixty-four percent of physicians used sputum specimens, rather than protected specimen brushes or bronchoalveolar lavage, as the diagnostic source of bacterial cultures in cases of suspected aspiration pneumonia . Microbiological assessment of aspiration pneumonia revealed the absence of any predominant pathogen, although Staphylococcus aureus and Pseudomonas aeruginosa were cited in 40.1% of combined responses, whereas anaerobes represented the fifth most prevalent cultured bacteria . In cases of suspected and confirmed aspiration, 51.9% and 77.7% of respondents, respectively, would prescribe an antimicrobial agent in the absence of a definitive infectious process, with administration of dual antimicrobial therapy increasing from 28.9% to 46.0% in suspected vs . confirmed cases of aspiration . In the treatment of aspiration pneumonia, 27.6% of physicians preferred pathogen-specific therapy, whereas the remainder (72.4%) selected an empirical antibiotic regimen based on prior clinical experience . Overall, a beta-lactam/beta-lactamase inhibitor, followed by a cephalosporin, aminoglycoside in combination, or clindamycin, was most often selected for empirical therapy of all defined aspiration-related clinical diagnoses . CONCLUSIONS: Our study revealed a divergent approach to antimicrobial treatment of cases of aspiration in the intensive care unit . Further investigation is warranted to determine why empirical antimicrobials are initiated frequently for noninfectious stages of aspiration.

J Ethnopharmacol, 2002 Feb, 79(2), 213 - 20
Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo; Cimanga K et al.; The chemical composition of essential oils from 15 aromatic medicinal plant species growing in the Democratic Republic of Congo have been studied . More than 15 constituents in an amount higher than 0.1% were identified in each essential oil . 1,8-cineole, alpha and beta-pinene, p-cymene, myrcene, gamma-terpinene, alpha-terpineol and limonene were prevalent constituents in almost more than 10 selected plant species . Results from the antibacterial testing by the diffusion method indicate that all essential oils (5 microl per disc) inhibited the growth of selected bacteria at different extents . The most active antibacterial essential oils were those of the leaves of Eucalyptus camadulensis and Eucalyptus terticornis (12-30 mm zone diameter of inhibition) . They showed particularly a most potent inhibition of Pseudomonas aeruginosa growth (15-16 mm), followed by Eucalyptus robusta (12 mm) . Essential oils from the leaves of Eucalyptus alba, Eucalyptus citriodora, Eucalyptus deglupta, Eucalyptus globulus, Eucalyptus saligna, Eucalyptus robusta, Aframomum stipulatum, Cymbopogon citratus, Ocimum americanum and that of the seeds of Monodora myristica showed also a good antibacterial activity (10-18 mm) . Eucalyptus propinqua, Eucalyptus urophylla and Ocimum gratissimum essential oils were the less active samples against the selected bacteria . No correlation between the amount of major constituents such as 1,8-cineol, alpha-pinene, p-cymene, cryptone or thymol and the antibacterial activity was observed.

Arch Microbiol, 2001 Dec, 177(1), 47 - 53 Epub 2001 Oct 30.
Pseudomonas aeruginosa corneal infection affects cholinergic enzymes in rat lacrimal gland; Dannelly KH et al.; Since the increased use of extended-wear contact lenses, Pseudomonas aeruginosa has emerged as a primary etiological agent of ulcerative keratitis . Clinical isolates have been classified into two types: cytotoxic and non-cytotoxic . This study revealed significant immune and neuro-enzymatic changes elicited by the two types of P . aeruginosa in the lacrimal gland of rats . The humoral immune response in the lacrimal gland to the non-cytotoxic strain was significantly lower than to the cytotoxic strain, possibly due to the immunogenicity of the extracellular toxin; however, the same effect was not seen in the serum . Choline acetyltransferase and acetylcholinesterase are known to be responsible for synthesis and degradation of acetylcholine, respectively, binding receptors on acini and plasma cells, modulating their activity, and constituting the principle regulator of tear secretion . Following infection, neuro-enzyme activities were significantly modified to reduce the concentration of acetylcholine and therefore potentially reduce secretion from the glands . The data lead to the hypothesis that P . aeruginosa may have the potential to reduce the protective barrier provided by the lacrimal gland to benefit pathogenicity . It was also observed that the neuro-enzyme response of the lacrimal glands of uninfected eyes of the test animals was the same as that of infected eyes, implying that the signal may be relayed by common lymphoidal tissue or the central nervous system and a measurable response returned to both eyes.

Chest, 2002 Jan, 121(1), 88 - 94
Exhaled and sputum nitric oxide in bronchiectasis: correlation with clinical parameters; Tsang KW et al.; STUDY OBJECTIVES: Although there has been tremendous attention on endogenous nitric oxide (NO) production in many respiratory and systemic diseases, little is known on NO production in bronchiectasis . DESIGN AND SETTING: We determined exhaled and sputum NO levels in 109 patients with stable bronchiectasis (71 women; mean +/- SD age, 58.2 +/- 14.1 years) and 78 control subjects (39 women; mean age, 56.7 +/- 12.1 years) by using an automatic chemiluminescence analyzer . MEASUREMENTS AND RESULTS: There was no significant difference in exhaled NO between patients with bronchiectasis and control subjects (p = 0.11) . Bronchiectasis patients with Pseudomonas aeruginosa infection had a significantly lower exhaled, but not sputum, NO levels than their counterparts and control subjects (p = 0.04 and p = 0.009, respectively) . Exhaled NO correlated with 24-h sputum volume in P aeruginosa-infected patients (r = - 0.36; p = 0.002) . After adjustment for sputum volume and number of bronchiectatic lung lobes, P aeruginosa-infected patients still had lower exhaled NO levels than their counterparts (p = 0.01) . There was no correlation between exhaled NO with FEV(1), FVC, and the number of bronchiectatic lung lobes (p > 0.05) . Sputum NO levels were not different between patients and control subjects (p = 0.64), and had no correlation with clinical parameters . CONCLUSION: Exhaled NO appears to be reduced among bronchiectasis patients with P aeruginosa infection independent of other clinical parameters, and further studies on the potential mechanisms and pathogenetic implications of this reduction should be pursued.

Chest, 2002 Jan, 121(1), 81 - 7
Exhaled H(2)O(2) in steady-state bronchiectasis: relationship with cellular composition in induced sputum, spirometry, and extent and severity of disease; Loukides S et al.; STUDY OBJECTIVES: To determine the concentration of exhaled H(2)O(2) in patients with bronchiectasis, and to study the relationship between levels of exhaled H(2)O(2), extent of disease, symptoms score, spirometry, and cellular composition obtained from induced sputum; furthermore, to account for possible confounding effects of inhaled corticosteroids (ICS) usage, long-term oral antibiotic treatment, and chronic colonization with Pseudomonas aeruginosa . DESIGN: Cross-sectional study . PATIENTS: Thirty patients with steady-state bronchiectasis . RESULTS: Mean (95% confidence interval {CI}) exhaled H(2)O(2) levels were significantly elevated in patients with bronchiectasis compared to normal subjects: 1.1 (0.87 to 1.29) microM vs 0.3 (0.19 to 0.36) microM, respectively (p < 0.0001) . Patients treated with ICS had similar values as steroid-naive patients . The group of patients with P aeruginosa colonization showed a significantly increased concentration of H(2)O(2) compared to the group without P aeruginosa colonization . Patients receiving long-term oral antibiotic treatment had significantly higher values of H(2)O(2) compared to those not receiving antibiotics . There was a significant positive correlation between H(2)O(2) and either the percentage of neutrophils in induced sputum or the extent of the disease as defined by high-resolution CT . A significant negative correlation was found between H(2)O(2) and FEV(1) percent predicted . Finally, there was a significant positive correlation between H(2)O(2) and the symptoms score . CONCLUSIONS: Patients with bronchiectasis in stable condition showed increased levels of exhaled H(2)O(2) . The above-mentioned levels were not decreased either by ICS or long-term oral antibiotic treatment, but were significantly affected by chronic colonization with P aeruginosa . H(2)O(2) levels could be an indirect index of neutrophilic inflammation, impairment of lung function, and extension and severity of the disease.

Chest, 2002 Jan, 121(1), 55 - 63
Long-term benefits of inhaled tobramycin in adolescent patients with cystic fibrosis; Moss RB; STUDY OBJECTIVE: To determine the effect of long-term suppression of Pseudomonas aeruginosa on lung function and other clinical end points in adolescent patients with cystic fibrosis (CF) . DESIGN: Two identical, randomized, placebo-controlled trials followed by three open-label follow-on trials . SETTING: Sixty-nine CF study centers in the United States . INTERVENTIONS: Active drug consisting of a 300-mg tobramycin solution for inhalation (TSI) . PATIENTS: One hundred twenty-eight adolescent CF patients (aged 13 to 17 years) with P aeruginosa and mild-to-moderate lung disease (FEV(1) percent predicted > or = 25% and < or = 75%) . MEASUREMENTS: Pulmonary function, P aeruginosa colony forming unit density, incidence of hospitalization and IV antibiotic use, weight gain, and aminoglycoside toxicity were monitored . RESULTS: At the end of the first three 28-day cycles of TSI treatment, patients originally randomized to TSI and placebo treatments exhibited improvements in FEV(1) percent predicted of 13.5% and 9.4%, respectively . FEV(1) percent predicted was maintained above the value at initiation of TSI treatment in both groups . At the end of the last "on-drug" period (92 weeks), patients originally randomized to TSI and placebo treatments showed improvements of 14.3% and 1.8%, respectively . Improvement in pulmonary function was significantly correlated with reduction in P aeruginosa colony forming unit density (p = 0.0001) . The average number of hospitalizations and IV antibiotic courses did not increase over time . TSI treatment was associated with increased weight gain and body mass index . P aeruginosa susceptibility to tobramycin decreased slightly over time, but this was not correlated with clinical response . CONCLUSIONS: TSI treatment improved pulmonary function and weight gain in adolescent patients with CF over a 2-year period of long-term, intermittent use.

Chest, 2002 Jan, 121(1), 48 - 54
Iron deficiency in cystic fibrosis: relationship to lung disease severity and chronic Pseudomonas aeruginosa infection; Reid DW et al.; BACKGROUND: Iron deficiency (ID) is common in patients with cystic fibrosis (CF) and may be related to GI factors and chronic inflammation . Pseudomonas aeruginosa (PA) infection is predominantly responsible for chronic lung suppuration in patients with CF, but its survival is critically dependent on the availability of extracellular iron, which it obtains via highly efficient mechanisms . OBJECTIVE: To determine whether ID in CF patients is directly related to the severity of suppurative lung disease . DESIGN: We determined the iron status of 30 randomly selected adult CF patients (13 women) and assessed the relationship to lung disease severity and GI factors by determining their daily sputum volume, FEV(1) percent predicted, C-reactive protein (CRP) level, erythrocyte sedimentation rate, and degree of pancreatic supplementation . Additionally, we measured the sputum concentrations of iron and ferritin in a randomly selected subgroup of 13 of the 30 subjects . SETTING: Adult CF Service in a tertiary-care center . RESULTS: Seventy-four percent of subjects experienced ID (ie, serum iron levels < or = 12 micromol/L and/or transferrin saturation levels < or = 16%) . There was no relationship found with the degree of pancreatic supplementation . The daily sputum volume was strongly associated with low serum iron levels, transferrin saturation, ferritin/CRP ratio, and FEV(1) percent predicted (p < 0.05) . Serum iron levels and transferrin saturation were negatively related to CRP (r = -0.8 and r = -0.7, respectively; p < 0.01) and erythrocyte sedimentation rate (r = -0.5 and r = -0.4, respectively; p < 0.05) . FEV(1) percent predicted was positively related to serum iron level (r = 0.5; p < 0.01), transferrin saturation (r = 0.4; p < 0.05), and ferritin/CRP ratio (r = 0.7; p < 0.05) . Sputum iron concentration (median, 63 micromol/L; range, 17 to 134 micromol/L) and ferritin concentration (median, 5,038 microg/L; range, 894 to 6,982 microg/L) exceeded plasma levels and negatively correlated with FEV(1) percent predicted (r = -0.6 and r = -0.5, respectively; p < or = 0.05) . CONCLUSION: In our CF patients, ID was directly related to the increased severity of suppurative lung disease but not to the degree of pancreatic insufficiency . Iron loss into the airway may contribute to ID and may facilitate PA infection.

