Pseudomonas aeruginosa

Antimicrob Agents Chemother, 1996 Aug, 40(8), 1785 - 9
Gentamicin concentrations in human subcutaneous tissue; Lorentzen H et al.; Wound infections frequently originate from the subcutaneous tissue . The effect of gentamicin in subcutaneous tissue has, however, normally been evaluated from concentrations in blood or wound fluid . The aim of the present study was to investigate the pharmacokinetic properties of gentamicin in human subcutaneous adipose tissue by a microdialysis technique . Seven healthy young volunteers each had four microdialysis probes placed in the fat (subcutaneous) layer of the abdominal skin . After the administration of a 240-mg gentamicin intravenous bolus, consecutive measurements of the drug concentrations in serum and subcutaneous interstitial fluid were obtained simultaneously for 6 h . The tissue gentamicin concentration peaked after 10 to 30 min . The peak concentration in the tissue was 6.7 +/- 2.0 mg.liter-1 (standard deviation), equivalent to 39.1% of the peak concentration in serum . The area under the concentration-versus-time curve for the first 6 h in the tissue was 1,281 +/- 390.0) mg.min liter-1, equivalent to 59.7% of the area under the concentration-versus-time curve in serum . It is concluded that the microdialysis technique can be used to make dynamic and quantitative measurements of the gentamicin concentration in human subcutaneous tissue . In this adipose tissue, the peak concentrations of gentamicin were approximately seven times the MIC for Pseudomonas aeruginosa and 33 times the MIC for Staphylococcus aureus after the administration of an intravenous bolus of 240 mg, indicating the presence of sufficient concentrations in the adipose tissue to be effective against common bacteria.

Am J Vet Res, 1996 Aug, 57(8), 1233 - 48
Effects of fumonisin-containing culture material on pulmonary clearance in swine; Smith GW et al.; OBJECTIVE: To determine the potential effects of feeding tumonisin-containing culture material on the pulmonary clearance of blood-borne particulates and bacteria in swine . ANIMALS: 21 healthy male pigs randomly assigned to control and treated groups . PROCEDURE: Control pigs were fed a standard grower ration while culture material containing fumonisins (20 mg of hydrolyzed fumonisin B1/kg of body weight/d) was added to the feed of treated pigs for 7 days . On day 8, pigs were anesthetized with halothane and catheterized, using a sterile cut-down procedure . 18 hours after recovery from anesthesia, Monastral Blue or Pseudomonas aeruginosa was infused into the right atrium of treated and control pigs for 30 minutes and pulmonary clearance was determined . RESULTS: Pigs that were fed fumonisin-containing culture material had a significantly (P < 0.05) decreased ability to clear Monastral Blue and P aeruginosa . Ultrastructural examination of the lung indicated that uptake of copper pigment by pulmonary intravascular macrophages was decreased in treated pigs . CONCLUSIONS AND CLINICAL RELEVANCE: Fumonisins, even when fed to pigs at sub-lethal concentrations, can inhibit pulmonary intravascular macrophages from removing particulate matter and bacteria from the circulation, thus potentially predisposing swine to infectious disease.

Planta Med, 1996 Aug, 62(4), 374 - 5
Quantitative assay of photoinduced antibiotic activities of naturally-occurring 2,2':5',2"-terthiophenes; Ciofalo M et al.; Nine naturally-occurring 2,2':5',2"-terthiophenes were quantitatively evaluated for their in vitro photoinduced growth inhibitory activity against three bacterial strains . All the compounds proved active towards Staphylococcus aureus . As regards Escherichia coli, the unsubstituted 2,2':5'2"-terthiophene (MIC = 0.62 microgram/ml at 4j/cm2 fluence), was found to be the only compound active . None of the compounds tested displayed activity on Pseudomonas aeruginosa at the highest concentration tested.

Can J Microbiol, 1996 Aug, 42(8), 859 - 62
Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa; Hughes EE et al.; Antisera were produced in mice immunized with 18 synthetic peptide conjugates representing various regions throughout the length of the outer membrane protein F molecule of Pseudomonas aeruginosa and analysed by flow cytometry to identify those antisera capable of binding to the surface of whole cells of P . aeruginosa . Antibodies to peptides 9, 18, 10, and 4 were significantly cell-surface reactive . The maximum median percentage of antibody-binding cells in this assay was 36.6% . Over six different determinations, peptide 9 antisera binding to the cells ranged from 16.9 to 57.0% of the cell population . We propose that the surface accessibility of protein F epitopes varies during the cell cycle.

Microbiology, 1996 Aug, 142 ( Pt 8), 2145 - 51
Flagellin gene and protein variation amongst clinical isolates of Pseudomonas aeruginosa; Winstanley C et al.; Flagellin gene sequences from 64 clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa were amplified by PCR and subjected to RFLP analysis by using seven restriction enzymes to digest the amplified products . Using this approach the isolates were assigned to one of 13 groups . The method was rapid, reproducible and applicable to all isolates . In contrast, serotyping failed to satisfactorily resolve 49% of the strains tested . The vast majority of clinical isolates generated amplified products of 1.02 kb (type a) or 1.25 kb (type b) . Electron microscopical analysis revealed evidence fax some . flagellar structural variation between P . aeruginosa strains . This study provides further evidence that the flagellin gene is a widely applicable and useful genetic marker for studying genetic variation within populations of closely related bacteria.

Microbiology, 1996 Aug, 142 ( Pt 8), 2137 - 44
Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein; Yoneyama H et al.; Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa forms small channels, as assayed by the liposome swelling method . We report here that OprC functions as a channel-forming and copper-binding protein . OprC purified to homogeneity formed a channel in planar lipid bilayers with an ion conductance of about 200 pS in 1 M NaCl . Cloning and sequencing of the gene encoding OprC revealed that it specified a polypeptide comprising 723 and 668 amino acid residues for the precursor and mature polypeptides (M(r) 73,372), respectively . The amino acid sequence of OprC showed the highest degree of similarity with that of NosA of Pseudomonas stutzeri (65% sequence identity) which conveys Cu2+ to intracellular acceptor(s) . OprC showed high copper-binding activity (Kd = 2.6 microM) in aqueous solution containing surfactant . The expression of OprC appeared to be repressed by exogenous Cu2+ and derepressed by anaerobiosis in the presence of nitrate . These results suggest that OprC might be involved in copper utilization.

Photochem Photobiol, 1996 Aug, 64(2), 334 - 9
Lethal effect induced in Pseudomonas aeruginosa Exposed to Ultraviolet-A radiation; Fernandez RO et al.; Ultraviolet-A (365 nm, 120 kJ/m2/h) exposure caused cell death in Pseudomonas aeruginosa at doses at which Escherichia coli cell viability was not affected . We have not found that UVA induced growth delay or any other sublethal effect . Irradiated suspensions of P . aeruginosa showed a marked reduction in membrane-bound succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activities . Succinate-driven respiration and several nutrient transport systems were also inhibited . Whereas SDH and LDH activities were independent of the irradiation conditions, cell viability, respiration and transport systems were protected when irradiation was performed in an N2 atmosphere . A similar protective effect was observed when cells were grown in media containing glycerol or when preirradiation bacterial growth was carried out at 30 degrees C (instead of 37 degrees C) . Results suggest that UVA induces a differential damaging effect on several biochemical functions of P . aeruginosa . The UVA- induced photodamage may fall into two categories: indirect damage mediated by oxygen (cell killing and inhibition of respiration and transport systems) and direct damage to SDH and LDH (apparently not oxygen dependent) . These enzymes and leucine transport appear not to be involved in the lethal effect described herein because they were altered despite viability-preserving conditions

J Appl Bacteriol, 1996 Aug, 81(2), 212 - 6
Note: microbial resistance of wool fabric treated with bis-Quats compounds; Infante MR et al.; In this paper, the antibacterial activity against Bacillus pumilus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa of the wool fabrics treated with new antimicrobial bis-quaternary surfactants, DABK and DABB, is studied . The activity was established on the basis of the agar diffusion and protective antibacterial test results and on the basis of scanning electron microscopy (SEM) observation . The results were compared with the HTAB, a monoquaternary surfactant of conventional use . The results from the agar diffusion and protective antibacterial tests do not enable us to confirm whether these compounds are potentially useful antimicrobial agents for the protection of textiles . However, SEM observations show clearly the efficacy of these compounds to protect the wool fabrics against the micro-organisms . SEM has been a useful technique for the assessment of antibacterial activity in textiles.

J Bacteriol, 1996 Aug, 178(16), 4997 - 5004
Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis; Schurr MJ et al.; The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P . aeruginosa to oxidative stress and extreme temperatures . AlgU also controls conversion of P . aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients . Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster . Here we analyze the biochemical basis of conversion to mucoidy . MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity . MucB, another negative regulator of AlgU, was localized in the periplasm . MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy . These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU . Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P . aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.

J Bacteriol, 1996 Aug, 178(16), 4990 - 6
Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA; Xie ZD et al.; Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients . One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P . aeruginosa chromosomal map . The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family . On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy . Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P . aeruginosa strains isolated from cystic fibrosis patients . We expressed and purified the protein products from the mucA and mucB open reading frames . The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo . Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.

J Bacteriol, 1996 Aug, 178(16), 4942 - 7
Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster; Ruepp A et al.; Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter . Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium) . Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found . They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein . The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides . The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available . Transcripts from H . salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses . It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter . The data indicate that expression of the arc gene cluster and its regulation differ in H . salinarium and P . aeruginosa.

Infect Immun, 1996 Aug, 64(8), 3259 - 66
Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria; Johansen KA et al.; Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis) . Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M . tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities . In contrast, only PLD activity was detected in cell extracts of M . smegmatis . Neither activity was detected in cell-free culture supernatants from these organisms . We and others recently identified two open reading frames in M . tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa . In contrast to the plc genes in P . aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp . Both the mpcA and the mpcB genes were individually cloned in M . smegmatis, and PLC activity was expressed from each gene in this organism . Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M . bovis, M . bovis BCG, and M . marinum but not in several other Mycobacterium species, including M . smegmatis, M . avium, and M . intracellulare . TLC analysis using radiolabeled substrates indicated that M . bovis and M . marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M . bovis BCG cell extracts . Sphingomyelinase activity was also detected in whole-cell extracts of M . tuberculosis, M . marinum, M . bovis, and M . bovis BCG, but this activity was not detected in extracts of M . smegmatis . Sphingomyelinase activity was detected in cell extracts from M . smegmatis harboring either recombinant mpcA or mpcB . These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains.

Infect Immun, 1996 Aug, 64(8), 3154 - 60
Characterization of elastase-deficient clinical isolates of Pseudomonas aeruginosa; Hamood AN et al.; Elastase production in Pseudomonas aeruginosa is regulated by the lasR, lasI, rhlR, and rhlI genes . Recently, we have analyzed several clinical isolates of P . aeruginosa for the production of elastase and other extracellular virulence factors . Four of these isolates (CIT1, CIW5, CIW7, and CIW8) produced no elastolytic activity . We have characterized these isolates with respect to their elastase-deficient phenotype . Elastase was detected by immunoblotting experiments using elastase-specific antiserum . We also determined the presence of IasB and IasR mRNAs by Northern (RNA) blot hybridization experiments using lasB and lasR internal probes, respectively . None of the four elastase-deficient strains produced either the elastase protein or the lasB mRNA . Complementation experiments (using plasmids carrying either the lasB or the lasR gene) were conducted to determine if the isolates carry defective lasB or lasR genes . The presence of either a lasB or a lasR plasmid in CIW7 and CIW8 resulted in the production of very low levels of elastase and lasB mRNA . Neither elastase nor lasB mRNA was detected in CIT1 and CIW5 carrying the lasB plasmid . The presence of the lasR plasmid in CIT1 and CIW5 resulted in the production of lasB mRNA and elastase protein in CIW5 only . All elastase-deficient strains produced detectable levels of lasR mRNA which were enhanced in the presence of the lasR plasmid . The Pseudomonas autoinducer (which is encoded by lasI) was also produced by all strains . CIT1 produced both hemolysin and alkaline protease but was defective in pyocyanin production . These results suggest that (i) CIT1 may contain a defect in a lasB-regulatory gene, (ii) CIW5 carries a defect within lasR, and (iii) the defect in isolates CIW7 and CIW8 affects the efficiency of lasB transcription.

Am J Respir Cell Mol Biol, 1996 Aug, 15(2), 283 - 91
Alginate, the slime exopolysaccharide of Pseudomonas aeruginosa, binds human leukocyte elastase, retards inhibition by alpha 1-proteinase inhibitor, and accelerates inhibition by secretory leukoprotease inhibitor; Ying QL et al.; The interaction of alginate from Pseudomonas aeruginosa ATCC 39324 with human leukocyte elastase was studied by determining the effects of the polysaccharide on the amidolytic activity of the enzyme toward a range of synthetic peptide substrates of different length . The data support a model in which each elastase molecule interacts with a total of 19 uronic acid units on the alginate, primarily through electrostatic forces . Binding of alginate results in occlusion of distal subsites, most likely S4 and S5, of the enzyme's extended substrate-binding domain . As a result, alginate alone appears to be a weak inhibitor of the hydrolysis of long synthetic peptide substrates and {14C}elastin by elastase . Alginate also has effects on the antielastase function of naturally occurring protease inhibitors in the lung: It reduces the association rate of elastase and alpha 1-proteinase inhibitor, whereas it increases the association rate of elastase and secretory leukoprotease inhibitor . In the presence of 36 micrograms/ml alginate, the median concentration found in sputum from cystic fibrosis patients infected with mucoid strains of P . aeruginosa, the second-order rate constant for inhibition of elastase by secretory leukoprotease inhibitor is 2.6-fold greater than that for alpha 1-proteinase inhibitor . Alginate has only a minor effect on the antielastase activities of elafin and a recombinant form of the isolated C-terminal domain of secretory leukoprotease inhibitor . Based on these findings, alginate may be an important factor in determining the local distribution of leukocyte elastase and perturbing the overall protease-antiprotease balance in the infected lungs of cystic fibrosis patients.

J Med Microbiol, 1996 Aug, 45(2), 110 - 9
The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa; Barth AL et al.; Many isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are auxotrophic and require amino acids for growth . A quantitative assay was used to determine the total content of free amino acids of sputum sol-phase extracts from CF and non-CF patients to assess the presence of amino acids in the airway . CF patients colonised with auxotrophic P . aeruginosa had a higher sputum amino-acid content (mean 6.77 mg/ml) than those colonised with prototrophs (mean 3.77 mg/ml); overall, CF specimens (mean 5.70 mg/ml) had a higher amino-acid content than non-CF samples (2.52 mg/ml) . The amino-acid profile of sputum extracts was assessed by one-dimensional thin layer chromatography (TLC) . Several amino acids were identified in the extracts, in particular, leucine, isoleucine, phenylalanine, tyrosine, alanine, serine and methionine or valine or both . All sputum specimens except two (which contained < 1.5 mg of amino acids/ml), promoted the growth, of 34 auxotrophic strains of P . aeruginosa from CF patients in a minimal medium . These results indicate, therefore, that amino acids are plentiful in the sputum of CF patients and are able to supply the requirements of auxotrophic strains . It is suggested that the increased amino-acid content in the airways of CF patients plays a significant role in the selection and maintenance of nutritionally deficient P . aeruginosa.