Chest, 2002 Jan, 121(1), 40 - 7
Nasal polyps in cystic fibrosis: clinical endoscopic study with nasal lavage fluid analysis; Henriksson G et al.; STUDY OBJECTIVES: Nasal polyps frequently appear in patients with cystic fibrosis (CF) . The aims of this study were to focus on what problems (symptoms, endoscopic findings, and laboratory correlates) nasal polyps cause the CF patient, and how these correlate to the total health situation of this patient group . PATIENTS AND STUDY DESIGN: The clinical histories, endoscopic investigations of the nasal cavity, and analyses of nasal lavage fluid of 44 patients with CF complicated with nasal polyposis have been compared with those of 67 CF control subjects . The patients were examined at annual control examinations (with pulmonary tests, working capacity, liver tests, and bacterial and blood tests) from 1995 to 1996 at Stockholm Cystic Fibrosis Center, Huddinge University Hospital . All patients were > 2 years of age . The endoscopic findings were related to the actual pulmonary function, inflammatory blood parameters, colonizing pathogens, antibodies (Staphylococcus aureus and Pseudomonas aeruginosa), and genotype . RESULTS: The patients with nasal polyps differed with respect to chronic colonization of P aeruginosa in sputum samples and had a higher occurrence of serum antibodies against the same species . The two groups did not differ in pulmonary functions, inflammatory parameters, or genotype . The polyps found were mainly small (within the meatus media) and gave no significant increase in ongoing clinical symptoms such as rhinorrhea, nasal obstruction, or hyposmia . Neither was any significantly marked finding concerning the nose (mucosal swellings, secretion, etc.) made in the polyp patients . The patients with CF scored slightly lower in a smell identification test in comparison with the healthy control group . The nasal lavage fluid was analyzed (in 93 of the 111 patients) for the occurrence of P aeruginosa (by polymerase-chain reaction {PCR}), interleukin {IL}-5, IL-8, and lysozyme . The lysozyme and IL-8 content was equal in the two CF groups but increased in comparison with the healthy control group . P aeruginosa was not detected with PCR in any nasal lavage fluid . No measurable levels of IL-5 in the nasal lavage were found . CONCLUSIONS: There was a higher frequency of chronic colonization of P aeruginosa in the lower respiratory tract in patients with nasal polyps . Otherwise, nonsevere nasal polyposis was not an indicator of lower respiratory tract morbidity in CF patients.

Antimicrob Agents Chemother, 2002 Feb, 46(2), 566 - 9
Integron-located oxa-32 gene cassette encoding an extended-spectrum variant of OXA-2 beta-lactamase from Pseudomonas aeruginosa; Poirel L et al.; Pseudomonas aeruginosa clinical isolate CY-1, which was resistant to ceftazidime, harbored a conjugative ca . 250-kb plasmid that contained a class 1 integron with two gene cassettes encoding OXA-32, an OXA-2- type beta-lactamase, and the aminoglycoside acetyltransferase AAC(6')Ib(9) . OXA-32 differed from OXA-2 by an Leu169Ile amino acid substitution (class D numbering) . Site-directed mutagenesis established that Ile169 is responsible for resistance to ceftazidime but not to cefotaxime.

Antimicrob Agents Chemother, 2002 Feb, 46(2), 333 - 43
SmeC, an outer membrane multidrug efflux protein of Stenotrophomonas maltophilia; Li XZ et al.; A homologue of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa, smeABC, was cloned from Stenotrophomonas maltophilia by using, as a probe, a PCR product amplified from this organism with primers based on the mexB sequence . The smeABC genes were hyperexpressed in a mutant strain displaying resistance to several antimicrobials, including aminoglycosides, beta-lactams, and fluoroquinolones . Deletions in smeC but not smeB compromised this resistance, suggesting that SmeC contributed to the multidrug resistance of the mutant as part of another, as-yet-unidentified multidrug efflux system . Consistent with SmeC functioning independently of SmeAB, a promoter activity was identified upstream of smeC . Upstream of the smeABC genes, a putative two-gene operon, smeSR, encoding homologues of bacterial two-component regulatory systems was identified . The cloned smeR gene activated expression of a smeA-lacZ fusion, indicating that SmeR positively regulates expression of the smeABC genes . Consistent with this, the multidrug resistance of the SmeABC-hyperexpressing mutant was compromised by deletion of smeR . Intriguingly, SmeC expression in S . maltophilia paralleled a beta-lactamase activity provided by a C-terminally truncated L2 enzyme, which was apparently responsible for the beta-lactam resistance of the SmeABC-hyperexpressing mutant . This represents the first report of coregulation of an efflux resistance determinant and a beta-lactamase.

Pharmacotherapy, 2002 Jan, 22(1), 81 - 7
The effect of an antimicrobial formulary change on hospital resistance patterns; Empey KM et al.; A university hospital formulary change that was designed to reduce the use of the third-generation cephalosporins ceftazidime and cefotaxime and replace them with the so-called "fourth-generation" cephalosporin cefepime was evaluated . A retrospective review of antibiotic use and antimicrobial resistance during two 6-month periods before and after the formulary change was performed . All hospital patients with vancomycin-resistant Enterococcus (VRE), ceftazidime-resistant Klebsiella pneumoniae (CRKP), methicillin-resistant Staphylococcus aureus (MRSA), piperacillin-resistant Pseudomonas aeruginosa (PRPA), and ceftazidime-resistant P . aeruginosa (CRPA) infections were included in the study Ceftazidime use decreased from 9600 g to 99 g, and cefotaxime use decreased from 6314 g to 732 g, which represented a combined decrease of 89% . Use of cefepime increased from 0 g to 5396 g . Infections from CRKP decreased from 13% to 3%, PRPA infections decreased from 22% to 14%, and CRPA infections decreased from 25% to 15% (p<0.05 for all) . Infections from MRSA dropped insignificantly, and VRE infections increased significantly . Substituting cefepime for ceftazidime and cefotaxime while reducing the overall use of cephalosporins appears to decrease rates of CRKP, PRPA, and CRPA.

Pediatr Nephrol, 2001 Dec, 16(12), 1015 - 8
Meropenem pharmacokinetics in children and adolescents receiving hemodialysis; Goldstein SL et al.; The emergence of multi-drug-resistant bacteria is of great concern to the care of pediatric end-stage renal disease (ESRD) patients who receive either hemodialysis or peritoneal dialysis via a catheter . Infections with gram-negative organisms, especially Pseudomonas aeruginosa, are difficult to eradicate and often necessitate catheter removal . Meropenem, a broad-spectrum antibiotic of the carbapenem class of beta-lactams, is effective against most gram-positive and gram-negative bacteria and has enhanced activity against P . aeruginosa . We studied the pharmacokinetics of meropenem during and between hemodialysis treatments in seven pediatric patients . Meropenem was given as a single dose of 20 mg/kg (maximum 500 mg) before and after two separate hemodialysis treatments . Meropenem administration was tolerated without any adverse effects . Hemodialysis effectively cleared meropenem in a manner that correlated with percent urea reduction . Median drug half-life was 7.3 h off dialysis (range 4.9-11.7 h) . The dose of 20 mg/kg was not sufficient to produce an acceptable interdialytic pharmacodynamic profile of 70% duration with a meropenem concentration >4 microg/ml, the MIC90 of meropenem for P . aeruginosa . Dosing simulations revealed that a daily dose of 25 mg/kg or an alternate day dose of 40 mg/kg would result in an acceptable pharmacodynamic profile . Both simulated doses achieved acceptable peak concentrations.

Am J Physiol Lung Cell Mol Physiol, 2002 Feb, 282(2), L285 - 90
Role of interleukin-1 in the pulmonary immune response during Pseudomonas aeruginosa pneumonia; Schultz MJ et al.; Pneumonia is associated with elevated concentrations of the proinflammatory cytokine interleukin (IL)-1 in the pulmonary compartment . To study the role of IL-1 in the pathogenesis of Pseudomonas pneumonia, IL-1 receptor type 1 gene-deficient (IL-1R -/-) mice and wild-type mice were intranasally inoculated with Pseudomonas aeruginosa . The absence of the IL-1 signal attenuated the outgrowth of Pseudomonas in lungs, as reflected by an increasing number of colony-forming units (cfu) during Pseudomonas pneumonia in wild-type mice and a concurrently decreasing number of cfu during pulmonary infection in IL-1R -/- mice (P < 0.05, IL-1R -/- mice vs . wild-type mice) . Influx of neutrophils was decreased in bronchoalveolar lavage fluids in IL-1R -/- mice compared with wild-type mice . Similarly, lung levels of cytokines (tumor necrosis factor-alpha, IL-6) and chemokines (macrophage inflammatory protein-2 and KC) were lower in IL-1R -/- mice 24 h postinoculation . Consistent with results obtained in IL-1R -/- mice, treatment of wild-type mice with IL-1R antagonist also diminished outgrowth of Pseudomonas when compared with wild-type mice treated with vehicle (P < 0.05) . These results demonstrate that an absence or reduction in endogenous IL-1 activity improves host defense against Pseudomonas pneumonia while suppressing the inflammatory response.

Cytokine, 2001 Nov 21, 16(4), 160 - 8
The effects of infection of thermal injury by Pseudomonas aeruginosa PAO1 on the murine cytokine response; Rumbaugh KP et al.; Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death . Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P . aeruginosa PAO1 . Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1 beta, macrophage inflammatory protein (MIP)-1 beta and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) expression . The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines {stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-alpha}; induced the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5 h and the expression of additional cytokines, including TGF-beta, TNF-beta, lymphotoxin beta (LT-beta), interferon gamma (IFN-gamma), and IFN-beta by 40 h post-burn/infection . While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40 h post-burn/infection . These results suggest that in P . aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P . aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism .

Dis Markers, 2001, 17(4), 285 - 93
Pseudomonas aeruginosa and a proteomic approach to bacterial pathogenesis; Sherman NE et al.; Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and can cause a variety of diseases in compromised patients . The genome of P . aeruginosa strain PAO1 has been reported to contain 5570 potential proteins . The value of this genomic database is that new proteins can be recognized to use as diagnostic markers, novel drug targets, and to better understand the physiology of this organism . However, similar to what has been observed in other sequenced bacterial genomes, approximately one third of the potential proteins have no known function . This is somewhat surprising given the long-standing interest in P . aeruginosa as an opportunistic pathogen . Obviously new tools, in addition to sequence similarity analysis, are needed to determine the role of these proteins . Proteomics using two-dimensional gel electrophoresis followed by mass spectrometry to detect and identify P . aeruginosa proteins represents a novel approach to address this gap.