Curr Microbiol, 1996 Aug, 33(2), 109 - 17
Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo; McClure CD et al.; Patients with cystic fibrosis often have chronic and ultimately lethal pulmonary infections with Pseudomonas aeruginosa . In order to understand why these bacteria resist pulmonary clearance, we have investigated the interaction of P . aeruginosa and phagocytic cells . In an earlier study we reported that sub-lytic concentrations of two glycolipids produced by P . aeruginosa (the mono- and dirhamnolipids) caused structural changes in human monocyte-derived macrophages, and at lower concentrations inhibited the phagocytosis of Staphylococcus epidermidis by these cells . In the present study we demonstrate that rhamnolipids also inhibit the in vitro phagocytosis of both P . aeruginosa and Saccharomyces cerevisiae by thioglycollate-elicited mouse peritoneal macrophages . Using lucifer yellow to label the lysosomal compartments of macrophages, we determined that rhamnolipids interfere with the internalization of attached particles and reduce the level of phagosome-lysosome fusion of internalized targets within macrophages . We also demonstrate that physiologically relevant concentrations of rhamnolipids injected intratracheally into rat lungs inhibited the response of alveolar macrophages to a challenge of zymosan particles in vivo . These studies further demonstrate the profound inhibitory effects of P . aeruginosa rhamnolipids on macrophage function and are consistent with our hypothesis that the in situ production of these rhamnolipids directly contributes to the persistence of this pathogen in cystic fibrosis patient lungs.

J Biotechnol, 1996 Jul 18, 48(1-2), 9 - 14
Large scale recovery and purification of periplasmic recombinant protein from E . coli using expanded bed adsorption chromatography followed by new ion exchange media; Johansson HJ et al.; Expanded bed chromatography was used for the recovery and purification of modified Pseudomonas aeruginosa exotoxin A . The exotoxin accumulates in the periplasmic space of E . coli BL21 (lambda DE3), was released from the cells by osmotic shock and captured by applying the open cell suspension directly to an anion exchanger (STREAMLINE DEAE) using an expanded bed (STREAMLINE) column . Processing of 4.5 kg of E . coli using the expanded bed process was 3 times faster and did not require clarification of the bacterial extract, in comparison with the conventional purification method . Also, the recovered protein solution was 3 times more concentrated and the yield slightly higher.

Biochemistry, 1996 Jul 16, 35(28), 9042 - 51
In vitro enzyme activation and folded stability of Pseudomonas aeruginosa exotoxin A and its C-terminal peptide; Beattie BK et al.; Pseudomonas aeruginosa exotoxin A(ETA) and its C-terminal, enzymatically active fragment (PE40, 375 residues) were studied by high-performance size-exclusion chromatography, steady-state and stopped-flow fluorescence spectroscopy, and circular dichroism spectroscopy . Both proteins have been overexpressed and purified by high-performance liquid chromatography . The effect of various activation conditions (pH, urea, and DTT) on enzymatic activity was studied . Upon enzymatic activation, structural changes induced within both proteins' structures were monitored, and these changes were correlated with concomitant alterations in the catalytic activity of the proteins . The pH optimum of enzymatic activity for both ETA and PE40 was between 7.0 and 8.0, decreasing to nearly zero at acidic (pH 5.0) and basic (pH 11-12) values . Analysis of the pH titration data revealed the presence of two distinct pKa values which implicate a His residue(s) (likely His-440 and -426) and a Tyr or Lys residue (possibly Tyr-481) . The identity and possible role of an active site Lys residue is not known . Additionally, a significant increase in the Stokes radii of both proteins was detected when the pH was lowered from 8.0 to 6.0 . The enzymatic activity of PE40 was not affected by urea or DTT, and its Stokes radius decreased monotonically with increasing urea concentration in the presence of DTT . In contrast, the enzymatic activity of ETA peaked when the protein was preincubated with 4.0 M urea, and this coincided with a large transition (increase) in the protein's Stokes radius between 3 and 5 M urea . Furthermore, loss of helical secondary structure of both PE40 and ETA commenced at approximately 2 M urea and progressively diminished at higher denaturant concentrations . The unfolding of both proteins in urea (and DTT) was reversible, and the free energies of unfolding were determined by both circular dichroism and fluorescence spectroscopy and were found to be 13.7 +/- 2.9 and 9.8 +/- 3.4 kJ/mol, respectively, for ETA and were 17.8 +/- 6.8 and 7.5 +/- 3.6 kJ/mol, respectively, for PE40 . The refolding rate of PE40 was relatively rapid {t 1/2(1) = 27 s, t 1/2(2) = 624 s}, which was in stark contrast to the refolding rate of ETA (t 1/2 = several hours) . The relative refolding rates of PE40 and ETA help to explain the mechanism of in vitro enzyme activation and assay.

Curr Opin Ophthalmol, 1996 Aug, 7(4), 17 - 21
The clinical use of contact lenses and collagen shields; van Setten GB; Contact lens wear before and after photorefractive or phototherapeutic keratectomy with excimer lasers has become an increasingly interesting subject . No negative side effects of contact lens wear prior to photorefractive keratectomy have yet been shown and refractive errors postoperatively do not seem to constitute a major practical problem . Normal side effects include the risk of bacterial infection and keratitis . The major pathogen in this context is still Pseudomonas aeruginosa, although increased insight into the mechanisms of bacterial adhesion and improvements in cleaning procedures now allow more effective preventive care of contact lenses themselves and of contact lens cases . Tear fluid research using leucotriene C4 as a possible indicator for subclinical inflammation has led to interesting results, and the importance of the arachidonic pathway and its metabolites has become more evident . These advances contribute considerably to the safe use of contact lenses in clinical ophthalmology, although major innovations such as the multifocal contact lenses have not reached the stage of perfection.

Mikrobiologiia, 1996 Jul-Aug, 65(4), 540 - 5
{Characterization of B-type bacteriophages adsorbed on Pseudomonas aeruginosa pili}; Zhilenkov EL et al.; Pilus-dependent B-morphotype bacteriophages isolated from various sources were studied . The adsorption of phages on Pseudomonas aeruginosa pili was proven . Electron-microscopic examination of the morphology of phages was carried out, and the adsorption properties were partially described . It was suggested that adsorption apparatuses of different pilus-dependent B-phages are alike with respect to structure and function.

Indian J Med Sci, 1996 Jul, 50(7), 239 - 43
Activity of third generation cephalosporins against Pseudomonas aeruginosa in high risk hospital units; Puri J et al.; Ceftazidime and Cefoperazone are the two third generation cephalosporins with anti-pseudomonal activity . They have been frequently used in the I.C.U.s . in the developed countries but their use in the Indian hospitals has begun relatively recently . We studied the in-vitro susceptibility of 139 Pseudomonas aeruginosa isolates that were multiple drug resistant from the Resuscitation Unit/I.C.U . (61 strains), Burns Unit (48 strains), Surgical Post-operative unit (24 strains), Nephrology unit (6 strains) of our hospital to these two cephalosporine over a period of about 18 months . Antibiotic susceptibility was studied using Kirby Bauer's disc dibusion method . Out of a total of 139 strains of multiple drug resistant Pseudomonas aeruginosa tested, 17.9% were found resistant each to Ceftazidime and Cefoperazone separately and 10% were found resistant to both antibiotics.

Genetika, 1996 Jul, 32(7), 914 - 21
{Cloning and study of the expression of Pseudomonas aeruginosa cip1 gene by phage-transposon D3112 in a homologous host and in Escherichia coli}; Bidenko EM et al.; Regulatory gene cipl of Pseudomonas aeruginosa transposable phage D3112 was cloned, and its expression was studied in P . aeruginosa and Escherichia coli . Overexpression of the cipl gene prevents transcription and replication of phage D3112 DNA and also lysogenization of bacteria P . aeruginosa PAO1 by phage D3112 . The direction of cipl gene transcription within the vector was determined in the study of cipl gene expression, dependent on its orientation toward the gene lacZ promoter . The expression of the cloned cipl gene inhibited the specific TCS phenotype of E . coli (RP4 :: D3112) cells . The functional homology of the cipl gene of phage D3112 and the negative regulator ner of E . coli Mul phage was discussed.

Z Gastroenterol, 1996 Jul, 34(7), 434 - 7
{Antibiotic-induced prolonged cholestasis: suspected induction by ceftibuten}; Combe C et al.; We report on a 43-year-old female diabetic patient who was treated with ceftibuten because of Pseudomonas aeruginosa induced otitis externa . Thereafter she developed a prolonged seven-months-persisting irreversible cholestasis . Liver puncutre revealed a canalicular and hepatocellular cholestasis without liver cell necrosis or bile duct injury . After seven-months the patient died because of antibiotic-resistant Pseudomonas-septicemia.

Thorax, 1996 Jul, 51(7), 733 - 8
Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry; Petersen TD et al.; BACKGROUND: Antibodies against chromosomal beta-lactamase of Pseudomonas aeruginosa (a beta ab) are markers of the development of resistance of P aeruginosa to beta-lactam antibiotics in patients with cystic fibrosis and chronic lung infection . The role of these antibodies in patients with chronic lung infection with P aeruginosa was further investigated by correlating the a beta ab IgG subclasses with pulmonary function in patients with cystic fibrosis . METHODS: Immunoglobulin G (IgG) and IgG subclass a beta ab were investigated by western blotting and quantified by laser scanning densitometry . A longitudinal study on 43 consecutive patients with cystic fibrosis who developed chronic lung infection with P aeruginosa was performed . RESULTS: IgG subclass a beta ab appeared in all patients with chronic infection with P aeruginosa . Eleven years after the onset of infection all the patients had IgG1, 79% had IgG4, 56% IgG2, and only 16% of the patients had IgG3 a beta ab . The IgG1 and IgG4 a beta ab appeared first, and more than 50% of the patients were IgG1 and IgG4 a beta ab positive within 2-3 years of the onset of infection, but IgG2 positivity only appeared after seven years and IgG3 remained absent from most of the patients . The median a beta ab levels increased during chronic infection: 100-fold for IgG1, 22-fold for IgG2, and 45-fold for IgG4 . A 16-fold increase in the IgG3 a beta ab levels was detected in the six patients who developed IgG3 a beta ab . In the first four years of the chronic infection the a beta ab titres were higher in patients with good lung function than in those with poor lung function . CONCLUSIONS: The association of a weak IgG3 and a strong IgG4 a beta ab response suggests that the contribution of a beta ab antibodies to lung diseases mediated by immune complexes might be less important than other antipseudomonal antibodies . A beneficial neutralising effect of the a beta ab antibodies on the antibiotic destroying enzymes may be an additional factor.

Haematologica, 1996 Jul-Aug, 81(4), 335 - 8
Rapid liver failure related to chronic C hepatitis in an HIV seropositive hemophilic patient with severe immunodepression; Dragoni F et al.; We report the case of a young HIV seropositive patient with severe hemophilia A who presented rapid liver failure related to his chronic C hepatitis . The patient had been receiving factor VIII:C clotting factor concentrates (mean 60,000 U/year) since 1975 . In 1984 alanine aminotransferase presented abnormal levels . The CD4 lymphocyte count in 1991 was normal and ultrasonographic scan showed normal liver morphology . In 1991 the patient were found to be seropositive for HCV antibodies as detected by the ELISA method and confirmed by the RIBA method . One year later, a progressive increase in policlonal gamma-globulin and a decrease in the CD4+ lymphocyte count to below 500/muL were detected in concomitance with ultrasonographic evidence of a progressive increase in the longitudinal diameters of the liver and spleen and signs of liver inhomogeneity . A significant inverse correlation was observed between the increase in the longitudinal diameter of the liver and the decline in albumin levels, and between the increase in the longitudinal diameter of the liver and the drop in platelet count . Elevated levels of ammonemia, gamma-glutamyl transpeptidase, alkaline phosphatase and IgA were detected . Moreover, decreased levels of the C4 and C3 complement fractions were documented . At this time (1994), esophagogram and esophagogastroscopy evidenced varicosities in the lower esophageal section (stage F1) . The patient died in 1995 March at the age of 29 years of sudden septic shock related to Pseudomonas aeruginosa infection.

J Antimicrob Chemother, 1996 Jul, 38(1), 133 - 7
A mechanism of rifamycin inhibition and resistance in Pseudomonas aeruginosa; Yee YC et al.; We sought to study the nature of rifampicin resistance in Pseudomonas aeruginosa . We hypothesized that the rifamycin regions of RNA polymerase are conserved in P . aeruginosa and that rifampicin resistance is mediated by a mutation in the rpoB gene encoding the beta subunit of RNA polymerase . Transcription assays showed that 50 nM of rifampicin inhibited transcription > 99% in a clinical isolate (MIC = 32 mg/L) and only < 40% in the rifampicin resistant mutant (MIC = 1000 mg/L) . DNA sequencing revealed that the rifampicin regions are conserved in P . aeruginosa and the rifampicin regions of the rifampicin-resistant strain contained a mutation . Sodium hexametaphosphate lowered rifamycin MIC in a rifamycin-resistant mutant four-fold and in the clinical isolate 32-fold, suggesting that P . aeruginosa has a natural membrane barrier to rifamycins.

J Antimicrob Chemother, 1996 Jul, 38(1), 39 - 45
Carbapenem resistance in Pseudomonas aeruginosa from cystic fibrosis patients; Ballestero S et al.; The evolution of imipenem resistance was evaluated in Pseudomonas aeruginosa sequentially isolated from 42 patients with cystic fibrosis . Susceptibility was determined using a commercial microdilution system and imipenem resistance was confirmed by the agar dilution technique . Resistance to imipenem increased during the years from 1988 to 1992 . A total of 12 patients (28.5%) carried resistant strains (11.6% of the total P . aeruginosa isolates) but only two of them were treated with the carbapenem . The other patient under imipenem treatment did not harbour resistant isolates . Sixty-four per cent of the imipenem resistant isolates were also meropenem resistant and showed low susceptibility to the other beta-lactams and tobramycin and amikacin . Twenty-one strains were selected for biochemical study . Imipenem susceptible strains showed normal OprD in two strains and diminished OprD in two more . Five strains with MIC of imipenem of 4-8 mg/L lacked OprD while another two had a band with decreased density . All strains with MIC higher than 8 completely lacked this band in western-blot analysis . Imipenem MICs of 0.5-2 mg/L only slightly increased to 1-4 mg/L when a pattern of beta-lactamase derepression was observed . While those with imipenem MICs between 8-16 mg/L increased the imipenem MIC to 16-64 mg/L in the population with a beta-lactamase derepression phenotype.

Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1996 Jul-Aug, 37(4), 283 - 5
Perforated appendicitis in a 4-month-old infant; Ko YS et al.; A 4-month-old male infant was admitted to our hospital because of poor intake and mild abdominal distention for 1 day . Fever and watery diarrhea had occurred 4 days prior to admission, but subsided 2 days later after taking oral medications . A physical examination showed an acute ill-looking baby with a soft and mildly distended abdomen . The bowel sound was hypoactive and no obvious abdominal tenderness was found . Normal leukocyte and differential counts were noted in initial laboratory examinations; however, the serum level of C reactive protein was extremely high (31.4 mg/dL) . Progressive abdominal distention and bilious vomiting occurred . Serial plain films of abdomen showed ileus with a fixed gas pattern and an abdominal echo revealed intraperitoneal fluid accumulation . Under the impression of intestinal perforation, an emergency laparotomy was performed . A perforated appendicitis with turbid fluid in the peritoneal cavity was noted during surgery . A pus culture grew Pseudomonas aeruginosa which was sensitive to Ceftazidime only . Triple antibiotics consisting of Prostaphlin, Metronidazole, and Ceftazidime were administered for 2 weeks . The patient was discharged 3 weeks later without any complications . Appendicitis in infancy is a rare condition and associated with a high frequency of perforation and peritonitis . Diagnosis is often difficult because of variable and nonspecific clinical manifestations.