Protein Sci, 2002 Feb, 11(2), 301 - 12
Toward genomic identification of beta-barrel membrane proteins: composition and architecture of known structures; Wimley WC; The amino acid composition and architecture of all beta-barrel membrane proteins of known three-dimensional structure have been examined to generate information that will be useful in identifying beta-barrels in genome databases . The database consists of 15 nonredundant structures, including several novel, recent structures . Known structures include monomeric, dimeric, and trimeric beta-barrels with between 8 and 22 membrane-spanning beta-strands each . For this analysis the membrane-interacting surfaces of the beta-barrels were identified with an experimentally derived, whole-residue hydrophobicity scale, and then the barrels were aligned normal to the bilayer and the position of the bilayer midplane was determined for each protein from the hydrophobicity profile . The abundance of each amino acid, relative to the genomic abundance, was calculated for the barrel exterior and interior . The architecture and diversity of known beta-barrels was also examined . For example, the distribution of rise-per-residue values perpendicular to the bilayer plane was found to be 2.7 +/- 0.25 A per residue, or about 10 +/- 1 residues across the membrane . Also, as noted by other authors, nearly every known membrane-spanning beta-barrel strand was found to have a short loop of seven residues or less connecting it to at least one adjacent strand . Using this information we have begun to generate rapid screening algorithms for the identification of beta-barrel membrane proteins in genomic databases . Application of one algorithm to the genomes of Escherichia coli and Pseudomonas aeruginosa confirms its ability to identify beta-barrels, and reveals dozens of unidentified open reading frames that potentially code for beta-barrel outer membrane proteins.

Biochemistry, 2002 Jan 22, 41(3), 1060 - 9
Effects of metal ligation and oxygen on the reversibility of the thermal denaturation of Pseudomonas aeruginosa azurin; Sandberg A et al.; Thermodynamic equilibrium transition models in DSC are only applicable to reversible processes, but reversibility of the thermal transitions of proteins is comparatively rare because of intermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures . The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversible thermal denaturation, both as holo- and apoprotein {Engeseth, H . R., and McMillin, D . R . (1986) Biochemistry 25, 2448-2455} . In this study, however, we demonstrate that this beta-barrel protein of Greek key topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligated to the protein . We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusively oxidized in a transition metal catalyzed oxidation reaction with dissolved O(2) at high temperatures . Both Cu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as well as the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant . Correspondingly, apoazurin mutants C112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-state transitions . Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due to autoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.

Biochemistry, 2002 Jan 22, 41(3), 1001 - 13
Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa; Blackburn NT et al.; Lytic transglycosylases cleave the beta,1-->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products . The genes encoding two hypothetical lytic transglycosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query . The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences . Expression studies of the two genes in E . coli in the presence of {(3)H}palmitate resulted in the labeling of only one of the two enzymes . This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies . Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation . This MltB derivative (named sMltB) was shown to not label with {(3)H}palmitate, and it was overexpressed in soluble form . The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB) . Both sMltB and SltB were purified to apparent homogeneity by a combination of affinity (Ni(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies . The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay . Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3 . The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products . At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalytically efficient, as reflected by its k(cat)/K(M) value, than sMltB.

Adv Exp Med Biol, 2001, 501, 223 - 32
Development of a topical vaginal microbicide: lessons learned from human milk; Isaacs CE et al.; Vaccines are not presently available to prevent adherence and transmission of many common pathogens at mucosal surfaces . As a result, sexually transmitted diseases were one of the most commonly reported infections in the US in 1999 . New methods are needed to reduce the spread of mucosal infections . Providing nonspecific protective factors, such as lipids and retinoids found in human milk to mucosal surfaces could reduce mucosal infection caused by viruses, e.g., herpes simplex virus-1 (HSV-1) and bacteria, e.g., Pseudomonas aeruginosa . Human milk lipids enzymatically modified to produce monoglycerides were antimicrobial and inactivated enveloped viruses, as well as gram-positive and gram-negative bacteria . Enveloped viruses were inactivated in seconds following contact with antimicrobial lipids, and P . aeruginosa infectivity was reduced by 99.9% after 2 hours . Transmission of pathogens at mucosal surfaces can also be prevented using retinoids that inhibit viral replication . In a human embryonic intestinal cell line the retinoic acid (RA) derivatives all-trans-RA and 9-cis-RA (10 microg/mL) decreased the production of HSV-1 and Echo-6 viruses by 1-2 log10 over a 48-hour period . In addition, all-trans-RA inhibited HSV-1 replication in Vero cells as effectively as interferon beta, reducing viral production by 2.5log10 . These studies indicate that lipids and retinoids could be part of a topical microbicide to prevent mucosal infections.

Chemotherapy, 2001 Dec, 47(6), 421 - 9
Effects of parenterally administered ciprofloxacin in a murine model of pulmonary Pseudomonas aeruginosa infection mimicking ventilator-associated pneumonia; Kaneko Y et al.; BACKGROUND AND METHODS: We compared the bacteriological, pharmacological and histopathological effects of parenterally administered ciprofloxacin (CPFX) to those of imipenem/cilastatin (IMP/CS) and cefozopran (CZOP) in a murine model of mucoid Pseudomonas aeruginosa pneumonia mimicking ventilator-associated pneumonia . RESULTS: The minimum inhibitory concentrations (MICs) of CPFX, IMP and CZOP were 1.0, 1.0 and 4.0 mg/l, respectively . Treatment with CPFX resulted in a significant decrease in the number of viable bacteria {control, IMP/CS, CZOP and CPFX (mean +/- SEM): 5.02 +/- 0.20, 4.96 +/- 0.38, 5.44 +/- 0.13 and 3.27 +/- 0.02 log(10) colony-forming units lung, respectively} . Histopathological examination revealed that inflammatory changes in the CPFX-treated group were less marked than in other groups . Of the drugs analyzed, the pharmacokinetic parameters of area under the time-concentration curve (AUC)/MIC, AUC exceeding MIC and the time that lung concentrations of drug remained above the MIC were highest for CPFX . CONCLUSION: Our results suggest that parenterally administered ciprofloxacin is effective in ventilator-associated P . aeruginosa pneumonia .

Chemotherapy, 2001 Dec, 47(6), 396 - 408
Antimicrobial resistance surveillance of gram-negative bacteria isolated from intensive care units of four different hospitals in Turkey . Evaluation of the prevalence of extended-spectrum and inducible beta-lactamases using different E-test strips and direct induction methods; Kocazeybek BS; BACKGROUND: The aim of this study was to determine the antimicrobial resistance of gram-negative rods (GNRs) isolated from surgical intensive care units and to establish the prevalence of extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) . We also wished to determine the widespread beta-lactam substrate in ESBL-positive GNRs by two different E-test strips and to discuss the value of the routine utilization of these substrates together . METHODS: Out of 348 nosocomial gram-negative strains isolated with similar methods, 236 strains with resistance to the beta-lactam group of antimicrobials using the E-test method were included in this study . Two different strips were used for the detection of ESBLs: cefotaxime/cefotaxime + clavulanic acid (CT/CTL) and ceftazidime/ceftazidime + clavulanic acid (TZ/TZL) . For IBLs, the double-disk method was used . RESULTS: The order of frequency of the strains, starting with the most frequent, was Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa . In the 236 strains, the ESBL positivity rate was found to be 19.5% with TZ/TZL and CT/CTL strips, while it was 13.2% for IBL in 348 strains . Seventy-one percent of ESBL-positive strains gave parallel results with TZ/TZL and CT/CTL . ESBL positivity with only TZ/TZL or only CT/CTL was found to be 18 and 8% . CONCLUSIONS: Our study showed that except for imipenem, amikacin and ciprofloxacin, there was a high resistance to other antimicrobials, and multiresistance rates were increased in the strains in which ESBLs and IBLs were detected . In particular, the increasing prevalence of ESBLs in K . pneumoniae and IBLs in P . aeruginosa emphasizes the importance of the problem of infection control and antibiotic administration policies . Although it was seen that the prevalence of substrate templates in the detection of ESBL positivity was similar, we think that it is more useful to use two different strips together to obtain precise results .

Mikrobiol Z, 2001 May-Jun, 63(3), 22 - 9
{Reorganization of the strain's cellular lipids--degradation of anionic surfactants during "detergent" stress}; Stavskaia SS et al.; Changes in the fatty acid composition and ultrastructure of Pseudomonas aeruginosa 1C strain--a destructor of alkyl sulphates under the effect of the stress evoked by sodium dodecyl-sulphate have been studied . It has been established that the "detergent" stress changes the ratio of the cell fatty acids (FA) towards the increase of their nonsaturation . Ultrastructural changes in the cells are revealed earlier than biochemical ones . Several stages in stress development have been distinguished . The escape from the stress state is performed both at the population level and at the level of individual cells, highly-resistant to dodecyl sulphate.

Microb Pathog, 2002 Jan, 32(1), 27 - 34
Pseudomonas aeruginosa-induced lung injury: role of oxidative stress; Suntres ZE et al.; Pseudomonas aeruginosa is a Gram-negative pathogen that can cause lung injury in immunocompromised patients, primarily by inducing a release of host-derived mediators responsible for the influx of phagocytes to the lung . These phagocytes exert their antimicrobial actions by releasing toxic metabolites, including reactive oxygen species and proteases, which can also cause cell injury . This study was carried out to assess the pulmonary oxidant-antioxidant status of male adult Sprague-Dawley rats infected with different numbers of P . aeruginosa (10(4)-10(7)cfu/animal) . Intratracheal instillation of P . aeruginosa resulted in lung injury, as evidenced by increases in wet lung weight and decreases in the lung activities of angiotensin converting enzyme and alkaline phosphatase, enzymes localized primarily in pulmonary endothelial and alveolar type II epithelial cells, respectively . The P . aeruginosa -induced lung injury was directly related to the infiltration of neutrophils, as indicated by increases in myeloperoxidase activity . The challenge of animals with P . aeruginosa resulted in increases in lipid peroxidation and decreases in glutathione content, which were associated with the indices of lung injury and neutrophil infiltration . Such a challenge also resulted in weakening the antioxidant defence system, as evidenced by decreases in superoxide dismutase, catalase and glutathione peroxidase activities . These data suggest that changes in the pulmonary oxidant-antioxidant status may play an important role in the P . aeruginosa -induced lung injury .

Diagn Microbiol Infect Dis, 2001 Dec, 41(4), 191 - 6
Unit differences in pathogen occurrence arising from the MYSTIC program European database (1997-2000); Mendes C et al.; The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal global antimicrobial surveillance study that compares the activity of meropenem and comparator antimicrobial agents against pathogens isolated from intensive care, neutropenic or cystic fibrosis patients, and general wards . Data from the different European MYSTIC Program units (1997-2000) showed that the most prevalent isolates tested overall were methicillin-susceptible Staphylococcus aureus (MSSA; in accordance with study design methicillin-resistant S . aureus was not tested), Pseudomonas aeruginosa and Escherichia coli . In all the unit types, E . coli (approximately 20% having an extended spectrum beta-lactamase phenotype) and MSSA were highly susceptible to meropenem (97-99% susceptibility) . Isolates of MSSA showed lower levels of susceptibility to ciprofloxacin (61-77% susceptibility) in both cystic fibrosis and neutropenia patients, and particularly high levels of resistance to ceftazidime (38% susceptibility) in cystic fibrosis units . Ciprofloxacin (54% susceptibility) and gentamicin (46% susceptibility) demonstrated low levels of activity against P . aeruginosa (frequently encountered in cystic fibrosis units) . Meropenem and piperacillin/tazobactam were the most active agents against P . aeruginosa in all the unit types . Carbapenems and piperacillin/tazobactam have sustained > 90% susceptibility rates overall against the most frequently isolated pathogens . The analysis of specific units that house patients with a high-risk of contracting antimicrobial-resistant pathogens remains very important for the optimal selection of empiric regimens.

J Microbiol Methods, 2002 Mar, 49(1), 75 - 87
Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis; Bockelmann U et al.; A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow) . River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed . Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique . In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix . The analysis could be performed on different levels of complexity . The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies . Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells . Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.

J Microbiol Methods, 2002 Mar, 49(1), 69 - 74
Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water; Ramalho R et al.; Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent . In the present study, the survival of P . aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4) . Initial counts, before starvation, were the same in all media tested . Following this period, P . aeruginosa became sensitive to PAB with added cetrimide . The addition of FeSO(4) did not improve the recovery of stressed P . aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C . The modified Pseudomonas agar medium was also tested with several P . aeruginosa strains, other species of Pseudomonas, and other genera . Only P . aeruginosa strains (pyocyanin positive) produced the typical colonies . Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P . aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P . aeruginosa in mineral water.

Biomacromolecules, 2001 Winter, 2(4), 1133 - 6
Biological activities of carbohydrate-branched chitosan derivatives; Morimoto M et al.; Two types of biological activities of the carbohydrate-branched chitosan derivatives were investigated . One is the specific interaction with lectins and bacterium . The other is activation of canine polymorphonuclear leukocyte (PMN) cells . The specific bindings of the L-fucose-branched chitosan derivative with Ulex europaeus agglutinin I (UEA-I) and the N-acetyl-D-glucosamine-branched chitosan derivative with Concanavalin A (Con A) were confirmed by a surface plasmon resonance technique . The specific aggregation of the fluorescence-labeled L-fucose-branched chitosan derivative with Pseudomonas aeruginosa was observed by fluorescent microscopic observation . The aggregation would be attributed to the specific binding between the L-fucose-branched chitosan derivative and PA-II receptor on the cell surface of P . aeruginosa . The influence of the chitosan derivatives on the active oxygen species generation from canine PMN cells was also investigated by the luminol-aided chemiluminescence method . The chemiluminescence responses depended on the degree of substitution and water solubility of the chitosan derivatives . The water-insoluble chitosan derivatives would stimulate the PMN cells by a phagocytosis mechanism, and the water-soluble ones would sensitize the PMN cells by a priming mechanism.

Infect Control Hosp Epidemiol, 2001 Oct, 22(10), 640 - 6
In vitro and in vivo efficacy of catheters impregnated with antiseptics or antibiotics: evaluation of the risk of bacterial resistance to the antimicrobials in the catheters; Sampath LA et al.; OBJECTIVE: To compare the efficacy of a new antiseptic catheter containing silver sulfadiazine and chlorhexidine on the external surface and chlorhexidine in the lumens to an antibiotic catheter impregnated with minocycline and rifampin on its external and luminal surfaces . DESIGN: Experimental trial . METHODS: Antimicrobial spectrum of catheters was determined by zones of inhibition . Resistance to luminal colonization was tested in vitro by locking catheter lumens with Staphylococcus epidermidis or Staphylococcus aureus culture after 7 days of perfusion . In vitro development of resistance to the antiseptic or antibiotic combination used in catheters was investigated . In vivo efficacy was tested (rat subcutaneous model) by challenge with sensitive or antibiotic-resistant bacteria . RESULTS: Antiseptic and antibiotic catheters exhibited broad-spectrum action . However, antibiotic catheters were not effective against Candida species and Pseudomonas aeruginosa . Both catheters prevented luminal colonization . Compared to controls, both test catheters resisted colonization when challenged with S aureus 7 and 14 days' postimplant (P<.05) . Repeated in vitro exposure of S epidermidis culture to the antibiotic and antiseptic combinations led to small increases in the minimum inhibitory concentration (15 times and 2 times, respectively) . Unlike the antibiotic catheter, the in vitro and in vivo activity of the antiseptic catheter was unaffected by the resistance profile of the test organism . Antiseptic catheters were more effective than antibiotic catheters in preventing colonization by rifampin-resistant S epidermidis in vivo (P<.05) . CONCLUSIONS: Antiseptic and antibiotic catheters exhibit similar efficacy; however, when challenged with a rifampin-resistant strain, the antibiotic catheter appeared to be more susceptible to colonization than the antiseptic device.

Chin Med J (Engl), 2000 Dec, 113(12), 1142 - 6
Successful treatment of invasive burn wound infection with sepsis in patients with major burns; Chai J et al.; OBJECTIVE: To investigate the clinical characteristics of invasive burn wound infection with sepsis in patients with major burns and to summarize the successful experiences in the treatment of such patients . METHODS: Eight patients with major burns, complicated by invasive burn would infection and sepsis were consecutively admitted to our hospital from September 1997 to October 1998 . Among them, 6 patients developed multiple organ dysfunction syndrome (MODS) and 2 developed septic shock . The plasma concentrations of IL-6, IL-8, TNF alpha and lypopolysaccharide (LPS) were assayed before and after surgical intervention, as well as when the patient's vital signs became stable . RESULTS: The patients' conditions usually deteriorated abruptly when extensive invasive burn wound infection emerged . While multi-microbial infection was usually found, Pseudomonas aeruginosa was the predominant bacteria isolated from the subeschar tissue . The plasma concentrations of IL-6, IL-8, TNF alpha and LPS before surgical intervention were significantly higher than those after surgical intervention (P < 0.05) . The lowest levels of the inflammatory mediators were observed when the patients' conditions became stable, and the values were significantly lower than those before surgical intervention (P < 0.001) . CONCLUSION: Since the main cause of burn wound sepsis is the presence of a large area of infected burn wound, they should be excised and covered as early as possible . LPS and pro-inflammatory mediators play an important role in the pathogenesis of burn sepsis . Although favorable results should be attributed to comprehensive treatment, we believe that early, aggressive and thorough surgical excision of infected burn wounds, followed by sound and complete coverage of the area, play a crucial role.

J Biomed Mater Res, 2002 Mar 5, 59(3), 438 - 49
Control of wound infections using a bilayer chitosan wound dressing with sustainable antibiotic delivery; Mi FL et al.; A novel bilayer chitosan membrane was prepared by a combined wet/dry phase inversion method and evaluated as a wound dressing . This new type of bilayer chitosan wound dressing, consisting of a dense upper layer (skin layer) and a sponge-like lower layer (sublayer), is very suitable for use as a topical delivery of silver sulfadiazine (AgSD) for the control of wound infections . Physical characterization of the bilayer wound dressing showed that it has excellent oxygen permeability, that it controls the water vapor transmission rate, and that it promotes water uptake capability . AgSD dissolved from bilayer chitosan dressings to release silver and sulfadiazine . The release of sulfadiazine from the bilayer chitosan dressing displayed a burst release on the first day and then tapered off to a much slower release . However, the release of silver from the bilayer chitosan dressing displayed a slow release profile with a sustained increase of silver concentration . The cultures of Pseudomonas aeruginosa and Staphylococcus aureus in agar plates showed effective antimicrobial activity for 1 week . In vivo antibacterial tests confirmed that this wound dressing is effective for long-term inhibition of the growth of Pseudomonas aeruginosa and Staphylococcus aureus at an infected wound site . The results in this study indicate that the AgSD-incorporated bilayer chitosan wound dressing may be a material with potential antibacterial capability for the treatment of infected wounds .

Clin Infect Dis, 2002 Feb 1, 34(3), 340 - 5 Epub 2001 Dec 26.
Risk factors for imipenem-resistant Pseudomonas aeruginosa among hospitalized patients; Harris AD et al.; Risk factors for the nosocomial recovery of imipenem-resistant Pseudomonas aeruginosa (IRPA) were determined . A case-control study design was used for the comparison of 2 groups of case patients with control patients . The first group of case patients had nosocomial isolation of IRPA, and the second group had imipenem-susceptible P . aeruginosa (ISPA) . Control patients were selected from the same medical or surgical services from which case patients were receiving care when isolation of IRPA occurred . Risk factors analyzed included antimicrobials used, comorbid conditions, and demographic variables . IRPA was recovered from 120 patients, and ISPA from 662 patients . Imipenem (odds ratio {OR}, 4.96), piperacillin-tazobactam (OR, 2.39), vancomycin (OR, 1.80), and aminoglycosides (OR, 2.19) were associated with isolation of IRPA . Vancomycin (OR, 1.64), ampicillin-sulbactam (OR, 2.00), and second-generation cephalosporins (OR, 2.00) were associated with isolation of ISPA . Antibiotics associated with ISPA are different from antibiotics associated with IRPA . The OR for imipenem as a risk factor for IRPA is less than that reported from studies in which control group selection was suboptimal.

Pediatrics . 2002 Jan;109(1):E13.
Increased prevalence of mutations in the cystic fibrosis transmembrane conductance regulator in children with chronic rhinosinusitis; Raman V et al.; OBJECTIVE: Chronic rhinosinusitis results in significant morbidity in the pediatric population; however, no predisposing factor is found in many cases . Cystic fibrosis (CF) is a recognized cause of chronic rhinosinusitis . Although the carrier frequency for CF ranges from 3% to 4% in the general white population, the prevalence of mutations in the CF transmembrane conductance regulator (CFTR) among children with chronic rhinosinusitis is unknown . Our objective was to study the frequency of CFTR mutations among children with chronic rhinosinusitis . METHODS: Fifty-eight white children who were from the St Louis metropolitan area and had chronic rhinosinusitis, none of whom satisfied diagnostic criteria for CF, underwent sweat testing and genotyping for CFTR mutations using an assay that detects 90% of mutations seen in this ethnic group . RESULTS: Seven of the 58 patients (12.1%) tested harbored CFTR mutations as compared with the expected rate of 3% to 4% in this ethnic group . Five patients had the DeltaF508, 1 had the R117H, and 1 had the I148T mutation . Only 1 of the 7 children had a borderline abnormal sweat test . Two of the 58 patients experienced recurrent Pseudomonas aeruginosa rhinosinusitis, and both were DeltaF508 heterozygotes . Three other children with no detectable CFTR mutation had borderline elevated sweat-test results . The CFTR intron 8 5T polymorphism was found at a frequency comparable to that reported for the general population . CONCLUSION: There is an increased occurrence of CFTR mutations in children who have chronic rhinosinusitis and do not meet diagnostic criteria for CF, usually in the setting of a normal sweat chloride . These results suggest a role for CFTR mutations in predisposition to chronic rhinosinusitis.

Invest Ophthalmol Vis Sci, 2002 Jan, 43(1), 189 - 97
The role of Langerhans cells in Pseudomonas aeruginosa infection; Hazlett LD et al.; PURPOSE: Previous experimental studies have shown that extended-wear contact lens usage results in a centripetal migration of Langerhans cells from the conjunctiva into the central cornea . To test the consequences of this, Langerhans cells were induced into the cornea before Pseudomonas aeruginosa infection in BALB/c mice that are normally resistant (the cornea heals) and in C57BL/6 mice that are susceptible (the cornea perforates) to bacterial challenge . METHODS: Mean clinical scores, slit lamp examination, adenosine diphosphatase (ADPase), and acid phosphatase staining as well as immunostaining with DEC-205, B7-1, CD4, and interleukin-2 receptor (IL-2R) antibodies and histopathologic, RT-PCR, and delayed-type hypersensitivity (DTH) analyses were used to examine the effects on bacterial disease after polystyrene bead induction of Langerhans cells into the cornea before bacterial challenge . RESULTS: No difference in disease response was observed in bead- versus sham-treated C57BL/6 mice after bacterial infection; however, significant differences leading to corneal perforation were seen in BALB/c mice that included an increased number of Langerhans cells in the central cornea at 1 and 6 days after infection, an increased number of B7-1+ (mature) Langerhans cells at 6 days after infection, CD4+ and IL-2R+ T cells at 5 days after infection, enhanced DTH, and increased mRNA levels for IFN-gamma in cornea and cervical lymph nodes . Alternately, levels of IL-4 were significantly higher in the cornea and cervical lymph nodes of sham- versus bead-treated animals . CONCLUSIONS: These data provide evidence that Langerhans cells are critical in the innate immune response to P . aeruginosa and provide new information regarding the mechanisms governing resistance versus susceptibility to bacterial infection with this opportunistic pathogen.