Protein Eng, 1996 Jul, 9(7), 611 - 6
Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system; Guidi-Rontani C; In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613) . We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli . We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells . Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine . These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A . These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.

Mol Microbiol, 1996 Jul, 21(1), 97 - 110
Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production; Hamood AN et al.; Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes . In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P . aeruginosa . In an iron-deficient medium, the presence of a plasmid carrying ptxR in P . aeruginosa PAO1 resulted in a four-to fivefold increase in exotoxin A synthesis . No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease . Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb Kpnl-Bg/II fragment . Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR) . The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system . The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators . The amino-terminus region of PtxR contains a putative helix-turn-helix DNA-binding motif . Specific ptxR-deletion mutants in P . aeruginosa strains PAO1 and PA103 were constructed . In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity . However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1 . Transcriptional studies, using both toxA-lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription . These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.

New Microbiol, 1996 Jul, 19(3), 221 - 6
Pseudomonas aeruginosa proteases: purification procedures for an enzymatic standard; Cristallo A et al.; Pseudomonas aeruginosa may cause severe infections in debilitated patients . Strains of this microorganism produce several extracellular space proteins, some of which are believed to be virulence factors . There are experimental correlations between the ability to produce proteases and virulence . Treatment of bacteria with subinhibitory concentrations of antimicrobial agents frequently increases bacterial phagocytosis, intracellular killing, and suppresses the production of bacterial virulence factors, including extracellular enzymes . We suggest a simple method for production, purification and quantitation of Pseudomonas aeruginosa extracellular proteases suitable for use in investigations of their role as virulence factors.

Bioorg Med Chem, 1996 Jul, 4(7), 1089 - 95
The hemA gene encoding glutamyl-tRNA reductase from the archaeon Methanobacterium thermoautotrophicum strain Marburg; Hungerer C et al.; In archaea the first general tetrapyrrole precursor 5-aminolevulinic acid (ALA) is formed via the tRNA-dependent five-carbon pathway from glutamate . We have cloned the hemA gene encoding the central enzyme of the pathway glutamyl-tRNA reductase from the methanogenic archaeon Methanobacterium thermoautotrophicum by complementation of an Escherichia coli hemA mutant to ALA prototrophy . An 1194 bp open reading frame that encodes a 398 amino acid polypeptide with the calculated M, 44,509 was detected . The deduced amino acid sequence showed 20-35% amino acid identity to bacterial HemAs with the highest identity score to the Pseudomonas aeruginosa HemA . An identity of approximately 22% was found to plant HemAs . Glutamyl-tRNA reductase activity was shown for the M . thermoautotrophicum HemA after overexpression in E . coli and partial purification . The enzymatic reaction catalyzed by the partially purified enzyme revealed a temperature optimum of 65 degrees C at an optimal pH of 7.0 . The reductase utilized preferentially NADPH for the reduction of the activated carboxyl group . The presence of ATP and GTP showed no obvious influence on catalysis.

J Reprod Med, 1996 Jul, 41(7), 534 - 6
Pseudomonas aeruginosa as an unusual cause of intraamniotic infection, fulminant neonatal sepsis and neonatal death . A case report; O'Boyle JD et al.; BACKGROUND: Intraamniotic infection may be caused by a wide variety of microorganisms . Significant maternal and neonatal morbidity have been associated with both subclinical and clinical infection . CASE: We identified a case in which Pseudomonas aeruginosa was isolated as the cause of intraamniotic infection, fulminant neonatal sepsis and neonatal death . CONCLUSION: Though P aeruginosa is an unusual cause of intraamniotic infection, it is important because of its high virulence.

Artif Organs, 1996 Jul, 20(7), 798 - 800
Bacterial challenge of NISSHO ultrafilter ETF 609: results of in vitro testing; Krautzig S et al.; In hemodialysis, a certain degree of bacterial contamination on the dialysate side is a regular finding . Concern has been growing that this contamination may lead to a chronic inflammatory response in the patient . Ultrafiltration of dialysate can be used to reduce bacterial content and levels of cytokine-inducing substances upstream of the patient's dialyzer . The aim of this study was to test in vitro the rejection capacity of a polysulfone hollow-fiber ultrafilter (ETF 609, NISSHO Co., Osaka, Japan) challenged with bacterial filtrates derived from Pseudomonas aeruginosa PA103 . Results showed a reduction of interleukin-1 beta-inducing activity (measured on peripheral blood mononuclear cells) from 5,035 +/- 394 pg/ml prefilter to nondetectable levels postfilter and endotoxin levels (limulus amebocyte lysate assay) of 4,167 +/- 1,079 versus 12 +/- 2 pg/ml, respectively . In conclusion, ultrafiltration of dialysate with the polysulfone ultrafilter ETF 609 leads to a potent reduction of cytokine-inducing activity.

Antimicrob Agents Chemother, 1996 Jul, 40(7), 1633 - 9
Prevalence of resistance to three fluoroquinolones: assessment of levofloxacin disk test error rates and surrogate predictors of levofloxacin susceptibility . AST Surveillance Group; Fuchs PC et al.; More than 3,000 consecutive clinical bacterial isolates from 10 U.S . medical centers were subjected to standard broth microdilution and disk diffusion tests to determine their susceptibilities to levofloxacin, ofloxacin, D-ofloxacin, and ciprofloxacin . Levofloxacin was confirmed to be twice as active as ofloxacin and to have activity comparable to that of ciprofloxacin, with minor variations in activity against some species . The prevalence of resistant isolates was 7.1% to levofloxacin, 9.3% to ciprofloxacin, and 11.2% to ofloxacin . The susceptibilities of some species to the quinolones were less than those reported in previous studies . Pseudomonas aeruginosa isolates had the greatest variability in their susceptibilities to the three drugs between the participating centers . Two proposed zone size breakpoints for levofloxacin disk tests yielded similar low error rates . Ofloxacin and ciprofloxacin susceptibility test results correlated reasonably well with those of levofloxacin and could be used as surrogate indicators of levofloxacin susceptibility, but that resulted in some serious errors, and thus, direct testing of levofloxacin susceptibility is preferable . Replicate testing of standard quality control strains confirmed the established and proposed quality control parameters for all three quinolones tested.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 209 - 14
The effect of the length of a malarial epitope on its antigenicity and immunogenicity in an epitope presentation system using the Pseudomonas aeruginosa outer membrane protein OprF as the carrier; Wong RS et al.; This study showed that the antigenicity of a malarial epitope increased with the length of the epitope when inserted at positions aa26 (amino acid position 26) and aa196, but not at aa213, of the Pseudomonas aeruginosa major outer membrane protein OprF (326 amino acids) . Immunization studies showed that a 19-aa epitope was significantly more immunogenic than a 7-aa epitope when inserted at aa26 of OprF, while neither an 11- nor a 19-aa epitope fused to the C-terminus of glutathione S-transferase was immunogenic.

J Bacteriol, 1996 Jul, 178(14), 4297 - 300
Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa; Bleves S et al.; Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa . Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features . Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane . To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization . We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.

J Bacteriol, 1996 Jul, 178(14), 3996 - 4003
Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities; Hassett DJ et al.; Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase . At present, the nature of the regulation of these enzymes in P . aeruginosa Is not understood . To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin . Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions . The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron . Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant . Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype . Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD . All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake . Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity . Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain . In summary, the results indicate that mutations in the P . aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P . aeruginosa . We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.

J Nat Prod, 1996 Jul, 59(7), 668 - 70
Antibacterial neoclerodane diterpenoids from Ajuga lupulina; Chen H et al.; The whole plants of Ajuga lupulina afforded five compounds, including three new clerodane diterpenes, lupulins A-C (1-3), whose structures were elucidated by spectral methods . Among these compounds, lupulins A (1) and B (2) as well as the acid hydrolysate (5) of lupulin D (4) showed antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

Infect Immun, 1996 Jul, 64(7), 2774 - 81
Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigmaE); Yu H et al.; A discerning feature of Pseudomonas aeruginosa strains causing chronic endobronchial infections in cystic fibrosis is their conversion into the mucoid, exopolysaccharide alginate-overproducing phenotype . This morphologically prominent change is caused by mutations which upregulate AlgU (sigma(E)), a novel extreme-stress sigma factor with functional equivalents in gram-negative organisms . In this work, we investigated the role of algU in P . aeruginosa sensitivity to reactive oxygen intermediates, killing by phagocytic cells, and systemic virulence of this bacterium . Inactivation of algU in P . aeruginosa PA01 increased its susceptibility to killing by chemically or enzymatically generated halogenated reactive oxygen intermediates and reduced its survival in bactericidal assays with J774 murine macrophages and human neutrophils . Surprisingly, inactivation of algU caused increased systemic virulence of P . aeruginosa in mouse models of acute infection . The increased lethality of the algU-deficient strain was also observed in the endotoxin-resistant C3H/HeJ mice . Only minor differences between algU+ and algU mutant cells in their sensitivity to human serum were observed, and no differences in their lipopolysaccharide profiles were detected . Intriguingly, while inactivation of algU downregulated five polypeptides it also upregulated the expression of seven polypeptides as determined by two-dimensional gel analyses, suggesting that algU plays both a positive and a negative role in gene expression in P . aeruginosa . While the observation that algU inactivation increases systemic virulence in P . aeruginosa requires further explanation, this phenomenon contrasts with the apparent selection for strains with upregulated AlgU during colonization of the cystic fibrosis lung and suggests opposing roles for this system in chronic and acute infections.

J Bacteriol, 1996 Jul, 178(13), 3809 - 17
Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa; Alm RA et al.; Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces . In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage . Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P . aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA* . The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli . Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes . However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis . The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.

Am J Respir Crit Care Med, 1996 Jul, 154(1), 124 - 9
Respiratory tract colonization and infection in patients with chronic tracheostomy . A one-year study in patients living at home; Harlid R et al.; The high rate of complications, especially respiratory tract infection (RTI), reported in patients with chronic tracheostomy (CT) has discouraged physicians from using this method . However, previous studies of CT have concerned mainly hospitalized patients . We have followed the bacterial colonization patterns of the upper and lower respiratory tract and recorded all RTIs in 39 outpatients with CT during a 12-mo period . Patients were colonized with one or more potential pathogens at the stomal site and in the trachea in 95% and 83%, respectively, of all sampling occasions . Staphylococcus aureus, gram-negative enteric bacteria (GNEB), and Pseudomonas aeruginosa were the most common colonizing bacteria at these sites . Seventy percent of bronchial-protected brush cultures were negative, despite simultaneous heavy colonization of the stomal site or the trachea . Only 18 of 39 (46%) patients were treated with antibiotics because of RTIs on a total of 30 occasions during the study year . Of these, only five episodes of pneumonia in four patients were registered, corresponding to an incidence of about 10 per 100 person years . We conclude that outpatients with chronic tracheostomy can be managed with a low risk for developing severe RTIs, despite massive airway colonization with potentially pathogenic bacteria.

Am J Respir Cell Mol Biol, 1996 Jul, 15(1), 132 - 40
Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A; Azghani AO; Lung injury in bacterial infection is a multifactorial phenomenon that involves bacterial metabolites and host factors . Primary isolates of type II pneumocytes and established cultures of Madin-Darby canine kidney (MDCK) cells were used to study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular permeability . The results indicate that elastase (PE) and exotoxin A (Exo A) have different, but complementary, actions that diminish epithelial barrier function . We measured transepithelial electrical resistance (TER) and permeability coefficient for mannitol (Pm) across cell monolayers plated on tissue culture membranes . Application of 100 ng/ml of Exo A to the basal side decreased TER from 1,405 +/- 106 to 462 +/- 50 ohm (omega) and increased Pm for mannitol 6-fold in 16 h (P < 0.05) . Application of Exo A to the apical side did not affect either TER or Pm . In contrast, PE (6.5 U/ml) applied either apically or basolaterally reduced TER to 353 +/- 66 omega and increased Pm by 10-fold within 90 min (P < 0.05) . The increase in permeability correlated with the number of bacteria that traversed the epithelial monolayers . Fluorescent staining and western immunoblot analysis of toxin-treated cells showed that two tight junctional proteins, ZO-1 and ZO-2, were depleted in monolayers treated with enzymatically active PE . The junctional proteins decreased in cells treated overnight with Exo A but were not depleted . Neither agent diminished cell viability as measured by trypan blue staining or release of radioactivity from 51 Cr-labeled cells . Elastase from P . aeruginosa thus seems to increase alveolar epithelial permeability by damaging tight junction-associated proteins . Exo A, through its effect on protein synthesis, may render the cells unable to restore the junctional proteins and thus the functional junctions.

Med Pediatr Oncol, 1996 Jul, 27(1), 62 - 3
Ecthyma gangrenosum occurring at sites of iatrogenic trauma in pediatric oncology patients; Murphy O et al.; We report two cases of ecthyma gangrenosum which occurred at sites of iatrogenic trauma . The first case developed due to metastatic seeding with Pseudomonas aeruginosa during an episode of septicaemia and the second case occurred as a primary skin lesion . Both required prolonged courses of antibiotics and one patient died . The different pathogenic mechanisms and outcomes associated with this condition are discussed.

Eur J Pharmacol, 1996 Jun 27, 307(2), 191 - 9
Evidence for tumor necrosis factor alpha as a mediator of the toxicity of a cyclooxygenase inhibitor in Gram-negative sepsis; Campanile F et al.; To investigate the effect of cyclooxygenase inhibition in experimental Gram-negative sepsis, indomethacin was administered to mice at different times (1 or 5 days, or 1 h) before sublethal infection with an intravenous inoculum of Pseudomonas aeruginosa Early indomethacin exposure did not alter the outcome of infection, yet treatment at the time of bacterial challenge resulted in a high mortality rate . Polymerase chain reaction-assisted mRNA amplification in the spleens of infected mice revealed that tumor necrosis factor alpha (TNF-alpha) messenger was selectively expressed by the drug-treated and infected mice during the 24 h preceding death . Higher TNF-alpha levels were found in sera from these mice, whose macrophages produced increased levels of nitric oxide in vitro . Both pentoxifylline, an inhibitor of TNF-alpha synthesis, and an inhibitor of nitric oxide production improved survival in the indomethacin-treated and infected mice, although no such effect followed the administration of TNF-neutralizing antibodies . These data support the notion that cyclooxygenase inhibitors may exert both positive and negative effects in Gram-negative sepsis, the latter presumably involving overproduction of TNF-alpha.

FEMS Microbiol Lett, 1996 Jun 15, 140(1), 15 - 22
Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl)homoserine lactone; Stead P et al.; A range of Pseudomonas spp . and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor L-N-(3-oxohexanoyl)homoserine lactone . In one of these, P . aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner . The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 micrograms/ml of the autoregulator . In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.

MMWR Morb Mortal Wkly Rep, 1996 Jun 14, 45(23), 491 - 4
Outbreaks of postoperative bacterial endophthalmitis caused by intrinsically contaminated ophthalmic solutions--Thailand, 1992, and Canada, 1993; Cloning of the Pseudomonas aeruginosa gene encoding CDP-diglyceride synthetase; Department of Fermentation Technology, Hiroshima University, JapanThe CDP-diglyceride synthetase (CDS)-encoding gene (cds) from Pseudomonas aeruginosa PAO1 was cloned and sequenced . The gene possessed an open reading frame of 813 bp capable of encoding a putative polypeptide of 271 amino acids (aa) (28 699 Da) . The deduced aa sequence of CDS revealed a 67% similarity (45% identity) to Escherichia coli CDS.

Gene, 1996 Jun 12, 172(1), 163 - 4
Construction of improved vectors for protein production in Pseudomonas aeruginosa; Watson AA et al.; We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19 . The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence . In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA . The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.

Orv Hetil, 1996 Jun 9, 137(23), 1259 - 62
{Diagnostic value of C-reactive protein levels in children with bone marrow transplantation}; Pinter E et al.; Serum quantitative C-reactive protein concentrations were measured in 16 bone marrow transplanted children at 202 occasions during and after the transplant period . Serum C-reactive protein concentrations were moderately increased in patients with viral and protozoon infections (5-67 mg/l) . High values were measured in patients with bacterial and fungal infections . The C-reactive protein level was between 15-102 mg/l in Coag . neg . Staphylococcus sepsis, and 160-178 mg/l in Pseudomonas aeruginosa infection, when blood cultures were positive . Values of 154-358 mg/l was found with Candida sepsis . C-reactive protein levels were 10-17 mg/l in 7 acute GvHD episodes, only one of the patients had high level (325 mg/l) in GvHD . In these cases the condition was very severe and affected the total surface of the skin and the gastrointestinal tract also . C-reactive protein becomes a valuable aid as laboratory parameter in the diagnosis of bone marrow transplant recipients with suspected bacterial infection and in monitoring of therapeutic efficiency.

Exp Eye Res, 1996 Jun, 62(6), 641 - 50
Bacterial proteases and adherence of Pseudomonas aeruginosa to mouse cornea; Gupta SK et al.; The goal of this study was to test whether bacterial exoproducts, such as elastase or alkaline protease contribute to the initial binding of Pseudomonas aeruginosa to mouse corneal epithelium . Each protease, purified from P . aeruginosa, when applied exogenously at concentrations of either 25 or 50 ng ml-1, elevated binding of Pseudomonas to mouse cornea in organ culture . Polyclonal antibodies against bacterial alkaline protease, but not elastase, interfered with bacterial binding and reduced significantly the number of organisms bound to cornea in an organ culture binding inhibition assay . Zymographic analysis of conditioned media from additional organ culture experiments showed that the P . aeruginosa strain used, which is highly virulent in cornea in vivo, secretes detectable levels of alkaline protease, but not elastase in vitro and that secretion was enhanced if the corneal epithelium was wounded . Lastly, how alkaline protease enhanced bacterial binding to the corneal epithelium of the organ cultured eye was examined . Data from this study suggest that exposure of lipase-sensitivity epithelial receptors represents at least one mechanism.

J Indian Med Assoc, 1996 Jun, 94(6), 230 - 3
Study of burn sepsis with special reference to Pseudomonas aeruginosa; Nagesha CN et al.; Fifty cases of burn of different degrees were subjected to clinical and microbiological studies . A total of 60 isolates were obtained . Of these, 40 (80.0% incidences) were Ps aeruginosa, 8 (16.0 incidences) Staph pyogenes, 6 (12.0% incidences) Kl pneumoniae, 4 (80.0% incidences) Esch coli and 2 (4.0% incidences) C albicans . Monobacterial cultures showed isolations in 41 cases (82.0%) and 34 (68.0%) of them were Ps aeruginosa . At the time to admission 42 cases (84.0%) were infected and during one week of hospitalisation another 8 cases (16.0%) were infected yielding an overall infection rate of 100% . The commonest organism on admission and after hospitalisation was Ps aeruginosa with isolation rates of 60.0% (30) and 20.0% (10) respectively . Gram-negative bacilli, predominantly Ps aeruginosa were found in the lower part of the body with an incidence of 74.0% (37) . Staph pyogenes was found in the upper half showing an incidence of 12.0% (6) next to 20.0% (10) incidence of Ps aeruginosa . The incidence of burn infection was high in patients with deep and major burn wounds, the bacterial isolates being 76.0% (38) and 80.0% (40) respectively . Silver sulphadiazine exhibited antimicrobial action in the range of 14 to 390 microM/ml, while cerium sulphadiazine had no inhibitory effect even up to 667 microM/ml on pseudomonas isolates . Zinc sulphadiazine was effective in inhibiting the growth of 10 isolates tested in 40 to 297 microM/ml range.

Eur J Clin Microbiol Infect Dis, 1996 Jun, 15(6), 459 - 64
Study of Pseudomonas aeruginosa serotype O12 isolates with a common antibiotic susceptibility pattern; Talarmin A et al.; A multicentre European study of Pseudomonas aeruginosa serotype O12 isolates with a common antibiotic resistance pattern was conducted . Resistance to beta-lactams and aminoglycosides was observed in 24 of the 25 isolates, as often reported in Europe, and all 25 isolates were significantly more susceptible to fosfomycin than 189 isolates of other serotypes (72% vs . 13.2%) . The mutational frequency of serotype O12 was similar to that of other serotypes and thus could not explain the susceptibility to fosfomycin . As a number of epidemiological studies using various methods, especially ribotyping with EcoRI, have shown that most strains are similar, it has been suggested that a single strain of this serotype is widespread . However, in this study ribotyping with EcoRI and PvuII distinguished seven clones among 24 ticarcillin resistant serotype O12 isolates, although one ribotype predominated (67%) . Thus the hypothesis of spread of one clone across Europe cannot explain the common resistance phenotype observed in different clones of serotype O12 . Resistance of beta-lactams and aminoglycosides might be explained by greater receptiveness for transposable resistance mechanisms, and susceptibility to fosfomycin by increased permeability of the outer membrane.

J Antimicrob Chemother, 1996 Jun, 37(6), 1155 - 64
Adaptive resistance to tobramycin in Pseudomonas aeruginosa lung infection in cystic fibrosis; Barclay ML et al.; Aminoglycoside antibiotics have been shown to induce adaptive resistance in Pseudomonas aeruginosa in vitro and in a mouse model of infection, but adaptive resistance has not been described in human infections . Seven patients with cystic fibrosis were treated with inhaled tobramycin to determine whether adaptive resistance occurred in P . aeruginosa in their sputum . In three patients who had not recently taken antibiotics, 80 mg tobramycin was administered by nebuliser and resulting peak sputum tobramycin concentrations were 90-240 mg/L (elimination half-life 1.9-2.1 h) . Adaptive resistance was detected in P . aeruginosa 1-4 h after the dose of tobramycin . Moderate resistance was present at 24 h and full susceptibility returned between 24 and 48 h . In four other patients on long-term twice-daily inhaled aminoglycoside treatment, adaptive resistance was present before, and 4 h after, 80 mg of tobramycin administered by nebuliser . The presence and time course of adaptive resistance in humans may have implications for improving aminoglycoside dosing regimens.

Mol Microbiol, 1996 Jun, 20(5), 965 - 79
Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis; Sundin GW et al.; We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa . The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity) . We have previously reported that P . aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk . Hyperexpression of ndk from the clone pGWS95 in trans in the P . aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20 . Hyperexpression of ndk from pGWS95 in the P . aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type . Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P . aeruginosa . The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates . However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP . We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP . The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

J Hosp Infect, 1996 Jun, 33(2), 145 - 51
Pseudomonas aeruginosa outbreak associated with a contaminated blood-gas analyser in a neonatal intensive care unit; Garland SM et al.; Over a 10 month period in a neonatal intensive care unit there was an outbreak of infection caused by Pseudomonas aeruginosa (resistant to ticarcillin, timentin) which involved 24 newborns . There was extensive morbidity and mortality (38%) associated with the infections, which presented as septicaemia (N = 6) (five succumbed and four had coexisting pneumonia), pneumonia (N = 6), meningitis (one, died), conjunctivitis (N = 1), otitis externa (N = 1), conjunctivitis plus otitis externa (N = 1) . In addition there were two pseudosepticaemias and six colonized infants, three of whom were treated for the presence of P . aeruginosa in endotracheal aspirates . There was always at least one baby colonized or infected with P . aeruginosa during the outbreak . Environmental surveillance and genomic DNA fingerprinting of isolates identified the blood gas analyzer port as the likely reservoir for the outbreak . Further spread of the organism did not occur after commencement of staff education on vigilant and careful handwashing, especially after use of the blood-gas analyser.

Shock, 1996 Jun, 5(6), 440 - 5
Differential effects of prolonged septicemia on isolated pulmonary arteries and veins from sheep; Nelson SH et al.; Isolated third-order pulmonary arteries and veins from sheep were examined for the effects of septicemia on norepinephrine-induced contractions, nitric oxide (NO)-mediated dilation, and basal cyclic GMP levels . The groups studied were as follows: control sheep (n = 7); sheep given live Pseudomonas aeruginosa (Ps, n = 6) for 48 h; and sheep given NG-mono-methyl-L-arginine during the last 24 h of Ps infusion (Ps-L-NMMA, n = 4) . The norepinephrine-induced contractions were significantly greater (p < .05) in arteries from septic (Ps and Ps-L-NMMA) sheep . Basal cyclic GMP levels were similar in all of the arteries . The norepinephrine-induced contractions were significantly depressed (p < .05) in veins from septic (Ps and Ps-L-NMMA) sheep . Basal cyclic GMP levels in veins from Ps sheep were markedly elevated (p < .01) . N omega-nitro-L-arginine methyl ester (L-NAME) ex vivo decreased cyclic GMP in both arteries and veins . Removal of endothelium enhanced contractions and decreased cyclic GMP in arteries and veins only from control sheep . The results show that septicemia differently affects the pulmonary artery and vein . The enhanced vasoconstriction of the artery is due to decreased endothelium-dependent NO release; the attenuated vasoconstriction of the vein is associated with NO-mediated increased cyclic GMP levels.

Immunol Cell Biol, 1996 Jun, 74(3), 258 - 64
Anti-receptor antibodies inhibit Pseudomonas aeruginosa binding to the cornea and prevent corneal perforation; Hobden JA et al.; A polyclonal antibody (pAb) against gangliotetraosylceramide (asialo GM1), a glycolipid to which bacterial pili and LPS bind, and a mAb against a 66 kDa pilus-binding protein purified from adult mouse corneal epithelium were used to determine if antibodies against host receptors for bacterial adhesins could inhibit bacterial binding to wounded corneal epithelium and protect ocularly challenged mice from corneal perforation when topically applied . Bacteria were mixed with anti-66 kDa mAb, a mixture of anti-asialo GM1 pAb and anti-66 kDa mAb, an irrelevant control mAb (anti-human histocompatibility Ag HLA-DR5) or PBS prior to application to scarified corneas in organ culture . The combination of the two antibodies or the anti-66 kDa mAb alone was effective in reducing bacterial adherence compared with either PBS or the antibody control . To determine if these antibodies were protective in vivo, corneas of C57BL/6J mice were scarified and inoculated with Pseudomonas aeruginosa . Eyes were treated topically with anti-asialo GM1 pAb, anti-66 kDa mAb, a mixture of the two or control mouse serum . More serum-treated corneas perforated compared to corneas from any other group (P < or = 0.005) by 30 days postinfection . Treatment with a combination of the two antibodies resulted in significantly less corneal pathology 30 days p.i . when compared to any other treatment (P < or = 0.005) . These data provide evidence that antibodies against host corneal receptors significantly inhibit bacterial binding in vitro and when applied topically in vivo, lessen the severity of ocular disease characteristic of P . aeruginosa keratitis.

Inflammation, 1996 Jun, 20(3), 243 - 62
Phagocytosis of Pseudomonas aeruginosa fails to elicit heat shock protein expression in human monocytes; Barazzone C et al.; Phagocytosis represents a powerful stress for the phagocytic cells . Phagocytosis of Staphylococcus aureus induces a stress response associated with the synthesis of specific heat shock/stress proteins (HSP) . Here we investigated the stress response of human monocyte-macrophages (m phi) to Pseudomonas aeruginosa, a bacterium found, as for S . aureus, in the airways of patients suffering cystic fibrosis . P . aeruginosa activated in m phi the production of both extra- and intracellular O2-; increased Interleukin-1 beta and actin, but failed to induce host HSP . Neither S . aureus' exotoxins nor the scavenging property of P . aeruginosa's alginate, but the lower toxicity of P . aeruginosa and/or differential activation of proteine kinase C (PKC) by the two bacteria, might explain their differences in host HSP induction . While O2- is insufficient to induce HSP synthesis in m phi, hydroxyl radicals, generated in the presence of exogenous iron, is a likely additional signal, along with PKC activation, for HSP induction during bacterial phagocytosis.

Jpn J Antibiot, 1996 Jun, 49(6), 544 - 54
{Isolation rate of Pseudomonas aeruginosa from surgical infections and their susceptibilities}; Shinagawa N et al.; Pseudomonas aeruginosa isolated from surgical infections during the period from July 1982 to June 1995 were investigated in a multicenter study involving 19 hospitals in Japan, and the following results were obtained . 1 . Though the isolation rate of P . aeruginosa was not high from primary infections, it was more frequently isolated from postoperative infections throughout the study period . Enterococcus spp., P . aeruginosa and Staphylococcus aureus including MRSA were predominant among postoperative infections . From the postoperative cases that had previous antibiotic treatment, Enterococcus spp., MRSA and P . aeruginosa were more predominantly isolated than from those without previous treatments with antibiotics . 2 . Cefozopran, ceftazidime, cefsulodin, aztreonam, carumonam, gentamicin, amikacin and ofloxacin had strong activities against P . aeruginosa . We recognize recently that antibiotic-resistant strains of P . aeruginosa against imipenem and ofloxacin have been increasing year by year.

Nippon Geka Gakkai Zasshi, 1996 Jun, 97(6), 437 - 41
{Pneumonia after esophagectomy}; Matsubara T; Progress in operation and postoperative management techniques markedly decreased the frequency of severe pulmonary complications after esophagectomy . However, intractable pneumonia, once occurs, is still likely to be fatal . Intensive care should be given to maintenance of the favorable hemodynamic and respiratory status, airway toilet and appropriate use of antibiotics . We routinely put patients on mechanical ventilation during the oliguric period and suction airway secretions with bronchofiberscope twice a day for five days or more . Antibiotics is administrated against highly toxic strains immediately after the operation, and against target strains selected based on cultivative tests thereafter . In 300 patients who underwent esophagectomy with cervicothoracoabdominal dissection from 1985 to 1995, the hospital mortality rate was 4% (12 patients) . Pneumonia was a cause of death in 6 of them, who were all operated upon before 1989 . In the most recent two years, nine of 75 patients had postoperative pneumonia (two cases of aspiration pneumonia, two of interstitial pneumonia and one of tracheal perforation) . Postoperative tracheostomy was done in four patients . From the early stage after operation, bacterial culture tests of the pharyngeal smear and sputum commonly demonstrated agents which generally cause opportunistic infection, such as Candida spp., Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) . By site, MRSA was first detected from the pharyngeal smear or sputum in 30 of 38 MRSA-positive patients.