Ophthalmology, 2002 Jan, 109(1), 27 - 39; discussion 39-40
Adaptive effects of 30-night wear of hyper-O(2) transmissible contact lenses on bacterial binding and corneal epithelium: a 1-year clinical trial; Ren DH et al.; OBJECTIVE: To determine effects of lens type and oxygen transmissibility on human corneal epithelium during extended wear (EW) . DESIGN: Prospective, randomized, double-masked, single-center, parallel treatment groups, 1-year clinical trial . PARTICIPANTS: One hundred seventy-eight patients completed the study: (1) high-O(2) soft lens (6-night {N} EW) (n = 27); (2) hyper-O(2) soft lens (6N-EW, n = 33) or (30N-EW, n = 66); and (3) hyper-O(2) rigid gas-permeable lens (RGP) (30N-EW, n = 52) . INTERVENTION: Irrigation chamber to collect exfoliated corneal surface cells, confocal microscopy, and tear collection at baseline, 1, 3, 6, 9, 12 months of EW . MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness; (3) superficial epithelial cell area; (4) epithelial surface cell exfoliation; and (5) tear lactate dehydrogenase . RESULTS: Quantitative evidence demonstrated increased binding of PA to human exfoliated corneal epithelial cells during the first 3 months of soft lens EW; the control high-O(2) test lens showed significantly higher bacterial binding (P < 0.05) . Binding activity gradually decreased thereafter and returned to baseline after 9 and 12 months . The corneal epithelium demonstrated enlargement of surface cell size, thinning of central epithelium, and a significant decrease in surface cell shedding (P < 0.05) . Remarkably, there was subsequent partial adaptive recovery in cell shedding and epithelial thickness but not surface cell size . There was no significant difference between 6N and 30N continuous wear of the hyper-O(2) soft lens for all outcome measures . Importantly, hyper-O(2) RGP lens wear did not show significantly increased PA binding during 1 year . CONCLUSIONS: This study establishes three important new findings: (1) hyper-O(2) soft lens EW produces significantly less PA binding than the lower O(2) soft lens with no significant difference in PA binding with 6N versus 30N EW of the hyper-O(2) soft lens; (2) there is a remarkable adaptive recovery after 6 months with all soft lens wear with gradual return to prelens PA binding levels and partial recovery of other outcome measures for all test lenses EW except surface cell size; (3) 30N EW of the hyper-O(2) RGP lens produced no significant increases in PA binding over 1 year . Taken together, these results suggest that introduction of new hyper-O(2) transmissible lens materials into clinical use may offer safer EW, and future epidemiologic studies of ulcerative infectious keratitis should consider both lens type and time in lens EW in any incidence/risk analysis.

Expert Opin Investig Drugs, 2001 Aug, 10(8), 1409 - 22
Efflux in bacteria: what do we really know about it?
Ryan BM, Dougherty TJ, Beaulieu D, Chuang J, Dougherty BA, Barrett JF.
Efflux is the process in which bacteria transport compounds outside the cell which are potentially toxic, such as drugs or chemicals or compounds . Efflux pumps can be identified not only by biochemical, microbiological, or molecular means but with the availability of microbial genomic sequences, by the application of bioinformatics analysis of DNA sequences for key conserved structure motifs . Efflux has been identified as a relevant contributor to bacterial resistance in the clinic and is now recognised as one of the most important causes of intrinsic antibiotic resistance in bacteria, especially in Pseudomonas aeruginosa . With the recognition of efflux as a major factor in bacterial resistance, several companies have invested in the identification and development of bacterial efflux pump inhibitors . Among those, Microcide, Pfizer, Paratek and several academic laboratories are in the process of exploring efflux pump inhibitors from synthetic, natural products and peptidomimetics . Inhibiting bacterial efflux with a non-antibiotic inhibitor would restore activity of an antibiotic subject to efflux (similar to the use of beta-lactamase inhibitors to combat beta-lactamase production by bacteria) . The feasibility of such an approach has been experimentally demonstrated in vitro and in vivo for efflux reversal of levofloxacin.

J Food Prot, 2001 Dec, 64(12), 1943 - 8
Joint effect of nisin, CO2, and EDTA on the survival of Pseudomonas aeruginosa and Enterococcus faecium in a food model system; Cabo ML et al.; A study on the joint effect of either nisin or Nisaplin, headspace CO2 levels, and EDTA on the survival of Pseudomonas aeruginosa and Enterococcus faecium was carried out in a water-soluble fish muscle extract at 3 degrees C using a second-order rotatable factorial design . E . faecium was completely deactivated by all processing after 2 days of storage . In contrast, P . aeruginosa was much less susceptible to treatments, and cell death was satisfactorily described by two models . Nisin increased cell death, whereas Nisaplin (commercial form of nisin) was not suitable, as it caused undesirable interference, presumably due to its co-compounds . Interactions between Nisaplin or nisin and either EDTA or CO2 were found to be nonstatistically significant . Factors that could account for this unexpected lack of synergism are discussed . However, a statistically significant positive interaction was found between CO2 and EDTA . This finding could allow CO2 levels to be decreased and hence to reduce the main disadvantages of CO2 application, namely, exudation and acidification.

Korean J Intern Med, 2001 Sep, 16(3), 167 - 72
Mucin secretion in the rat tracheal epithelial cells by epidermal growth factor and Pseudomonas aeruginosa extracts; Song JS et al.; BACKGROUND: Hypersecretion of mucin due to goblet cell hyperplasia is frequently encountered in many chronic airway diseases, such as chronic bronchitis, bronchiectasis, bronchial asthma and cystic fibrosis . Even in normal individuals, viral infection or bacterial pneumonia frequently provoke huge amounts of bronchial secretions which may cause airway obstruction . The production of mucin was regulated by epidermal growth factor (EGF) in vitro . To know whether this EGF system regulates mucin secretion in vivo and Pseudomonas also stimulates the mucin secretion by the same pathway, we studied these relationships in the cultured rat tracheal epithelial cells . METHODS: Rat tracheal epithelial cells were obtained by pronase dissociation from the male Fisher 344 rats . When cells became confluent, they were divided into 6 groups and stimulated with either EGF for 24 hours or Pseudomonas extracts for 12 hours with or without selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478 . RESULTS: We found that both EGF and Pseudomonas extracts phosphorylated the tyrosine residue in the EGF receptor from the rat tracheal epithelial cells and this tyrosine phosphorylation was nearly completely blocked by selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478 . The mucin secretion was also stimulated by either EGF or Pseudomonas extracts but more strong secretion of mucin and MUC5AC gene expression in the rat tracheal epithelial cell was done by Pseudomonas extracts . CONCLUSION: These data suggest that Pseudomonas secretes the mucin by way of the EGF receptor and MUC5AC gene expression and the inhibitors of EGF receptor tyrosine phosphorylation would be useful to prevent the huge production of mucin due to Pseudomonas aeruginosa lung infection.

An Med Interna, 2001 Oct, 18(10), 521 - 4
{Pneumothorax in patients infected by the human immunodeficiency virus}; Martinez-Vazquez C et al.; OBJECTIVE: Patients with HIV who develop pneumothorax have been previously described . Pneumocystis carinii pneumonia (PCN) is the leading cause of this complication, but infection by other pulmonary microorganism, inhaled pentamidine therapy and lung invasive manoeuvres have also been associated with pneumothorax in HIV infected patients . METHOD: We review the most relevant clinical aspects of pneumothorax in HIV-infected persons, gathered in our hospital along eight years, before HAART therapy was started . During this time, 97 patients with PCN were diagnosed and 148 patients received prophylaxis with inhaled pentamidine . Only 14 episodes of pneumothorax in 13 patients, were recorded . In ten occasions pneumothorax was related to pulmonary invasive manoeuvres, pulmonary infections were found in three and was considered spontaneous in one . The pulmonary invasive manoeuvres were: subclavia vein catheterisation in six cases (one of them was diagnosed of proved PCN and the other has pneumococcal pneumonia); transbronchial biopsy in one patient (also with proved PCN), knife chest trauma in two cases and after fine needle aspiration of an axillary lymph node in one patient . RESULTS: The pulmonary infections associated with pneumothorax in three patients were: proved PCN (this patient was the only one in the group with inhaled pentamidine prophylaxis who developed pneumothorax), active pulmonary infection by mycobacterium tuberculosis and Pseudomonas aeruginosa pneumonia . A drainage chest tube was placed in 12 patients with complete resolution in nine . In the other two patients pleurodesis was necessary and surgical repair was carried out in the other one (who had pulmonary tuberculosis) . During the follow up six patients died (median time to death: 7 months) . Among patients who died, five had pulmonary infections when the pneumothorax was diagnosed: PCN in three cases, pulmonary tuberculosis and pseudomonas pneumonia in the other two; all of them with less than 100 CD4 lymphocytes . CONCLUSIONS: Pneumothorax is frequent in HIV-infected patients with PCN, but other lung infections and, above all pulmonary invasive manoeuvres, can cause this complication . In our experience, HIV-infected patients who develop pneumothorax have a bad prognosis.

Mikrobiologiia, 2001 Sep-Oct, 70(5), 607 - 10
{Effect of reduced concentrations of carbon, nitrogen, and phosphorus in a medium on growth of three dissociants of Pseudomonas aeruginosa}; Mil'ko ES et al.; The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate mu of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2, culture pH, and the population composition was studied . Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants had virtually identical mu . The maximal values of mu were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant) . The R and M dissociants showed the most rapid decrease in mu in the nitrogen-deficient medium containing 0.55% NaNO3 . By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively . In the case of the carbon-deficient medium, the drop in the culture pH was lower . After a 2.5-day incubation of the S dissociant in the phosphorus-deficient medium containing 0.028% NaH2PO4.2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.

Res Microbiol, 2001 Nov, 152(9), 799 - 804
In-frame fusion of a His-Cys motif into the Pseudomonas aeruginosa outer membrane OprI lipoprotein results in increased metal binding capacity by Escherichia coli; Bouia A et al.; OprI, a small outer membrane lipoprotein from Pseudomonas aeruginosa, can be produced in large amounts and anchored at the surface on Escherichia coli cells . A four-time repeated (His-Cys) motif was fused to the C-terminal part of OprI . After induction, E . coli cells harbouring the recombinant oprI gene became more sensitive to Cd and Co . The same cells, after IPTG induction, bound four to eight times more Cd and Cr than control cells expressing oprI alone.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 57 - 63
Rapid flow cytometry--Nile red assessment of PHA cellular content and heterogeneity in cultures of Pseudomonas aeruginosa 47T2 (NCIB 40044) grown in waste frying oil; Vidal-Mas J et al.; The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity ofbacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source . A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates (PHA) by two basic mechanisms: (i) rapid assessment of polyhydroxyalkanoate content and (ii) definition of flow cytometric cell sorting protocols to select high polyhydroxyalkanoate (PHA)-producing strains . We report a rapid (less than 30 min) flow cytometric assessment of PHAs in Pseudomonas aeruginosa 47T2 following Nile red staining: (i) to estimate cellular PHAs content; (ii) to study heterogeneity of the batch cultures producing PHAs and (iii) to establish the basis for sorting sub-populations with a high capacity to accumulate PHAs.