J Cardiovasc Pharmacol, 1996 Jun, 27(6), 901 - 7
Calcitonin gene-related peptide does not mediate the abnormal vascular reactivity observed in a rat model of acute Pseudomonas pneumonia; Fox GA et al.; Abnormal systemic and pulmonary vascular reactivity has been demonstrated in numerous models of sepsis and pneumonia . Furthermore, the attenuated hypoxic pulmonary pressor response observed in these animals probably is responsible for the ventilation/perfusion (V/Q) mismatching and consequent arterial hypoxemia . We hypothesized that excess release of endogenous vasodilators such as calcitonin gene-related peptide (CGRP) in pneumonia was responsible for the diminished hypoxic pressor response . Using the CGRP receptor antagonist CGRP (8-37), we examined the role of CGRP in the attenuated hypoxic pulmonary response in a rat model of acute Pseudomonas pneumonia . Sixteen Sprague-Dawley rats were instrumented for chronic hemodynamic monitoring and subsequently randomized to either Pneumonia (n = 8), induced by the instillation of 0.2 ml broth containing 2 x 10(8) colony-forming units (CFU)/ml Pseudomonas aeruginosa into the right lower lobe, or Sham (n = 8) procedure . Hemodynamic measurements and the hypoxic (FiO2 = 0.08) pulmonary pressor response were recorded at baseline, 48 h after the pneumonia or sham procedure and after the administration of 250 micrograms CGRP (8-37) (post-CGRP(8-37)) . The regional distribution of pulmonary blood flow was determined by the injection of radioactive microspheres . Forty-eight hours after the instillation of Pseudomonas, Pneumonia animals had significantly increased cardiac output (CO) as compared with Sham (193 +/- 7 vs . 154 +/- 7 ml/min, p < 0.05), slightly decreased mean arterial pressure (MAP 109 +/- 4 vs . 118 +/- 3 mm Hg, p = NS), and reduced total systemic vascular resistance (TSVR 0.57 +/- 0.03 vs . 0.78 +/- 0.05 mm Hg.min.ml-1, p < 0.05) . Pneumonia animals were further characterized by increased mean pulmonary artery pressure (MPAP) as compared with Sham (24 +/- 2 vs . 20 +/- 1 mm Hg, p < 0.05) animals, and an increased alveolar-arterial (A-a) oxygen gradient (31 +/- 3 vs . 20 +/- 4 mm Hg, p < 0.05) . The administration of CGRP (8-37) did not alter baseline hemodynamic variables and did not change the pressor response to hypoxia in either group . Furthermore, CGRP receptor blockade did not alter the distribution of blood flow in the lung during normoxia or hypoxia . These data suggest that although this model of acute pneumonia is characterized by an attenuated hypoxic pressor response, the mechanism does not appear to be mediated by excess release of the vasodilator CGRP.

Br J Biomed Sci, 1996 Jun, 53(2), 140 - 5
Cystic fibrosis and the pseudomonads; Wright KC; The microbiology of pulmonary disease in cystic fibrosis has altered over the past 10 years . The major pathogens in this disease are now Pseudomonas aeruginosa and, increasingly, Pseudomonas cepacia . P . aeruginosa respiratory infection in these patients is rarely eradicated and this is often the only pathogen found at post-mortem . The most important points in the pathogenesis of this infection are probably the protective role of the bacterial mucoid exopolysaccharide and the interaction of various other bacterial factors with the immune system of the body . P . cepacia has recently emerged as the common isolate from the lungs of cystic fibrosis patients . The actual role of this organism in the progression of lung disease is poorly understood . There has been some speculation about the role of cross-infection in the acquisition of both of these organisms . The treatment of these infections is problematical because of the altered antimicrobial pharmaco-kinetics within the cystic fibrotic lung and the resistant properties of the organisms involved . Approaches which have been suggested recently include immunological interventions and genetic therapy.

Korean J Ophthalmol, 1996 Jun, 10(1), 8 - 17
Efficacy of ciprofloxacin and dexamethasone in experimental pseudomonas endophthalmitis; Kim IT et al.; To determine injection time and effective dose of ciprofloxacin in endophthalmitis and to evaluate the effectiveness of dexamethasone . In rabbits, Pseudomonas aeruginosa (2 x 10(4) CFU/0.1 ml) was inoculated intravitreally . At 6, 12, 18, 24 hours postinoculation, single intravitreal doses of ciprofloxacin (300 micrograms/0.15 ml or 100 micrograms/0.05 ml) alone or with dexamethasone (400 micrograms) were given . Electrophysiological and histologic measures were utilized to rate drug effectiveness . 300 micrograms ciprofloxacin was effective in killing P . aeruginosa at 6 and 12 hours postinoculation, but one hundred ug ciprofloxacin was not effective . 300 ug ciprofloxacin had no significant effect in killing P . aeruginosa at 18 hrs and 24 hrs postinoculation . Eyes treated with dexamethasone (400 micrograms) and ciprofloxacin (300 micrograms) at 6 hours postinoculation did not differ from eyes treated with ciprofloxacin alone . Cultures from eyes treated with dexamethasone and ciprofloxacin at 12 hours postinoculation were positive . Cultures from eyes treated with ciprofloxacin alone were negative . The failure of treatment at 18 hrs and 24 hrs postinoculation may be due to either an increased rate of clearance of drugs from the eyes or a reduced bactericidal effect of ciprofloxacin which could be altered by acidic pH, degree of hypoxia or bacterial counts . Dexamethasone had no beneficial effect in the treatment of P . aeruginosa endophthalmitis in the early phase.

Kansenshogaku Zasshi, 1996 Jun, 70(6), 605 - 12
{Drug-resistance patterns of clinical isolates of Pseudomonas aeruginosa in regard to their lipopolysaccharide-chain sizes}; Hasegawa M et al.; Pseudomonas aeruginosa strains isolated from patients with different types of the infections each consisted of LPSs different in chain sizes . The drug-susceptibility patterns of these strains of P . aeruginosa were investigated to clarify the relationship between the LPS-compositions and susceptibility to some kinds of anti-pseudomonal drugs . The susceptibilities of nineteen strains (seven long-LPS strains, four short-LPS strains and eight LPS-deficient strains) to piperacillin, ceftazidime, gentamicin, norfloxacin and polymyxin-B were determined and these strains were classified into six types (Types I-VI) according to their drug-resistance patterns . Six of the eight LPS-deficient strains were found to be highly resistant to gentamicin alone (Type IV) . Four of the seven strains with the long-LPS and one strain with the short-LPS were resistant to three drugs such as piperacillin, ceftazidime and norfloxacin, and classified into Type I . These results indicated that the major part of the LPS-deficient strains and the considerable part of the long-chain LPS strains of P . aeruginosa tested had each characteristic profile in the drug resistance . The outer membrane proteins of thirteen strains, consisting of different types of LPS compositions, were analyzed by SDS-PAGE . The strains belonging to the same types of the drug-resistance patterns were found to have similar OMP-profiles, although a few exceptions were found . beta-lactamase and gentamicin-inactivating activities were determined for piperacillin-resistant and gentamicin-resistant strains, respectively . Of the piperacillin-resistant strains tested, the activity of beta-lactamase was high in one (No . 8) only, low in four and not found in four . The results showed that degrees of resistance of P . aeruginosa strains tested to piperacillin did not correlate to their producibility of beta-lactamase except one strain . Of the nine gentamicin-resistant strains tested, the gentamicin inactivating activity was high in one (No . 30) only, moderate in six and low in two . These results suggested that the significant levels of piperacillin- or gentamicin-resistance of P . aeruginosa isolated tested might be expressed each due to their decreased abilities for drug-permeabilities in addition to drug-inactivating activities such as beta-lactamase or gentamicin-modifying enzyme . In the case of some resistant strains, the resistance to piperacillin or gentamicin was not explained by the results of the present study . Therefore, we must investigate the possibility that other mechanisms participate in the resistance of these strains.

Antimicrob Agents Chemother, 1996 Jun, 40(6), 1438 - 41
Indole and (E)-2-hexenal, phytochemical potentiators of polymyxins against Pseudomonas aeruginosa and Escherichia coli; Kubo A et al.; Combinations of polymyxins and phytochemicals were tested for antimicrobial activity against two gram-negative bacteria . Various degrees of potentiation were found against Pseudomonas aeruginosa and Escherichia coli with (E)-2-hexenal and indole . Three-compound combinations were found to further increase the activity of polymyxin B sulfate and colistin methanesulfonate against both bacteria . Combinations with colistin against P . aeruginosa resulted in the highest degree of potentiation, with a 512-fold increase in colistin antimicrobial activity . These results indicate the potential efficacy of phytochemical combinations with antibiotics to enhance total biological activity.

Antimicrob Agents Chemother, 1996 Jun, 40(6), 1387 - 93
In vitro characterization of aminoglycoside adaptive resistance in Pseudomonas aeruginosa; Karlowsky JA et al.; Aminoglycoside adaptive resistance was characterized in one reference strain and four clinical isolates of Pseudomonas aeruginosa . Adaptive resistance was initiated with a 2-h gentamicin or tobramycin exposure at the MIC . Each P . aeruginosa strain demonstrated an adaptive-resistance period of between 8 and 12 h when tested with both aminoglycosides . Aminoglycoside adaptive resistance was shown to correlate with a decrease in {3H} gentamicin accumulation and a small (5%) but significant (P < 0.05) reduction in proton motive force . The mean generation time of P . aeruginosa during peak levels of adaptive resistance (i.e., maximum reductions in aminoglycoside killing) was not significantly different from that of control organisms (P < 0.05) . No changes in outer membrane protein or lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were noted when control, adaptively resistant, and postadaptively resistant cells were compared . Cytoplasmic membrane profiles of adaptively resistant cells, however, demonstrated several band changes when compared with control and postadaptively resistant cells . We conclude that the decrease in aminoglycoside accumulation associated with adaptive resistance in P . aeruginosa may be, in part, a function of reductions in proton motive force and/or cytoplasmic membrane protein changes . However, the importance of these changes requires further investigation.

J Appl Bacteriol, 1996 Jun, 80(6), 682 - 6
Note: development of an external quality assurance scheme for the detection and enumeration of Pseudomonas aeruginosa from water and comparison of results using modified King's A broth and a commercial agar; Place BM et al.; Two trials of the isolation and enumeration of a given strain of Pseudomonas aeruginosa from water are reported . In each trial participants received concentrated samples from two batches, one with low and one with high counts, to be diluted to 500 ml in sterile distilled or deionized water and examined for Ps . aeruginosa by membrane filtration . Membranes were incubated at 37 degrees C for 48 h on pads soaked in modified King's A broth (MKAB) and Unipath Pseudomonas Agar plus CFC supplement (PCFC) . The first trial involved eight Public Health Laboratories (PHL) and the organizers provided media from single batches . The second trial, involving 50 PHL, examined the feasibility of a large scale external quality assessment (EQA) distribution . Participants were invited to use the same two media and their usual medium if different . Average counts were close to expected and the spread of results was comparable to that observed from the EQA scheme for indicator organisms . From the results of the two trials a better isolation of the strain of Ps . aeruginosa under consideration was noted with PCFC compared with MKAB.

Infect Immun, 1996 Jun, 64(6), 2288 - 94
Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa; Fleiszig SM et al.; We have reported that some strains of Pseudomonas aeruginosa can enter corneal epithelial cells during experimental murine eye infection and when the cells are cultured in vitro . Following invasion, both the host cell and the intracellular bacteria can remain viable for up to 24 h . Others have reported that toxin-mediated damage of epithelial cells contributes to the pathogenesis of P . aeruginosa keratitis . To clarify the relationship between cell invasion and cytotoxicity, fourteen P . aeruginosa isolates were compared for their capacity to enter epithelial cells and for their ability to induce cytotoxicity . Bacterial invasion was quantified by gentamicin survival assays both in vivo and in vitro . Cytotoxicity was examined qualitatively by trypan blue exclusion assays and quantitatively by chromium release assays in vitro . A significant inverse correlation was found between the ability to induce cytotoxicity and epithelial cell invasion as measured by gentamicin survival assays . Both cytotoxic and noncytotoxic strains were identified among corneal and noncorneal isolates; all isolates that were not cytotoxic were capable of epithelial cell invasion . Efficient host cell invasion could not be demonstrated for cytotoxic strains; however, the gentamicin survival assay relies upon host cells retaining viability in order to yield useful results, and this may limit the effectiveness of this assay for testing epithelial cell invasion by cytotoxic strains . Since all of the corneal isolates that were tested were virulent in vivo, the results show that there are at least two different types of P . aeruginosa-induced disease, one caused by strains that are cytotoxic and the other involving bacteria that can enter epithelial cells and survive intracellularly without killing the host cell.

Infect Immun, 1996 Jun, 64(6), 2216 - 9
Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress; Polack B et al.; Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients . In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen . Two isoforms of superoxide dismutase are synthesized by P . aeruginosa; they differ by the metal present at their active site, which is either iron or manganese . To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P . aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient . The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain . It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat . These results suggest that MnSOD may participate in the adaptation of mucoid strains of P . aeruginosa to the stationary phase of growth in the lungs of CF patients.

Infect Immun, 1996 Jun, 64(6), 2130 - 6
Cloning and characterization of Pseudomonas aeruginosa fliF, necessary for flagellar assembly and bacterial adherence to mucin; Arora SK et al.; Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs . This process appears to be mediated by nonpilus adhesins which bind to mucin . To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P . aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin . All of these mutants were also found to be defective in motility . One such mutant, PAK-RR20, is characterized here . The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG . Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans . Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette . This mutant was also nonmotile and nonadhesive . However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon . The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P . aeruginosa were determined . Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P . aeruginosa with mutations in several regulatory genes . Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors . The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.

J Mol Evol, 1996 Jun, 42(6), 617 - 30
The distance between bacterial species in sequence space; Ambler RP; Despite the revolution caused by information from macromolecular sequences, the basis of bacterial classification remains the genus and the species . How do these terms relate to the variety of bacteria that exist on earth? In this paper, the inter- and intraspecies differences in amino acid sequence of several bacterial electron transport proteins, cytochromes c, and blue copper proteins are compared . For the soil and water organisms studied, bacterial species can be classed as "tight" when there is little intraspecies variation, or "loose" when this variation is large . For this set of proteins and organisms, interspecies variation is much larger than that within a species . Examples of "tight" species are Pseudomonas aeruginosa and Rhodobacter sphaeroides, while Pseudomonas stutzeri and Rhodopseudomonas palustris are loose species . The results are discussed in the context of the origin and age of bacterial species, and the distribution of genomes in "sequence space." The situation is probably different for commensal or pathogenic bacteria, whose population structure and evolution are linked to the properties of another organism.

Anal Biochem, 1996 Jun 1, 237(2), 216 - 23
Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase; Besson C et al.; Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung . In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities . Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P . aeruginosa elastase itself . In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group . Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1 . The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower . The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements) . Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.

J Bacteriol, 1996 Jun, 178(11), 3350 - 2
Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C; Makin SA et al.; Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs . The relative expression levels of these two LPS types in P . aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide . A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody . Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) {A+B-}) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression . B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.

J Bacteriol, 1996 Jun, 178(11), 3346 - 9
Membrane topology of the outer membrane protein OprH from Pseudomonas aeruginosa: PCR-mediated site-directed insertion and deletion mutagenesis; Rehm BH et al.; The 21-kDa outer membrane protein OprH from Pseudomonas aeruginosa is overexpressed under Mg2+ starvation conditions and when overproduced causes resistance to polymyxin B, gentamicin, and EDTA . By circular dichroism analysis, OprH revealed a calculated beta-sheet structure content of 47.3% . PCR-based site-directed deletion and epitope insertion mutagenesis was used to test a topological model of OprH as an eight-stranded beta-barrel . Three permissive and seven nonpermissive malarial epitope insertion mutants and four permissive and four nonpermissive deletion mutants confirmed the general accuracy of this model . Thus, OprH is the smallest outer membrane protein to date to be confirmed as a beta-stranded protein.

J Bacteriol, 1996 Jun, 178(11), 3314 - 21
Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the response regulator MtrA; Via LE et al.; A putative two-component system, mtrA-mtrB, was isolated from M . tuberculosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridization probe . The predicted gene product of mtrA displayed high similarity with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR . The predicted gene product of mtrB displayed similarities with the histidine protein kinases AfsQ2, PhoR, and EnvZ and other members of this class of proteins . Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa . MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using a heterologous histidine protein kinase, CheA, as a phosphoryl group donor . Mycobacterium bovis BCG, harboring an mtrA-gfp (green fluorescent protein cDNA) transcriptional fusion, was used to monitor mtrA expression in infected J774 monolayers . Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J774 macrophages . In contrast, the hsp60-gfp fusion displayed no change in expression under the growth conditions tested . These results suggest a potential role for mtrA in adaptation of the M . tuberculosis complex organisms to environmental changes which may include intracellular conditions.

J Bacteriol, 1996 Jun, 178(11), 3085 - 90
The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa; Huang H et al.; Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane . In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein . Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS . When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel . From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM . In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding . Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline . Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site . The disposition of these loop regions in the interior of the OprD channel is discussed.

J Bacteriol, 1996 Jun, 178(11), 3066 - 71
Identification and characterization of BpH2, a novel histone H1 homolog in Bordetella pertussis; Goyard S; A basic protein, BpH2, with an apparent molecular mass of 18 kDa was purified from Bordetella pertussis, and the corresponding gene, bph2, was cloned . Sequence analysis revealed some homology to the H1 class of eukaryotic histones and to AlgP protein of Pseudomonas aeruginosa . BpH2 binds both single- and double-stranded DNA in a nonspecific manner . Deletion of the corresponding gene in B . pertussis generated a BpH2 null mutant with an altered growth rate in which the expression of two virulence factors, adenylate cyclase-hemolysin (CyaA) and filamentous hemagglutinin (FhaB), was reduced . It is suggested that BpH2 may exhibit specific regulatory functions through its interaction with chromosomal DNA.

J Infect Dis, 1996 Jun, 173(6), 1415 - 21
Potential hazards of combination immunotherapy in the treatment of experimental septic shock; Opal SM et al.; Using an actual infection model of Pseudomonas aeruginosa sepsis in neutropenic rats, the potential utility of a combination anticytokine approach for the treatment of sepsis was tested . A dimeric tumor necrosis factor binding protein (TNF-BP) consisting of two soluble recombinant human TNF type 1 receptors linked with polyethylene glycol was used with recombinant human interleukin-1 receptor antagonist (IL-1ra) . Despite having levels of bacteremia and endotoxemia similar to the control group (survivors, 0/18), 30% of IL-1ra-treated animals survived (P < .05); 31% of TNF-BP-treated animals survived (P < .01) . Unexpectedly, the combination of IL-1ra plus TNF-BP proved to be uniformly fatal (survivors, 0/20) . Endotoxin (P < .0001) and bacteremia (P < .01) levels were >10-fold higher than levels in animals treated with IL-1ra alone, TNF-BP alone, or placebo . Disseminated microabscesses in major organs were found in animals treated with combination immunotherapy . Combination anticytokine therapy may exacerbate systemic infection and worsen outcome in experimental sepsis.

J Urol, 1996 Jun, 155(6), 2094 - 7
Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin; Ishibashi K et al.; PURPOSE: These studies were designed to determine the combined inhibitory effect of a human monoclonal antibody (MAb) and cefsulodin on Pseudomonas aeruginosa renal infection in a neutropenic condition . MATERIALS AND METHODS: Protection against the infection of mice was estimated by survival rate and bacterial numbers in the kidney and blood . Opsonophagocytic assay by human polymorphonuclear neutrophils (PMNs) and fluorescence activated cell sorter (FACS) analysis were also examined . RESULTS: Treatment of infected mice with MAb combined with a suboptimal dose of cefsulodin prevented the mice from developing pyelonephritis and bacteremia and resulted in a significantly higher survival rate than treatment with either MAb or cefsulodin alone (p < 0.01) . When bacteria were preexposed to cefsulodin, a significant enhancement in opsonophagocytic killing with MAb was observed . Fluorescence activated cell sorter analysis suggested that the bacteria incubated with 1/4 minimal inhibitory concentration (MIC) of cefsulodin showed greater binding of MAb to bacteria than the control . CONCLUSION: The combination therapy with human antilipopolysaccharide MAb and cefsulodin is useful for P . aeruginosa pyelonephritis in neutropenic hosts.

Presse Med, 1996 May 18, 25(17), 803 - 4
Breast abscess with lethal septicemia due to Pseudomonas aeruginosa in a patient with AIDS; Roca B et al.; OBJECTIVE: The incidence of Pseudomonas aeruginosa in HIV-infected patients has increased over the last years . We describe a case of pseudomonal breast abscess complicated with fatal septicemia in an AIDS patient . CASE REPORT: A 21-year-old woman was admitted for fever, chills, nausea, vomiting and pain in the breast . She had a swelling in the right breast of 3 days duration . HIV infection had been confirmed 6 years earlier . CD4 count was 2/mm3 . Surgical drainage produced a blue-green purulent discharge which grew Pseudomonas aeruginosa on culture . Despite cloxacilin, then ceftazidime and amikacin, initial improvement was followed 2 weeks later by nodular pulmonary infiltration with cavitation . P . aeruginosa was recovered from sputum and blood cultures, but stepwise resistance developed and the patient died 3 months after admission . DISCUSSION: Breast abscesses are infrequent in nonlactating women . P . aeruginosa is rarely involved, even in HIV patients . Due to the risk of resistance, prompt administration of appropriate antibiotics is required.

J Appl Bacteriol, 1996 May, 80(5), 540 - 4
Damage to Pseudomonas aeruginosa PAO1 bacteriophage F116 DNA by biocides; Maillard JY et al.; The mechanism of action of biocides against viruses has not been widely studied, although two main targets are viral proteins (capsids, enzymes) and the viral genome . This study was undertaken in order to investigate the efficacy of several disinfectants against the nucleic acid of the Pseudomonas aeruginosa PAO bacteriophage F116 . Of all the biocides tested, only peracetic acid affected significantly the phage genome . However, it is not clear whether the nucleic acid was damaged inside the phage capsid or when released into the surrounding medium.

Mikrobiologiia, 1996 May-Jun, 65(3), 352 - 6
{Morphologic and physiologic-biochemical characterization of Pseudomonas aeruginosa dissociation variants}; Mil'ko ES et al.; Populations of the hydrocarbon-oxidizing strain K-2 of Pseudomonas aeruginosa dissociate into R, S, and M variants . They differ in colony morphology, cell fine structure, and cell yields on synthetic medium with hexadecane and on nutrient broth mixed with wort (NB + W) . The best growth on NB + W was shown by the R variant, while the highest cell yield on hexadecane medium (exceeding that of the original culture) was attained by the S variant.

Chemotherapy, 1996 May-Jun, 42(3), 210 - 14
Meropenem permeation through the outer membrane of Pseudomonas aeruginosa can involve pathways other than the OprD porin channel; Perez FJ et al.; The outer membrane protein (OMP) OprD is the major channel through which carbapenems permeate the outer membrane of Pseudomonas aeruginosa . In this study, we analyzed the OMP profiles of several P . aeruginosa clinical isolates showing diminished susceptibility to imipenem while remaining susceptible to meropenem . All these isolates lacked OprD or showed a reduced expression of this porin . Susceptibility to meropenem was thus independent of the level of OprD expression, indicating that the antimicrobial could be taken up via an alternative route . The level of expression of OprC (70 kD) was also unrelated to meropenem susceptibility . Nevertheless, OMPs OprF and OprE were expressed by all isolates, suggesting that in the absence of OprD, these porins might be involved in the permeation of meropenem.

Chemotherapy, 1996 May-Jun, 42(3), 170 - 6
Comparison of the bactericidal action of amikacin, netilmicin and tobramycin in free and liposomal formulation against Pseudomonas aeruginosa; Omri A et al.; The rates at which free, cationic and anionic liposomal forms of amikacin, netilmicin and tobramycin kill Pseudomonas aeruginosa were studied in vitro . Control inocula with no antibiotic yielded 6.76, 9.53 and 9.74 log CFU/ml at 0, 6 and 24 h, respectively . Empty anionic or cationic liposomes had no effect on bacterial growth . The killing rates of free antibiotics against the bacterial strain were not enhanced by the addition of either empty anionic or cationic liposomes . After 6 and 24 h of exposure at 1, 2 and 4 times the minimum inhibitory concentrations, free amikacin, netilmicin and tobramycin demonstrated a more rapid bactericidal effect than encapsulated anionic or cationic liposomes . The killing rates of liposomal aminoglycosides were lower than those of free aminoglycosides at identical concentrations, suggesting that only fractions of the encapsulated drugs were released from liposomes.

Am J Physiol, 1996 May, 270(5 Pt 1), L819 - 28
Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections; Hashimoto S et al.; The mechanism for neutrophil (PMN) influx into infected airspaces of the lung is not known . To determine whether alveolar macrophage products are important in the initiation of chemotaxis, we depleted rats of alveolar macrophages by aerosolizing negatively charged oligolamellar liposomes complexed to clodronate disodium . Ninety-five percent of the alveolar macrophages were depleted, and lung injury and inflammation were minimized with this depletion technique . Rats depleted of alveolar macrophages were then anesthetized, and either 5 x 10(6) colony-forming units (CFU) or 5 x 10(7) CFU of Pseudomonas aeruginosa were instilled into the airspaces of these animals . When recombinant macrophage inflammatory protein MIP-2 was intratracheally instilled into rats depleted of alveolar macrophages, PMN were recruited to their airspaces . Nonetheless, PMN numbers were decreased in the lavage fluids when moderate or large inoculums of bacteria were instilled into depleted rats, although the PMN response appeared dose dependent . Levels of bioactive tumor necrosis factor-alpha and immunoreactive proteins CINC/gro (cytokine-induced PMN chemoattractant) in the lavage fluids obtained from infected rats depleted of alveolar macrophages were significantly decreased compared with the levels in the lavage fluids obtained from normal infected rats . MIP-2 mRNA expression, as detected by Northern analysis, was also decreased in the infected lungs of depleted rats, and the lavage fluid from these rats had significantly decreased chemotactic activity . Therefore these results suggest that alveolar macrophage products play a direct role in the initial recruitment of PMN into infected lungs.

Antonie Van Leeuwenhoek, 1996 May, 69(4), 331 - 5
Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa; West TP; Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated . Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P . aeruginosa growth as a nitrogen source when glucose served as the carbon source . Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaminase were shown to be active in ATCC 15692 . Compared to (NH4)2SO4-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources . Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P . aeruginosa.

Diagn Microbiol Infect Dis, 1996 May, 25(1), 27 - 33
Pseudomonas aeruginosa ribotyping: stability and interpretation of ribosomal operon restriction patterns; Nociari MM et al.; Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping . Several enzyme combinations, including the best ones proposed in literature, were utilized and the highest discrimination was reached by individual digestion with PvuII, HindII, and EcoRI or BamHI . Clinical isolates from outbreaks were clonally related as identified by this molecular approach . Restriction rDNA profiles were composed of strong and weak bands . Using 6 micrograms DNA we were able to demonstrate that PvuII, HindIII, and BamHI weak bands were reproducible . These weak bands should be considered not only to accomplish the highest discrimination but also to correctly assign isolate clonality . Conversely, we found that EcoRI weak bands were not reproducible and, therefore, are not recommended for ribotype analysis . Finally, profiles differing in one single band actually represented isolates of different genotype, as confirmed by further analysis using other molecular methods . In this report on P . Aeruginosa ribotyping of clinical isolates, criteria for band pattern interpretation are established.

Diagn Microbiol Infect Dis, 1996 May, 25(1), 1 - 8
Comparative evaluation of an automated ribotyping system versus pulsed-field gel electrophoresis for epidemiological typing of clinical isolates of Escherichia coli and Pseudomonas aeruginosa from patients with recurrent gram-negative bacteremia; Pfaller MA et al.; Ribotyping and macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE) are among the more useful molecular epidemiologic typing methods . Because these techniques are labor intensive, automation of one or more steps may allow clinical laboratories to apply molecular typing methods . We compared the recently developed automated ribotyping system, the RiboPrinter Microbial Characterization System (DuPont), with PFGE as a means of typing clinical isolates of E . coli and P . aeruginosa . A total of 22 E . coli and 24 P . aeruginosa were typed by both PFGE and the RiboPrinter . When compared with PFGE typing of E . coli and P . aeruginosa, the RiboPrinter was less sensitive in identifying different strains, particularly among the isolates of P . aeruginosa . The RiboPrinter was completely automated and allowed up to 32 isolates to be typed within an 8-hour period . The pattern of results obtained in this study suggests that a heirarchical approach to molecular typing using the RiboPrinter Microbial Characterization System plus PFGE might be feasible . The RiboPrinter Microbial Characterization System promises to be a very useful addition to the expanding molecular typing armamentarium.

Infection, 1996 May-Jun, 24(3), 238 - 41
Nosocomial septicemias due to methicillin-resistant staphylococcus aureus and Pseudomonas aeruginosa in a university hospital over a 12-year period--the same intractable infections; Mizushima Y et al.; Clinical features of nosocomial bacteremias due to methicillin-resistant Staphylococcus aureus (MRSA) (M group, n = 71) and Pseudomonas aeruginosa (P group, n = 25) in a university hospital during 1982-1993 were retrospectively analyzed . The majority of these patients had an underlying disease, and bacteremia occurred in hospital . There were no differences in the male to female ratio and the mean age of the patients between the two groups . The ratio of medical wards to surgical wards was higher in the P group (18/7 = 2.6) than for the M group (38/33 = 1.2) . P . aeruginosa was more frequently isolated from patients with hematological malignancies and MRSA with solid tumors . The percentage of MRSA among gram-positive bacteremia and of P . aeruginosa among gram-negative bacteria has shown a tendency to increase in recent years, and antibiotic sensitivity of these two organisms showed, on a whole, a tendency to decrease . Attention should be called to the increase of these two pathogens.

FEMS Immunol Med Microbiol, 1996 May, 14(1), 31 - 8
A polyreactive human anti-lipid A monoclonal antibody having cross reactivity to polysaccharide portions of Pseudomonas aeruginosa lipopolysaccharides; Yokota S et al.; A hybridoma cell line producing a human anti-lipid A monoclonal antibody (mAb), FKF-IF3 (IgM (k)) was obtained by cell fusion of Epstein-Barr virus-transformed cells and mouse myeloma . The mAb bound to not only Gram-negative bacterial lipid A, but also to polysaccharide portions of Pseudomonas aeruginosa lipopolysaccharides (LPS) . The mAb seemed to recognize two distinct regions of P . aeruginosa LPS other than lipid A, namely the outer core regions of some serotype strains and the O-polysaccharide region of serotype A strains . The mAb cross-reacted with N-acetyl-beta-glucosamine-conjugated bovine serum albumin, N-acetyl-beta-galactosamine-conjugated bovine serum albumin, myosin and actin, but not with other autoantigens such as ss- and ds-DNA, cardiolipin and glycosaminoglycans . The mAb conferred protective activity against a mouse pseudomonal infection model . The evidence suggested that the mAb was a naturally occurring polyspecific antibody that participated in defense against pseudomonal infections.

Eur J Clin Microbiol Infect Dis, 1996 May, 15(5), 402 - 5
Role of anti-pseudomonal antibiotics in the emergence of Stenotrophomonas maltophilia in cystic fibrosis patients; Denton M et al.; A retrospective case-control study of 12 patients positive for Stenotrophomonas maltophilia and 24 age-sex-matched controls revealed that in the year prior to initial isolation, colonised patients spent more days in hospital and received more days of oral ciprofloxacin, intravenous anti-pseudomonal antibiotics, and nebulised aminoglycosides . They were also more likely to have grown Pseudomonas aeruginosa at some time in the past, despite there being no difference in current chronic infection with this organism . The role of anti-pseudomonal antibiotics in promoting Stenotrophomonas maltophilia colonisation in cystic fibrosis is discussed.