J Chemother, 2001 Oct, 13(5), 546 - 54
Clonal spread of imipenem-resistant Pseudomonas aeruginosa in the intensive care unit of a Turkish hospital; Gulay Z et al.; Pseudomonas aeruginosa may cause life-threatening infections, especially in nosocomial settings . Although carbapenems are considered as one of the most effective alternatives in antipseudomonal therapy, resistance to the carbapenem group of antibacterials is a growing problem . In the first 6 months of 1997, P . aeruginosa isolates that were resistant to almost all antipseudomonal agents including imipenem were recovered from various specimens from intensive, care unit (ICU) patients . Isolates with the same antibiogram profile caused a small outbreak in May 1997 . A retrospective case-control study revealed that the major risk factors for infection/colonization with multiresistant P . aeruginosa were prolonged stay in the ICU (p<0.001), previous and lengthy imipenem usage (p<0.001 and p<0.0001, respectively), and mechanical ventilation (p<0.001) . Analytical isoelectric focusing of the sonicates prepared from the isolates showed that each isolate produced 1-5 beta-lactamases, enzymes with isoelectric points (pIs) of 5.1, 6.4, 8.5-8.7 being the most prevalent . DNA macrorestriction patterns of imipenem-resistant isolates were distinct from those of the imipenem-sensitive isolates recovered from ICU patients during the same interval and from the environmental isolates (controls) . Thus, our results indicate that colonized patients appear to be the major source for cross-contamination of other patients and if imipenem is selected for empirical therapy, emergence of resistant strains should be anticipated and appropriate precautions taken.

Int J Hyg Environ Health, 2001 Nov, 204(2-3), 139 - 42
Capability of mucoid Pseudomonas aeruginosa to survive in chlorinated water; Grobe S et al.; Mucoid strains of Pseudomonas aeruginosa are characterized by an overproduction of the extracellular polysaccharide alginate . When suspended into chlorinated swimming-pool water or drinking water samples, mucoid bacteria revealed enhanced survival compared with isogenic nonmucoid cells . Removal of slime from mucoid bacteria abolished chlorine resistance, addition of purified alginate to washed bacteria again enhanced survival . Thus, alginate-containing slime confers protection on P . aeruginosa against chlorine and may contribute to survival of these bacteria in chlorinated water systems.

Microb Drug Resist, 2001 Fall, 7(3), 273 - 5
In vivo selection of oxacillinase-mediated ceftazidime resistance in Pseudomonas aeruginosa; Leotard S et al.; Ceftazidime-susceptible and -resistant Pseudomonas aeruginosa strains were isolated from pulmonary specimens following a treatment with ceftazidime in a patient who developed a nosocomial pneumonia . The ceftazidime-susceptible and -resistant strains were clonally related and harbored a self-transferable approximately 155-kb plasmid . These isolates expressed two OXA-10-like oxacillinases, the narrow-spectrum OXA-35 and the expanded-spectrum OXA-19, respectively, differing by one amino acid substitution . This is the first example of in vivo selection of an extended-spectrum oxacillinase from a restricted-spectrum oxacillinase.

Med Dosw Mikrobiol, 2001, 53(1), 45 - 51
{Occurrence of beta-lactamase type ESBL and IBL in Pseudomonas aeruginosa rods}; Wolska MK et al.; The aim of the study was to determine extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among Pseudomonas aeruginosa strains . A total of 43 strains isolated from humans (6), hospital sink (1), fish (15), cattle (5), swine (5), dog (1), redder (1) fur animals (9) were studied . ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al . (8) . Clavulonate and tazobactam were used as the inhibitors of ESBL . Inducible beta-lactamases were determined using double disc method according to Sanders (15) . Cefoxitin was the inductor of these beta-lactamases . The susceptibility study was carried out using the disc diffusion method according to NCCLS standards . A total of 8 ESBL (18.6% of all strains) and 31 (72%) IBL producing strains were detected . The obtained results indicate the necessity of monitoring of ESBL- and IBL-producing strains of Pseudomonas aeruginosa.

Ann Surg, 2002 Jan, 235(1), 113 - 24
Interleukin-1alpha and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts; Erdag G et al.; OBJECTIVE: To determine whether the antibacterial properties of cultured composite keratinocyte grafts can be enhanced by cytokines that stimulate the innate immune response . SUMMARY BACKGROUND DATA: Use of composite grafts of cultured keratinocytes has been limited because of their susceptibility to burn wound microorganisms as a result of their lack of a vasculature and immune cells when transplanted . Moreover, use of topical antimicrobial agents is limited with these composite grafts because of cytotoxic effects . Keratinocytes, like all epithelial cells in the body, maintain a natural defense mechanism called the innate immune system . Some components of this system can be induced by cytokines . METHODS: The innate immune response of cultured composite keratinocyte grafts treated with various cytokines was assessed indirectly by measuring the levels of mRNA encoding antimicrobial peptides (human beta defensin-1 and -2, LL-37, and antileukoprotease) and antimicrobial proteins (lysozyme, bactericidal/permeability-inducing protein, and phospholipase A2) by reverse transcription-polymerase chain reaction and directly by measuring the ability of keratinocytes to inhibit the growth of added bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) . RESULTS: Treatment with interluekin-1alpha increased mRNA levels of antimicrobial peptides in keratinocytes on plastic dishes and in composite grafts . Interleukin-6 increased mRNA levels of antimicrobial proteins in composite grafts only . When added to composite grafts, both cytokines increased antibacterial activity against E . coli, P . aeruginosa, and S . aureus . Moreover, interleukin-1alpha and interleukin-6 did not impair the formation of a differentiated epidermis in vitro or after transplantation of the composite grafts . CONCLUSIONS: Treatment with interleukin-1alpha or interleukin-6 of cultured composite keratinocyte grafts stimulates the innate immune response of keratinocytes, enhances the antibacterial properties of these grafts, and may better prepare them to combat infections in contaminated burn wounds.

Nucleic Acids Res, 2002 Jan 1, 30(1), 50 - 1
EXProt: a database for proteins with an experimentally verified function; Ursing BM et al.; EXProt is a non-redundant protein database containing a selection of entries from genome annotation projects and public databases, aimed at including only proteins with an experimentally verified function . In EXProt release 2.0 we have collected entries from the Pseudomonas aeruginosa community annotation project (PseudoCAP), the Escherichia coli genome and proteome database (GenProtEC) and the translated coding sequences from the Prokaryotes division of EMBL nucleotide sequence database, which are described as having an experimentally verified function . Each entry in EXProt has a unique ID number and contains information about the species, amino acid sequence, functional annotation and, in most cases, links to references in MEDLINE/PubMed and to the entry in the original database . EXProt is indexed in SRS at CMBI and can be searched with BLAST and FASTA through the EXProt web page .

J Antimicrob Chemother, 2002 Jan, 49(1), 87 - 94
The antibacterial activity of triclosan-impregnated storage boxes against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Shewanella putrefaciens in conditions simulating domestic use; Braid JJ et al.; Antimicrobial resistance has increased over the past decade causing concern for public health . Domestic antimicrobial products containing triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether), a broad-spectrum antibacterial agent, were introduced in 1997 and have become popular among consumers . Cross-resistance to other antibacterial agents has been suggested as a possible consequence of their widespread use . Triclosan-impregnated plastic storage boxes were tested for activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Shewanella putrefaciens in various conditions, including some designed to simulate usual storage conditions . Results showed inhibition up to a factor of 106 of bacteria grown in direct contact with triclosan-impregnated plastic at 30 and 22 degrees C, but not at 4 degrees C . Triclosan resistance was not found to increase after repeated exposure in triclosan-impregnated boxes . Further investigation into the effect of triclosan-impregnated products on bacteria will increase understanding of domestic antimicrobial products and implications of their overuse.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 255 - 8
Nosocomial outbreak of carbapenem-resistant Pseudomonas aeruginosa with a new bla(IMP) allele, bla(IMP-7); Gibb AP et al.; Pseudomonas aeruginosa isolates from an outbreak in Canada were highly resistant to carbapenems and ceftazidime but not piperacillin . They produced a novel integron-associated metallo-beta-lactamase, designated IMP-7, with 91% identity to IMP-1 . bla(IMP-7) was not detected with standard bla(IMP)-specific primers, owing to mismatches in the forward primer.

FEMS Immunol Med Microbiol, 2001 Dec, 32(1), 33 - 6
Interaction of Pseudomonas aeruginosa galactophilic lectin PA-IL with pigeon egg white glycoproteins; Lerrer B et al.; Pseudomonas aeruginosa produces a galactophilic lectin, PA-IL, that resembles P-fimbrial adhesins of uropathogenic Escherichia coli strains in binding to human P blood group antigens . We examined, in the present study, its interaction with pigeon egg white glycoproteins carrying N-glycans with terminal Galalpha1-4Gal which inhibit the adhesion of P-fimbriae . For comparison, the lectin concanavalin A (Con A) and additional avian egg whites (of hen and quail) were also examined . The results obtained in both hemagglutination inhibition and Western blot analyses showed that PA-IL, unlike Con A, preferentially reacted with the pigeon egg white glycoproteins . These results, which confirmed PA-IL similarity in sugar specificity to E . coli P-fimbriae, demonstrated the advantage of this purified lectin for representing P-type and additional galactophilic microbial adhesins unavailable in purified stable form, in Western blot analyses.

Emerg Infect Dis, 2002 Jan, 8(1), 14 - 8
Surveillance for antimicrobial resistance in Croatia; Tambic Andrasevic A et al.; We describe the activities of the Croatian Committee for Antibiotic Resistance Surveillance and report surveillance results for 1999 . Twenty-two Croatian microbiology laboratories participated in the study . Resistance rates for the organisms isolated in different centers varied widely, but certain trends were apparent . Penicillin resistance in pneumococci (38%), methicillin resistance in Staphylococcus aureus (22%), the production of extended spectrum beta-lactamases by Klebsiella pneumoniae (21%), and imipenem resistance in Pseudomonas aeruginosa (11%) represent major resistance problems, especially in large hospitals . A comprehensive system of antimicrobial resistance surveillance, combined with training and external quality control programs, has identified high rates of resistance in key pathogens in some regions of Croatia . The program has heightened awareness of the problems of antimicrobial resistance and contributed to ongoing improvements in laboratory practice.

Infect Immun, 2002 Jan, 70(1), 360 - 7
In vivo rho GTPase-activating protein activity of Pseudomonas aeruginosa cytotoxin ExoS; Krall R et al.; ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain . In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed . Using a transient transfection system and delivery by P . aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells . Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation . GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton . Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location . Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol . Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated . Similar results were observed when ExoS was delivered into CHO cells by P . aeruginosa . These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.

Infect Immun, 2002 Jan, 70(1), 240 - 8
Bacterial lipoprotein-based vaccines induce tumor necrosis factor-dependent type 1 protective immunity against Leishmania major; Cote-Sierra J et al.; Immunity against Leishmania major requires rapid induction of a type 1 immune response in which tumor necrosis factor alpha (TNF-alpha) plays an essential role . Hence, vaccination strategies that simulate the protective immune response found in hosts that have recovered from natural infection provide a rational approach to combat leishmaniasis . One method for optimizing the qualitative and quantitative immune responses after vaccination is to use an adjuvant . In this study we demonstrate that the OprI lipoprotein (L-OprI) from Pseudomonas aeruginosa induces a long-term cellular (gamma interferon {IFN-gamma}) and humoral (immunoglobulin G2a) type 1 immune response against a truncated 32-kDa version (COOHgp63) of the 63-kDa major cell surface glycoprotein gp63 . By contrast, immunization with COOHgp63 either fused to OprI nonlipoprotein or with no adjuvant did not result in the induction of type 1 immune responses . The adjuvanticity of L-OprI is strongly dependent on its capacity to induce TNF-alpha, since generation of type 1 immune responses is clearly delayed and impaired in TNF-alpha(-/-) mice . Vaccination with L-OprICOOHgp63 fusion protein protected BALB/c mice against L . major infection for at least 19 weeks . Vaccinated mice were largely free of lesions or clearly controlled lesion size on termination of the experiment . The control of disease progression in mice vaccinated with L-OprICOOHgp63 was associated with enhancement of antigen-specific IFN-gamma production . These data indicate that bacterial lipoproteins constitute appropriate adjuvants to include in vaccines against diseases in which type 1 immune responses are important for protection.