Int J Artif Organs, 1996 May, 19(5), 276 - 83
Cytokine production by human peripheral blood mononuclear cells stimulated by a Pseudomonas aeruginosa culture filtrate: role of plasma and polymyxin B; Sundaram S et al.; The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out . In this study, we examined the production of interleukin-1 alpha (IL-1 alpha), interleukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate . Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized . The results of our study indicate that the Ps . aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components . The enhancing effect of plasma was most evident at low concentrations of the filtrate . The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma . There was a direct correlation between the production of IL-1 alpha and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced . There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes.

Pathol Biol (Paris), 1996 May, 44(5), 363 - 6
{Comparison of four methods for the determination of the susceptibility of 47 strains of Pseudomonas aeruginosa to ceftazidime and cefepime: influence of the choice of interpretative criteria}; Boucaud-Maitre Y et al.; 47 isolates of Pseudomonas aeruginosa were tested for susceptibility to ceftazidime and cefepime by four methods (agar disc diffusion, E TEST and API-ATB) . Concordance between the methods is good, but the percentage of strains susceptible or of intermediate susceptibility depends of the interpretative criteria (French Committee for Antibiogram or National Committee Control for Laboratory Standards) . An international agreement could be useful to compare the bacteriological and clinical studies.

Pathol Biol (Paris), 1996 May, 44(5), 337 - 40
{In vitro study of the bacteriostatic and bactericidal activity of meropenem against strains of Pseudomonas aeruginosa multiresistant to antibiotics}; Delisle F et al.; Meropenem is a new dehydropeptidase stable carbapenem which has an in vitro bacteriostatic activity superior to imipenem against Pseudomonas aeruginosa . Minimal inhibitory concentrations of meropenem (MEM) and imipenem (IPM) were determined on twelve strains: two were genetically characterized reference strains and ten isolated from patients . Killing curves were performed for MEM used at 1,2 and 4 fold the MIC and 32 mg/l; survivors were counted at baseline and after 5 and 24 hours of incubation by spiral plating . Five strains were susceptible to MEM and IPM; five were intermediate and two resistant to MEM; these seven strains were resistant to IPM . A weak bactericidal effect was obtained after 5 h at each concentration . Only one strain achieved a 3 log 10 reduction after 5 h at 2 x MIC . A regrowth of all the strains tested was observed after 24 h.

Pathol Biol (Paris), 1996 May, 44(5), 329 - 32
{Comparison of the activity of beta-lactams against Pseudomonas aeruginosa according to phenotypes of resistance}; Bert F et al.; The in vitro activity of ticarcillin, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, cefsulodin, ceftazidime, cefepime, cefpirome and aztreonam was evaluated against 130 isolates of Pseudomonas aeruginosa with various beta-lactam phenotypes . MICs were determined by the agar dilution method and inhibition method and inhibition zones by the disk test . The resistance mechanisms were characterized by the iodometric method . The activity of cefepime was greater than that of cefpirome whatever the resistance phenotype . Some ticarcillin-susceptible strains were intermediate to cefepime and cefpirome . Most strains with a penicillinase or "intrinsic resistance" phenotype were susceptible to ceftazidime but intermediate to cefepime and cefpirome . Only 10% of cephalosporinase-over-producing strains remained susceptible to cefepime.

Epidemiol Mikrobiol Imunol, 1996 May, 45(2), 56 - 8
{Postantibiotic effects and postantibiotic effects of subinhibitory levels of quinolines on Pseudomonas aeruginosa}; Hostacka A; The postantibiotic effect (PAE) (PA phase induced by 2x or 4x MIC) and postantibiotic effect of subinhibitory concentrations (PA SME) of three quinolones-enoxacin, norfloxacin and pefloxacin against Pseudomonas aeruginosa were determined . It was observed that the tested antibiotics at suprainhibitory concentrations 2x MIC induced PAEs of 2.1-6.7 h . Longer PAEs were observed after treatment with antibiotics at concentration 4x MIC (4.1-9.0 h) . Subinhibitory concentrations of quinolones added to bacterial suspensions in the postantibiotic phase (2x or 4x MIC) displayed longer delay of bacterial regrowth (PA SMEs) compared to PAEs . No regrowth of bacterial culture was observed even at 48 h in the case of pefloxacin (2x MIC + 0.3x MIC, 4x MIC +0.2x MIC, 4x MIC + 0.3x MIC) as well as norfloxacin (4x MIC + 0.2x MIC, 4x MIC + 0.3x MIC).

J Hosp Infect, 1996 May, 33(1), 63 - 70
Outbreak of severe Pseudomonas aeruginosa respiratory infections due to contaminated nebulizers; Cobben NA et al.; During the six months from January-June 1994, 10 cases of severe and 11 of less severe pulmonary infection caused by Pseudomonas aeruginosa were diagnosed in patients with chronic obstructive airways disease . Possible sources were evaluated . P . aeruginosa was isolated from four of the 22 nebulizers tested . The relationship of isolates from the patients and nebulizers was confirmed by sero- and phage-typing, and by arbitrarily-primed polymerase chain reaction (AP-PCR) . Three types were identified and the distribution of types in patients with severe infection was as follows (one patient had a multiple infection) . Type I was isolated from two nebulizers and from sputa, and/or blood and/or bronchial protected specimen brush samples or bronchial lavage fluid from four patients . Type II came from the sputa of three patients and a third nebulizer; and type III from sputa and/or blood of four further patients and another nebulizer . The data provided evidence for the relation between P . aeruginosa as a cause of infection and the contamination of the nebulizers . When nebulizer mouthpieces were changed every 24 h and sterilized between patients, no more contamination occurred, and the outbreak ceased.

J Antimicrob Chemother, 1996 May, 37(5), 993 - 8
The effect of rifampicin on adaptive resistance of Pseudomonas aeruginosa to aminoglycosides; Xiong YQ et al.; Using the dynamic chequerboard technique, we confirmed that rifampicin produces a synergistic bactericidal effect when combined with amikacin or netilmicin . Adaptive resistance was suppressed when rifampicin was added to aminoglycoside after, but not during, first exposure to amikacin or netilmicin . The effect of rifampicin on adaptive resistance could account for the synergy between rifampicin and aminoglycosides.

J Inorg Biochem, 1996 May 1, 62(2), 77 - 87
Isolation and characterization of the d1 domain of Pseudomonas aeruginosa nitrite reductase; Silvestrini MC et al.; Proteolitic digestion of nitrite reductase from Pseudomonas aeruginosa allows to obtain and purify a domain containing only the d1 heme and constituted by two noncovalently bound peptides . This d1 domain catayzes oxygen consumption, and binds carbon monoxide with a kinetic constant slightly higher than the parental dimeric holoenzyme . The capacity to oxidize the physiological substrate, cytochrome c551, is lost, even when the proteolytic c heme domain is added to this reaction mixture . This finding suggests that the two domains do not have a significant affinity for each other, and are kept together only by being part of the same polypeptide.

J Clin Microbiol, 1996 May, 34(5), 1129 - 35
Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis; Mahenthiralingam E et al.; Pseudomonas aeruginosa isolates recovered from chronically colonized patients with cystic fibrosis (CF) are phenotypically different from those collected from other patients or from the environment . To assess whether alterations in motility, mucoidy, and serum susceptibility represented an adaptation to chronic infection or replacement by a new strain, sequential P . aeruginosa isolates of known phenotype collected from 20 CF patients were typed by random amplified polymorphic DNA (RAPD) analysis . A total of 35 RAPD strain types were found among 385 isolates from 20 patients, and only two patients had P . aeruginosa strains of the same RAPD fingerprint . Eight strain pairs representative of the first eight RAPD types were also analyzed by SpeI macrorestriction followed by pulsed-field gel electrophoresis (PFGE); the strain types found by both fingerprinting techniques correlated exactly . In 11 of 20 patients, the RAPD types of serial P . aeruginosa isolates remained stable despite alterations in isolate motility, colonial morphology, and lipopolysaccharide phenotype . However, in isolates collected from one CF patient, a single band change in RAPD fingerprint and CeuI PFGE profile correlated with the appearance of an RpoN mutant phenotype, suggesting that the altered phenotype may have been due to a stable genomic rearrangement . Secretion of mucoid exopolysaccharide, loss of expression of RpoN-dependent surface factors, and acquisition of a serum-susceptible phenotype in P . aeruginosa appear to evolve during chronic colonization in CF patients from specific adaptation to infection rather than from acquisition of new bacterial strains.

Gastrointest Endosc, 1996 May, 43(5), 457 - 62
Quality improvement in gastrointestinal endoscopy: microbiologic surveillance of disinfection; Merighi A et al.; BACKGROUND: Cleaning and disinfection procedures play an essential role in the prevention of infection transmission in gastrointestinal endoscopy . In spite of published detailed guidelines, several variants and weak points still exist . METHODS: Cleaning-disinfection procedures were carried out according to "Working Party, Sydney 1990." A microbiologic surveillance protocol tested the contamination of endoscopes and of automatic washing machines . To assess and improve the efficacy of disinfection, we adopted a quality assurance program . RESULTS: During a 2-year follow-up, the outside surfaces of gastroscopes were contaminated in 60.5% and channels in 41.3%; the outside areas of colonoscopes were contaminated in 62.3% and channels in 40.3% . Isolated bacteria were gram-negative organisms, particularly Pseudomonas species, and gram-positive organisms, mostly Staphylococcus species . The water reservoirs of automatic washing machines were frequently contaminated by Pseudomonas aeruginosa . The disinfection of washing machines and alcohol rinsing of endoscopes after standard procedures significantly reduced the bacterial contamination . CONCLUSIONS: The microbiologic surveillance pointed out the main weak points that could be improved by the adoption of corrective interventions . Quality assurance is a feasible method to assess the efficacy of cleaning-disinfection, and its wide application would improve quality of care.

Pediatr Pulmonol, 1996 May, 21(5), 267 - 75
Bronchoalveolar lavage or oropharyngeal cultures to identify lower respiratory pathogens in infants with cystic fibrosis; Armstrong DS et al.; As collections of lower respiratory tract specimens from young children with cystic fibrosis (CF) are difficult, we determined whether oropharyngeal cultures predicted lower airway pathogens . During 1992-1994, 75 of 90 (83%) infants with CF diagnosed by neonatal screening had 150 simultaneous bronchoalveolar lavage (BAL) and oropharyngeal specimens collected for quantitative bacterial culture at a mean age of 17 months (range, 1-52) . Ten children undergoing bronchoscopy for stridor served as controls . Total and differential cell counts and interleukin-8 concentrations were measured in BAL fluid . A subset of bacterial pathogens were typed by pulsed field gel electrophoresis . A non-linear relationship with inflammatory markers supported a diagnosis of lower airway infection when > or = 10(5) colony-forming units/ml were detected . This criterion was met in 47 (31%) BAL cultures from 37 (49%) children . Staphylococcus aureus (19%), Pseudomonas aeruginosa (11%), and Hemophilus influenzae (8%) were the major lower airway pathogens . In oropharyngeal cultures, S . aureus (47%), Escherichia coli (23%), H . influenzae (15%), and P . aeruginosa (13%) predominated . The sensitivity, specificity, and positive and negative predictive values of oropharyngeal cultures for pathogens causing lower respiratory infections were 82%, 83%, 41%, and 97%, respectively . When there was agreement between paired oropharyngeal and BAL cultures, genetic fingerprinting showed some strains of the same organism were unrelated . We conclude that oropharyngeal cultures do not reliably predict the presence of bacterial pathogens in the lower airways of young CF children.

J Lipid Res, 1996 May, 37(5), 962 - 71
Effects of acute inflammation on plasma retinol, retinol-binding protein, and its mRNA in the liver and kidneys of vitamin A-sufficient rats; Rosales FJ et al.; The acute inflammatory response to tissue injury and infection is associated with low concentrations of plasma retinol and its specific transport proteins, retinol-binding protein (RBP) and transthyretin (TTR) . To examine the kinetics and mechanism of hyporetinemia, we have induced acute inflammation with lipopolysaccharide (LPS, from Pseudomonas aeruginosa) in rats with adequate stores of vitamin A . Twenty-four h after treatment with LPS (50 micrograms i.p . per 100 g body weight) or saline and food withdrawal, plasma retinol equalled 0.72 +/- 0.06 mumol/L (mean +/- SEM) in five LPS-treated rats versus 1.35 +/- 0.1 mumol/L in five saline-treated rats (P < 0.01) . Plasma, liver, and kidney RBP and TTR concentrations were also significantly reduced, but liver and kidney retinol concentrations did not differ between treatment groups . The relative abundance of RBP mRNA in liver (LPS treatment compared to saline treatment) was reduced as early as 12 h (0.44 +/- 0.15, n = 4 pairs, P < 0.02), and continued to be reduced at 24 h (0.57 +/- 0.12, n = 5 pairs, P < 0.02) . In the kidney this ratio did not change significantly due to LPS treatment . The relative abundance of cellular retinol-binding protein (CRBP) mRNA in liver and kidney also was not affected by LPS treatment . We infer from these data that inflammation-induced hyporetinemia results from a reduction in the hepatic synthesis of RBP and secretion of the retinol-RBP complex . Moreover, the results imply that plasma retinol concentration is a poor indicator of vitamin A status during inflammation.

Antimicrob Agents Chemother, 1996 May, 40(5), 1321 - 4
Kill kinetics and regrowth patterns of Pseudomonas aeruginosa exposed to concentration-time profiles of tobramycin simulating in vivo infusion and bolus dosing; Wood PJ et al.; Pseudomonas aeruginosa ATCC 27853 was exposed to tobramycin concentration-time profiles modelling in vivo bolus and infusion dosing . Dependence of bactericidal and bacteriostatic activity on the initial profile of peak concentration (bolus effect > infusion) and area under the antibiotic concentration-time curve was observed at peak concentration/MIC ratios of 10 or below.

Antimicrob Agents Chemother, 1996 May, 40(5), 1280 - 1
Ceftazidime kinetics of diffusion in inoculated agar plates; Arcelloni C et al.; The ceftazidime concentration in agar plates inoculated with Pseudomonas aeruginosa was determined by high-performance liquid chromatography at fixed points (3, 6, 9, 12, and 15 mm) from the disk center and at fixed times (2, 4, 6, 16, and 24 h) to study the antibiotic kinetics of diffusion . A statistical difference between the concentrations determined in the presence of microorganisms and in uninoculated plates after 16 and 24 h was evidenced and was probably ascribable to the drug hydrolysis carried out by the induced beta-lactamase.

Antimicrob Agents Chemother, 1996 May, 40(5), 1254 - 6
Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene, aph (3')-IIb, in Pseudomonas aeruginosa; Hachler H et al.; A novel, probably chromosomally encoded, aminoglycoside phosphotransferase gene was cloned on a 2,996-bp PstI fragment from Pseudomonas aeruginosa and designated aph (3')-IIb . It coded for a protein of 268 amino acids that showed 51.7% amino acid identity with APH (3')-II {APH(3') is aminoglycoside-3' phosphotransferase} from Tn5 . Two other open reading frames on the cloned fragment showed homology to a signal-transducing system in P . aeruginosa.