Microb Pathog, 2001 Dec, 31(6), 271 - 81
Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro; Miyajima S et al.; The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines . Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa . Effects of pseudomonal virulence factors {elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)}, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting . Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas . Pseudomonal elastase caused the most extensive damage to both cell types . RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9 . Exotoxin A had no effect on MMP expression . Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells . Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells . In conclusion, corneal destruction seen with P . aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase .

Pediatr Pulmonol, 2002 Jan, 33(1), 32 - 7
Effects of inhaled gentamicin prophylaxis on acquisition of Pseudomonas aeruginosa in children with cystic fibrosis: a pilot study; Heinzl B et al.; Inhaled antibiotics are an established treatment for chronic Pseudomonas aeruginosa (PA) infection in patients with cystic fibrosis (CF) . However, inhaled antibiotics might also have prophylactic potential to delay acquisition of PA in early stages of the disease . From 1986-1999, all CF patients at this center who experienced defined risk situations for acquisition of PA (28 patients) received inhaled gentamicin (80 mg BID for those < 12 months; 120 mg BID for those > 12 months) for a minimum of 3 years . Twelve patients had repeated risk situations and continued this prophylaxis without interruption during the entire study period (group 1) . In the remaining 16 patients, inhaled antibiotics were discontinued at various times for a variety of reasons (group 2) . None of the patients in group 1, but 7 in group 2, became chronically infected with PA (P = 0.01) . Lung function and chest X-ray scores were significantly worse in those 7 infected patients, when compared to the noninfected ones in both groups . This suggests that long-term-prophylaxis with inhaled gentamicin can effectively delay acquisition of PA and decrease disease progression in children with CF .

Biochem J, 2002 Jan 1, 361(Pt 1), 87 - 95
Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomonas aeruginosa MutS by preventing protein oligomerization; Pezza RJ et al.; MutS, a component of the mismatch repair system begins the DNA reparation process by recognizing base/base mismatches or small insertion/deletion loops . We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa and analysed the biochemical properties of the encoded protein . Complementation of the hypermutator phenotype of a P . aeruginosa mutS mutant strain indicated that the isolated gene was functional . When purified MutS was incubated at 37 degrees C in the absence of ligands, a rapid inactivation of the oligonucleotide binding capability and ATPase activity occurred . However, the presence of ATP, ADP or heteroduplex oligonucleotides, but not homoduplex oligonucleotides, prevented the protein from being inactivated . The analysis of the protein by native PAGE indicated that the active conformation state correlates with the presence of MutS dimer . Analysis by gel-filtration chromatography showed that the inactive protein formed by incubation at 37 degrees C in the absence of ligands corresponds to the formation of a high molecular mass oligomer . The kinetic analysis of the oligomer formation showed that the extent of the reaction was markedly dependent on the temperature and the presence of MutS ligands . However, the protein inactivation apparently occurred before the maximum extent of MutS oligomerization . Further analysis of the MutS oligomers by electron microscopy showed the presence of regular structures consisting of four subunits, with each subunit probably representing a MutS homodimer . It is concluded that MutS possesses an intrinsic propensity to form oligomeric structures and that the presence of physiological ligands, such as nucleotides or heteroduplex DNA, but not homoduplex DNA, plays an important role in keeping the protein in an active conformation by preventing protein oligomerization.

J Biol Chem, 2002 Feb 15, 277(7), 4722 - 30 Epub 2001 Dec 11.
WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide; Zhao X et al.; WaaP of P . aeruginosa is a crucial sugar kinase that phosphorylates HepI in the inner core region of lipopolysaccharide (LPS) . WaaP shares homology with eukaryotic protein kinases in the conserved functional motifs (I-IX), indicating that it is also a protein kinase . This interpretation is substantiated by several lines of evidence including the following: (i) site-directed mutagenesis on catalytic domain residues abrogated the protein kinase activity; (ii) positive reaction in immunoblotting with anti-phosphotyrosine monoclonal antibody PY20; (iii) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and proteolytic peptide mapping showing excess mass equivalent to eight phosphate substituents on the tyrosine residues in WaaP; and (iv) WaaP is capable of catalyzing tyrosine self-phosphorylation as well as phosphorylating an exogenous synthetic co-polymer poly(Glu, Tyr) . Thus, WaaP possesses dual kinase functions, and it utilizes a catalytic mechanism similar to that of the eukaryotic protein kinases . WaaP was localized to the cytoplasm, suggesting that phosphorylation of the LPS core occurred prior to translocation to the periplasm and attachment of O-antigen . A chemiluminescence-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the kinetics of the WaaP sugar kinase activity, and the results showed that the K(m) was 0.22 mm for ATP and 14.4 microm for hydrofluoric acid-treated LPS, V(max) was 408.24 pmol min(-1), and k(cat) was 27.23 min(-1).

J Bacteriol, 2002 Jan, 184(1), 323 - 6
WbjA adds glucose to complete the O-antigen trisaccharide repeating unit of the lipopolysaccharide of Pseudomonas aeruginosa serogroup O11; Dean CR et al.; Lipopolysaccharide from a wbjA mutant, deficient in a putative glycosyltransferase from Pseudomonas aeruginosa serogroup O11, was compared to that from an O-antigen polymerase mutant . Results suggest that WbjA adds the terminal glucose to complete the serogroup O11 O-antigen unit and identifies the biological repeating unit as {-2)-beta-D-glucose-(1-3)-alpha-L-N-acetylfucosamine-(1-3)-beta-D-N-acetylfucosamine-(1}.

Ophthalmologica, 2001 Nov-Dec, 215(6), 439 - 43
Effect of sulfur hexafluoride gas on antibacterial activity of antibiotics in vitro against agents causing endophthalmitis; Yanyali A et al.; BACKGROUND: We conducted a study to evaluate the effect of sulfur hexafluoride gas (SF(6)) on the antibacterial activity of antibiotics in vitro against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, which are common endophthalmitis-causing agents . METHODS: In this experimental study, antibiotic susceptibility tests were prepared according to the National Committee for Clinical Laboratory Standards macrobroth dilution method . Muller-Hinton broth was the test medium . Standard P . aeruginosa (ATCC 27853), S . epidermidis (ATCC 12228) and S . aureus (ATCC 29213) strains were studied . For P . aeruginosa ceftazidime, ofloxacin and tobramycin dilutions, and for S . epidermidis and S . aureus, cefazolin, ofloxacin and tobramycin dilutions were prepared identically in two sets of tubes . One set of tubes, into which pure SF(6) was injected, was defined as the SF(6) group . The other set of tubes, into which no SF(6) was injected, was taken as the control group . To determine the minimal bactericidal concentrations of the antibiotics, subcultures were made onto Muller-Hinton agar, and the colonies were counted after 18 h of incubation at 37 degrees C . RESULTS: The minimal inhibitory concentrations of the antibiotics in the SF(6) group were found to be identical with those of the control group . However, the minimal bactericidal concentrations of the antibiotics were found to be at least two dilutions lower in the SF(6) group than in the control group, except for the minimal bactericidal concentration of cefazolin for S . aureus, which was found to be one dilution lower in the SF(6) group . CONCLUSION: SF(6) was found to potentiate the in vitro antibacterial activity of ofloxacin and tobramycin against S . aureus, S . epidermidis and P . aeruginosa, ceftazidime against P . aeruginosa, and cefazolin against S . epidermidis . Experimental animal studies are required to determine the role of SF(6) in the management of endophthalmitis .

J Leukoc Biol, 2001 Dec, 70(6), 911 - 9
MIP-1alpha regulates CD4+ T cell chemotaxis and indirectly enhances PMN persistence in Pseudomonas aeruginosa corneal infection; Kernacki KA et al.; The role of macrophage inflammatory protein-1alpha (MIP-1alpha) in cell infiltration into Pseudomonas aeruginosa-infected cornea and subsequent disease was examined . Greater amounts of the chemokine (protein and mRNA) were found in the infected cornea of susceptible B6 ("cornea perforates") versus resistant BALB/c ("cornea heals") mice from 1 to 5 days postinfection . Treatment of BALB/c mice with recombinant (r) MIP-1alpha exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea . Treatment of BALB/c mice with rMIP-1alpha also induced recruitment of activated CD4+ T cells into the affected cornea, converting resistant to susceptible mice . Depleting CD4+ T cells in r-treated BALB/c mice significantly decreased PMNs in cornea tissue, suggesting that T cells regulate persistence of PMNs at this site . In B6 mice, administration of neutralizing MIP-1alpha polyclonal antibody also significantly reduced PMN numbers and pathology . Collectively, evidence is provided that MIP-1alpha directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.

J Biol Chem, 2002 Feb 15, 277(7), 5476 - 83 Epub 2001 Dec 05.
Blocking the secretion of hepatic very low density lipoproteins renders the liver more susceptible to toxin-induced injury; Bjorkegren J et al.; Recently, we generated mice lacking microsomal triglyceride transfer protein (MTP) in the liver (Mttp(Delta/Delta)) and demonstrated that very low density lipoprotein secretion from hepatocytes was almost completely blocked . The blockade in lipoprotein production was accompanied by mild to moderate hepatic steatosis, but the mice appeared healthy . Although hepatic MTP deficiency appeared to be innocuous, we hypothesized that a blockade in very low density lipoprotein secretion and the accompanying steatosis might increase the sensitivity of Mttp(Delta/Delta) livers to additional hepatic insults . To address this issue, we compared the susceptibility of Mttp(Delta/Delta) mice and Mttp(flox/flox) controls to hepatic injury from Escherichia coli lipopolysaccharides, concanavalin A, and Pseudomonas aeruginosa exotoxin A . At baseline, neither the Mttp(Delta/Delta) nor the Mttp(flox/flox) mice had elevated serum transaminases or histologic evidence of hepatic inflammation . After the administration of the toxins, however, the Mttp(Delta/Delta) mice manifested higher levels of transaminases and, unlike the Mttp(flox/flox) mice, developed histologic evidence of hepatic inflammation . The toxic challenge induced tumor necrosis factor-alpha to a similar extent in Mttp(Delta/Delta) and Mttp(flox/flox) mice, but other parameters of injury (e.g . chemokine transcript levels and lipid peroxides) were disproportionately increased in the Mttp(Delta/Delta) mice . Our results suggest that blocking lipoprotein secretion in the liver may increase the susceptibility of the liver to certain toxic challenges.

Int J Antimicrob Agents, 2001 Dec, 18(6), 567 - 70
In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa; Karakoc B et al.; The in-vitro activities of various antibiotics, either alone or in combination with amikacin were assessed using clinical isolates of Pseudomonas aeruginosa . The minimum inhibitory concentrations (MIC) of these antibiotics were determined by microbroth dilution method against 50 clinical strains . The MIC values showed that 96, 94, and 74% of the isolates were susceptible or moderately susceptible to amikacin, meropenem and ceftazidime, respectively . The in vitro activities of ceftazidime and meropenem in combination with amikacin were determined by microbroth chequerboard technique and results were interpreted using the fractional inhibitory concentration (FIC) index . With a FIC index of < or =0.5 as borderline, synergistic interactions were more frequent with ceftazidime (70.8%) than with meropenem (40%) . No antagonism was observed.

Med Clin (Barc), 2001 Dec 8, 117(19), 721 - 6
{Risk and prognostic factors of Pseudomonas aeruginosa bacteremia in critically ill patients}; Alvarez-Lerma F et al.; BACKGROUND: To determine risk and prognostic factors in patients admitted to the intensive care unit (ICU) in which an episode of bacteremia caused by Pseudomonas aeruginosa has been diagnosed . PATIENTS AND METHOD: Cohort, observational, prospective, multicenter study . Patients admitted to 30 ICUs in Spain in whom one or more pathogens were isolated from blood cultures were included . RESULTS: In a total of 16,216 patients admitted to the participating ICUs during the study period, 949 episodes of bacteremia were diagnosed In 77 cases (8.11%), P . aeruginosa was the causative pathogen, with an infection rate of 4.7 episodes per 1000 patients . Independent risk factors associated with P . aeruginosa bacteremia were as follows: respiratory infection focus (OR 3.92; 95% IC 2.33-6.59; p </= 0.0001), previous use of antibiotics (OR 2.13; 95% IC 1.18-3.81; p </= 0.0078), arterial catheter (OR 4.09; 95% IC 2.26-7.38; p </= 0.0001), and previous longer ICU stay (days) (OR 1.02; 95% IC 1.003-1.033; p = 0.0274).Crude mortality rate in patients with bacteremia caused by P . aeruginosa was 50.6% (39/77), whereas mortality rate of bacteremia caused by other pathogens was 38.6% (337/872) (p = 0.039) . This difference was also found for attributed mortality (31.2% {24/77} vs . 20.4% {178/872}, (p = 0.027) . In the multivariate analysis adjusted by respiratory infection focus, previous ICU stay, and age, crude mortality (OR 1.55; 95% CI 0.96-2.51; p = 0.071) and attributed mortality (OR 1.63; 95% CI 0.96-2.78; p = 0.0709) of P . aeruginosa bacteremia were higher than in bacteremia caused by other pathogens . In the multivariate analysis, risk factors significantly associated with crude mortality were respiratory infection focus (OR 4.13; 95% IC 1.15-14.76; p = 0.0293) and severe sepsis or septic shock (OR 4.96; 95% IC 1.23-20.09; p = 0.0248) . CONCLUSIONS: Bacteremia caused by P . aeruginosa admitted to the ICU have a higher crude and attributed mortality than bacteremias caused by other pathogens . Prognosis is associated with the presence of severe sepsis or septic shock and respiratory infection focus.

Eur J Biochem, 2001 Dec, 268(24), 6408 - 16
Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa; Dong Z et al.; C57BL/6J naive and immunized mice were intracorneally infected with Pseudomonas aeruginosa . Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis . The enzymatic activities of cathepsins B, D and L were measured by peptidase assays . Immunohistochemical staining was carried out to localize the expression of the cathepsins . Cathepsins B, D and L were detected in the normal cornea by RT-PCR . A peptidase assay revealed activities of all three cathepsins under normal physiological conditions . In naive mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P . aeruginosa and the peak of the induction appeared around day 6 postinfection . Immunoblot analysis showed increased expression of cathepsins B, D and L . The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naive mice . Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria . Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naive corneas infected with P . aeruginosa, which correlated well with the inflammatory response . Immunization of mice against P . aeruginosa attenuated the inducing effect on cathepsin expression caused by infection . The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P . aeruginosa infection.

Nature, 2001 Nov 29, 414(6863), 562 - 5
Isochorismate synthase is required to synthesize salicylic acid for plant defence; Wildermuth MC et al.; Salicylic acid (SA) mediates plant defences against pathogens, accumulating in both infected and distal leaves in response to pathogen attack . Pathogenesis-related gene expression and the synthesis of defensive compounds associated with both local and systemic acquired resistance (LAR and SAR) in plants require SA . In Arabidopsis, exogenous application of SA suffices to establish SAR, resulting in enhanced resistance to a variety of pathogens . However, despite its importance in plant defence against pathogens, SA biosynthesis is not well defined . Previous work has suggested that plants synthesize SA from phenylalanine; however, SA could still be produced when this pathway was inhibited, and the specific activity of radiolabelled SA in feeding experiments was often lower than expected . Some bacteria such as Pseudomonas aeruginosa synthesize SA using isochorismate synthase (ICS) and pyruvate lyase . Here we show, by cloning and characterizing an Arabidopsis defence-related gene (SID2) defined by mutation, that SA is synthesized from chorismate by means of ICS, and that SA made by this pathway is required for LAR and SAR responses.

J Mol Biol, 2001 Dec 7, 314(4), 923 - 35
{L29M} substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity; Chervyakova D et al.; Genetic analysis of RecA protein chimeras and their ancestors, RecAEc (from Escherichia coli) and RecAPa (Pseudomonas aeruginosa) had allowed us to place these proteins with respect to their recombinogenic activities in the following order: RecAPa>RecAX21>RecAX20=RecAEc . While RecAX20 differs from RecAEc in five amino acid residues with two substitutions ({S25A} and {I26V}) at the interface of subunit interactions in the RecA polymer, RecAX20 and RecAX21 differ only by a single substitution {L29M} present at the interface . Here, we present an analysis of the biochemical properties considered important for the recombinogenic activity of all four RecA proteins . While RecAX20 was very similar to RecAEc by all activities analysed, RecAX21 differed from RecAEc in several respects . These differences included an increased affinity for double-stranded DNA, a more active displacement of SSB protein from single-stranded DNA (ssDNA), a decreased end-dependent RecAX21 protein dissociation from a presynaptic complex, and a greater accumulation of intermediate products relative to the final product in the strand-exchange reaction . RecAPa was more tolerant than RecAX21 only to the end-dependent RecA protein dissociation . In addition, RecAPa was more resistant to temperature and salt concentrations in its ability to form a presynaptic RecAPa::ATP::ssDNA filament . Calculations of conformational energy revealed that the {L29M} substitution in RecAX21 polymer caused an increase in its flexibility . This led us to conclude that even a small change in the flexibility of the RecA presynaptic complex could profoundly affect its recombinogenic properties .

J Mol Biol, 2001 Dec 7, 314(4), 823 - 37
Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity; Wedekind JE et al.; Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm . In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively . Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively . The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH . Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin . The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure . Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding . The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication .

Commun Dis Public Health, 2001 Sep, 4(3), 205 - 8
Survey of Staphylococcus aureus contamination in a hospital's spa and hydrotherapy pools; Meldrum R; Twenty-seven percent of water samples taken from one spa and two hydrotherapy pools in one Welsh hospital over three months contained Staphylococcus aureus . Four per cent of samples were deemed unacceptable because they contained coliforms, E . coli or Pseudomonas aeruginosa . Aerobic colony counts varied between samples but no counts were > 100 cfu/ml . Disinfection and pH records indicated no significant problems with pool maintenance over the sampling period, and bather loads were generally low . Some samples positive for S . aureus were found after lengthy periods of pool inactivity, indicating persistence of the organism.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 17 - 23
MHYT, a new integral membrane sensor domain; Galperin MY et al.; MHYT, a new conserved protein domain with a likely signaling function, is described . This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane . In Synechocystis sp . PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098 . In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins . In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains . A ykoW null mutant of B . subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein . A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.

Scand J Infect Dis, 2001, 33(10), 738 - 43
Molecular epidemiology of a multiresistant Pseudomonas aeruginosa outbreak in a paediatric intensive care unit; Miranda G et al.; After isolation of multiresistant (MR) Pseudomonas aeruginosa from 3 hospitalized patients in a paediatric intensive care unit (PICU), a prospective surveillance programme was established to detect infected and/or colonized patients in the hospital . Isolates were examined by means of outer membrane protein (OMP) profiles, serotyping and DNA genomic analysis using pulsed-field gel electrophoresis (PFGE) . Fifty-five P . aeruginosa strains were isolated from 23 hospitalized patients during September and October 1997 . The median hospital stay before isolation of P . aeruginosa was 8 d . PFGE demonstrated that the same clone infected 14 patients, 4 of whom were not hospitalized in the PICU . Susceptibility patterns and OMP profiles correlated with PFGE results in 37.8% and 36.4% of cases, respectively . Serotype O11 correlated with pattern A in 77% of cases and serotype O4 correlated with unrelated strains in 75% of cases but did not discriminate between outbreak and unrelated isolates . Extensive investigation of cultures failed to identify a reservoir of P . aeruginosa . PFGE was superior to OMP analysis and serotyping for discriminating between strains . The possible mode of acquisition for most of the patients infected with the same clone was cross-contamination.

Int J Med Microbiol, 2001 Nov, 291(5), 387 - 93
Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis; Sener B et al.; Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients . The purpose of this study was to type the P . aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P . aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR) . Sequential P . aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated . The isolates were analyzed by RAPD-PCR using two arbitrary primers . Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method . RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype . Some CF patients were colonized with a rather constant P . aeruginosa flora, with strains of different phenotypes but of one genotype . However, some patients may be colonized with more than one genotype . The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center.

Invest Ophthalmol Vis Sci, 2001 Dec, 42(13), 3223 - 7
Expression of membrane-type matrix metalloproteinases 4, 5, and 6 in mouse corneas infected with P . aeruginosa; Dong Z et al.; PURPOSE: To investigate the expression and regulation of membrane-type matrix metalloproteinases (MT-MMPs) 4, 5, and 6 in the mouse corneas infected with Pseudomonas aeruginosa . METHODS: C57BL/6J mice were intracorneally infected with P . aeruginosa . The expression of MT4-, MT5-, and MT6-MMP was detected at both the mRNA and protein levels by RT-PCR and immunoblot analysis . Immunohistochemical staining was performed to localize the expression of MT4- and MT5-MMP in the mouse corneas . RESULTS: Expression of MT4- and MT5-MMP was detected in the normal (uninfected) cornea by RT-PCR and immunoblot analysis . When infected with P . aeruginosa, the corneas showed significant induction of each MT-MMP . Localization of MT4- and MT5-MMP revealed that the expression of MT5-MMP was restricted to the epithelial tissue in the normal cornea, whereas the induced expression of MT4- and MT5-MMP was predominantly in the substantia propria, which contained most of the infiltrating cells . MT6-MMP expression was not detected in the uninfected cornea but was upregulated in the infected corneas . CONCLUSIONS: Expression of MT4-, MT5-, and MT6-MMP was induced in corneas infected with P . aeruginosa . Immunohistochemistry showed predominant immunoreactivity of MT4- and MT5-MMP in the substantia propria . Previous histologic studies have revealed different patterns of inflammatory cell infiltration with an increased number of polymorphonuclear neutrophils (PMNs) during the early stage of inflammation and increased macrophages during the late stage . These results indicate a good correlation between the overexpression of the MT-MMPs in the infected corneas and the inflammatory response-that is, leukocyte infiltration-indicating that inflammatory cells such as macrophages and PMNs may play a role in the upregulation of MT-MMPs during corneal infection, which in turn can cause the destruction of corneal tissue.

EMBO J, 2001 Dec 3, 20(23), 6735 - 41
Involvement of the twin-arginine translocation system in protein secretion via the type II pathway; Voulhoux R et al.; The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria . The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton . This step is considered to be an extension of the general export pathway, i.e . the export of proteins across the inner membrane by the Sec machinery . Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane . These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton . Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

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