Crit Care Med, 1996 May, 24(5), 797 - 801
Impact of the resin blood culture medium on the treatment of critically ill patients; Levin PD et al.; OBJECTIVE: To assess the relevance, both clinical and bacteriologic, of the use of resin-containing blood culture media in blood cultures taken from critically ill patients receiving antibiotics . DESIGN: A prospective, open clinical trial . SETTING: The mixed medical surgical intensive care unit (ICU) of a 550-bed urban hospital . PATIENTS: All ICU patients admitted during a 3-month period (n = 49) with suspected sepsis requiring blood cultures as part of their laboratory investigations . INTERVENTIONS: The use of an aerobic resin-containing blood culture medium, in addition to the regular aerobic and anaerobic media for all blood cultures taken . MEASUREMENTS AND MAIN RESULTS: Each blood culture result was classified as to its clinical significance . Changes in patient management were recorded . Culture sets in which the resin-containing bottle provided the information central to the change in patient management were identified . Bacteriologically, the results from the resin-containing medium were compared with the results from the aerobic and anaerobic media . Of 266 blood culture sets, 103 (39%) were positive, growing 278 bacterial and fungal isolates . Clinically, the resin-containing medium alone provided relevant data leading to changes in patient management on three occasions . On two of these occasions, cultures from the regular media provided the same data within 72 hrs . Bacteriologically, 77 (29%) aerobic bottles, 55 (21%) anaerobic bottles, and 89 (33%) resin-containing bottles were positive (statistical comparison of percentages: aerobic vs . resin-containing bottles, nonsignificant; aerobic vs anaerobic bottles, p < .046; anaerobic vs . resin-containing bottles, p < .0027) . A similar proportion of pathogens was isolated from the resin-containing bottles only (9%) and aerobic bottles only (6%) . A higher proportion of contaminants was isolated from the resin-containing bottles only than the aerobic bottles only in the various sets (17% vs . 7%, p < .046) . The resin-containing bottle showed a trend toward increased detection of Staphylococcus aureus and Pseudomonas aeruginosa bacteremia . CONCLUSIONS: The resin-containing medium offers little clinical benefit to the majority of ICU patients . Bacteriologically, it seems to have a similar overall sensitivity as the regular aerobic medium (with the possible exception of a higher sensitivity for the isolation of S . aureus and P . aeruginosa), but a lower specificity . The wide-spread use of the resin-containing bottle cannot be recommended.

Microbiology, 1996 May, 142 ( Pt 5), 1181 - 90
Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO; Stintzi A et al.; Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid bank (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mutation in the 47 min region of the chromosome yielded one clone which restored yellow-green pigmentation and fluorescence when grown on iron-deficient medium . The relevant pMMB33-derivative cosmid, pPYP17, contained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 Sacl-CIal fragment conferring the same phenotype . This derivative, pPYP180, like pPYP17, also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of the P . aeruginosa PAO chromosomes . Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PAO chromosome, demonstrating that the restored apparent wild-type phenotype observed for the transconjugants was not the result of a true gene complementation . A gene interruption was obtained by replacing a 0.6 kb BgIll-BgIll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype, by a Hgr interposon (omega Hg) . After conjugational transfer and introduction of the mutagenized fragment into the PAO1 chromosome by gene replacement, pyoverdine-deficient mutants were recovered, indicating that the fragment indeed contained at least one gene involved in pyoverdine synthesis . The yellow-green fluorescent compound produced by such cells harbouring plasmids pPYP17 or pPYP180 differed from pyoverdine in several aspects and was consequently named pseudoverdine . Although pseudoverdine was able to complex iron, it was unable to restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid), or to mediate iron uptake into PAO1 . Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine, suggesting that it was structurally related to the chromophore of the pyoverdine molecule . The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthetic pathway of the pyoverdine chromophore.

APMIS, 1996 May, 104(5), 350 - 4
Effects of Chinese medicinal herbs on a rat model of chronic Pseudomonas aeruginosa lung infection; Song Z et al.; The aim of the study was to evaluate the effects of two kinds of Chinese medicinal herbs, Isatis tinctoria L (ITL) and Daphne giraldii Nitsche (DGN), on a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF) . Compared to the control group, both drugs were able to reduce the incidence of lung abscess (p < 0.05) and to decrease the severity of the macroscopic pathology in lungs (p < 0.05) . In the great majority of the rats, the herbs altered the inflammatory response in the lungs from an acute type inflammation, dominated by polymorphonuclear leukocytes (PMN), to a chronic type inflammation, dominated by mononuclear leukocytes (MN) . DGN also improved the clearance of P . aeruginosa from the lungs (p < 0.03) compared with the control group . There were no significant differences between the control group and the two herbal groups with regard to serum IgG and IgA anti-P . aeruginosa sonicate antibodies . However, the IgM concentration in the ITL group was significantly lower than in the control group (p < 0.03) . These results suggest that the two medicinal herbs might be helpful to CF patients with chronic P . aeruginosa lung infection, DGN being the most favorable.

Nippon Yakurigaku Zasshi, 1996 May, 107(5), 225 - 35
{Nephrotoxicity and drug interaction of vancomycin (2)}; Toyoguchi T et al.; Vancomycin hydrochloride (VCM) has an antibacterial action against Gram positive bacteria, e.g., Methicillin-resistant Staphylococcus aureus (MRSA) . In the clinical situation, there are patients with serious infections, being infected with not only MRSA, but also with Gram negative bacteria like Pseudomonas aeruginosa . Because VCM has the adverse reaction of nephrotoxicity, we are apprehensive about using VCM with other antibiotics, which might increase this problem . Therefore, the nephrotoxic effects and pharmacokinetics of VCM were examined in rabbits and compared with those in rabbits administered with VCM and other antibiotics . Responses indicating nephrotoxicity such as increases of serum creatinine concentration and BUN and morphological changes of the kidney were induced by the single injection of VCM at 300 mg/kg, i.v . In contrast, no abnormality of clinical data and morphological alteration were observed in the groups injected with VCM and imipenem (IPM)-cilastatin sodium (CS), flomoxef sodium (FMOX) or fosfomycin sodium (FOM) . This was not true for groups injected with VCM and ceftazidime, cefpimizole sodium (CPIZ) or cefoperazone sodium . Clearance of VCM increased obviously in the groups injected with VCM and IPM-CS, FMOX or FOM, but decreased in those given VCM and CPIZ . Since the renal concentrations of VCM in the groups that were administered VCM with IPM-CS, FMOX or FOM were lower than that in the control group, IPM-CS, FMOX and FOM may decrease the nephrotoxicity of VCM by inhibiting its uptake into the kidney.

J Leukoc Biol, 1996 May, 59(5), 639 - 47
Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice; Gamelli RL et al.; Glucose is the primary metabolic substrate of macrophages, which are critical components of the host response to injury and infection . We have carried out a series of studies to examine macrophage glucose uptake and the status of glucose transporter 1 (GLUT1) at both the mRNA and protein level . Peritoneal macrophages that were obtained from mice undergoing sham burned (S), 15%TBSA burn (B) +/- Pseudomonas aeruginosa burn infection (B + I) and lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) administration . {3H}deoxyglucose uptake was significantly increased (B, 157 +/- 9%; B + I, 243 +/- 19%; S + LPS, 231 +/- 24%; S + TNF-alpha, 379 +/- 18%; B + LPS, 230 +/- 13%; and B + TNF, 305 +/- 23%, P< 0.01 vs . sham) . GLUT1 mRNA and protein levels were increased as well (mRNA: B, 135 +/- 13%; B + I, 250 +/- 33%; S + LPS, 282 +/- 29%; S + TNF-alpha, 193 +/- 19%; B + LPS, 378 +/- 20%; and B + TNF-alpha, 204 +/- 16%; protein: B, 159 +/- 27%; B + I, 181 +/- 17%; S + LPS, 219 +/- 26%; S + TNF-alpha, 343 +/- 51%; B + LPS, 366 +/- 41%; and B + TNF-alpha, 415 +/- 44, P< 0.01 vs . sham) . Macrophages co-cultured with LPS or TNF-alpha in vitro demonstrated a similar response pattern . Following burn injury and infection, macrophages augment their cellular glucose uptake, which is facilitated by an increased GLUT1 mRNA and protein levels . TNF-alpha elicited by LPS may mediate this enhanced carbohydrate metabolism at the point of glucose entry into the cell.

Postgrad Med, 1996 May, 99(5), 153 - 4, 157-8
Otitis externa . Management in the primary care office; Mirza N; Otitis externa is a widespread problem that is most commonly caused by Pseudomonas aeruginosa . Pain, ear discharge, and edema of the ear canal are the main manifestations . The presence of granulation tissue is an ominous sign that usually indicates necrotizing otitis externa or even a neoplastic process . It is important for primary care physicians to be familiar with methods of ear cleaning and use of topical medications for otitis externa . It is equally vital to be aware of the importance of a timely referral to an otolaryngologist when a serious underlying cause is suspected.

J Bacteriol, 1996 May, 178(10), 2767 - 74
Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics; Kadurugamuwa JL et al.; Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin . Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J . L . Kadurugamuwa and T . J . Beveridge, J . Bacteriol . 177:3998-4008, 1995) . Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z . Li, A . J . Clarke, and T . J . Beveridge, J . Bacteriol . 178:2479-2488, 1996) . g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin . Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides . Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria . They also adhered to the cell wall of gram-positive bacteria . g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin . The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins . n-MVs were capable of killing cultures of P . aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells . g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells . These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.

Invest Ophthalmol Vis Sci, 1996 May, 37(6), 976 - 86
Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa; Zaidi TS et al.; PURPOSE . Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection . The authors identified bacterial ligands involved in adherence and entry of P . aeruginosa into corneal epithelial cells . METHODS . In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P . aeruginosa mutants . Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (approximately 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin . The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria . Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin . Similar assays were carried out with whole mouse eyes in situ . RESULTS . A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P . aeruginosa into corneal cells . Bacterial pili and flagella were not involved . Mutants of P . aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic . Complementation of the LPS productions defect by plasmid-borne DNA returned association and ingestion to near parental levels . Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells . Experiments using P . aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry . CONCLUSIONS . Corneal epithelial cells bind and internalized P . aeruginosa by the exposed LPS core.

Am J Respir Crit Care Med, 1996 May, 153(5), 1600 - 5
Local Pseudomonas instillation induces contralateral lung injury and plasma cytokines; Terashima T et al.; We investigated whether local bacterial instillation leads to lung injury in noninstilled lung regions and examined local and systemic cytokine accumulation . Rats were challenged by intrabroncheal instillation of Pseudomonas aeruginosa, 10(7) colony-forming units (CFU) (HD group, n = 11), 4 x 10(6) CFU (LD group, n = 10), or saline (control group, n = 12) . 99mTc-labeled macroaggregated albumin was added to the P . aeruginosa or saline solution for later documentation of the instilled area . At 4 h the right lung, including instilled segment, and the left lung were sampled . Lung injury was assessed by lung tissue to plasma 125I-labeled albumin (T/P) and lung wet-dry (W/D) ratios . We measured plasma and bronchoalveolar lavage fluid (BALF) levels of tumor necrosis factor (TNF) and cytokine-induced neutrophil chemoattractant (CINC) . HD bacterial instillation induced neutrophil recruitment and TNF and CINC elevation in BALF (p < 0.05) associated with increased T/P (p < 0.005) and W/D (p < 0.05) ratios in both instilled and the noninstilled lungs as compared with the saline-instilled and noninstilled controls . LD bacterial instillation induced neutrophil recruitment and TNF and CINC elevation only in the instilled lung (p < 0.05), and not in the noninstilled lung, and did not increase the T/P or W/D ratio . Plasma levels of TNF and CINC were increased in the HD, but not the LD, group when compared with the saline controls (p < 0.05) . These data indicate that, when the dose is high enough to cause an excess inflammatory response, local bacterial instillation leads to neutrophil sequestration, lung injury, and cytokine elevation in the noninstilled lung associated with systemic cytokine release.

J Virol, 1996 May, 70(5), 3319 - 24
Hybrid proteins between Pseudomonas aeruginosa exotoxin A and poliovirus 2Apro cleave p220 in HeLa cells; Novoa I et al.; Cleavage of p220, a component of the initiation factor eIF-4F, has been correlated with the inhibition of host translation during poliovirus infection . To obtain p220 cleavage in the absence of any other poliovirus gene products, hybrid proteins containing Pseudomonas aeruginosa exotoxin A and poliovirus protease 2Apro have been constructed . The addition of the hybrid molecules to cultured cells did not lead to substantial p220 cleavage . However, the simultaneous presence of the hybrid toxin with replicationally inactive chicken adenovirus particles results in efficient cleavage of p220 in the intact cells . Under these conditions, cellular translation continues unabated for several hours, arguing against a direct requirement for intact p220 in each round of the initiation of translation of cellular mRNAs.

J Bacteriol, 1996 May, 178(9), 2586 - 92
PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor; Heinrichs DE et al.; The product of the pchR gene, an AraC-like regulatory protein, is required for production of the FptA ferric pyochelin receptor in response to iron limitation and pyochelin (D . E . Heinrichs and K . Poole, J . Bacteriol . 175:5882-5889, 1993) . The influence of iron, pyochelin, PchR, and FptA on fptA and pchR gene expression was assessed with fptA-lacZ and pchR-lacZ transcriptional fusions . As was expected, the expression of fptA decreased dramatically following the inactivation of pchR by the insertion of an OmegaHg cartridge, although the effect (> 10-fold) was not as dramatic as that of pyochelin deficiency, which obviated fptA gene expression . Insertional inactivation of pchR in a pyochelin-deficient (Pch-) background restored fptA expression to levels observed in the pyochelin-producing (Pch+) PchR- strain, suggesting that PchR represses fptA expression in the absence of pyochelin . Consistent with this, the cloned gene caused a five-fold decrease in the expression of the fptA-lacZ fusion in Escherichia coli . pchR gene expression was inducible by iron limitation, a result in agreement with the previous identification of a Fur box upstream of the gene, although the magnitude of the induction was less than that observed for fptA in response to iron limitation . Expression of pchR was effectively absent in a pyochelin-deficient strain, and insertional inactivation of pchR in a Pch+ or Pch- background caused an increase in pchR gene expression . PchR, thus, negatively regulates its own expression . Two related heptameric sequences, CGAGGAA and CGTGGAT, were identified upstream of the putative -35 region of both fptA and pchR and may function as a binding site for PchR . Insertional inactivation of fptA caused a marked decrease in fptA expression in a Pch+ background and obviated the apparent repression of fptA expression in a Pch- background, reminiscent of the effect of a pchR mutation . The fptA mutant did not, however, exhibit a defect in pchR expression . Interestingly, fptA mutants were unable to grow in the presence of pyochelin, suggesting that FptA is the sole outer membrane receptor for ferric pyochelin . These data indicate that PchR functions as both an activator and a repressor in controlling the expression of fptA and pchR . The involvement of FptA in this control is unclear, although it may be important in mediating the pyochelin effect on fptA expression, possibly by modulating PchR activity.

J Bacteriol, 1996 May, 178(9), 2479 - 88
A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles; Li Z et al.; A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures . In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases . Immunogold labeling of thin sections of P . aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm . It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled . Since division is due to cell constriction in this P . aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled . Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm . Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J . L . Kadurugamuwa and T . J . Beveridge, J . Bacteriol . 177:3998-4008, 1995) . The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.

Arch Ophthalmol, 1996 May, 114(5), 576 - 80
Effect of adhered bacteria on the binding of Acanthamoeba to hydrogel lenses; Gorlin AI et al.; OBJECTIVES: To determine the effect of Pseudomonas aeruginosa and Staphylococcus epidermidis on the binding of Acanthamoeba species to hydrogel lenses . METHODS: Cells of amebae and bacteria were incubated with different types of hydrogel lenses . Densities of amebae that were bound to the lenses after rinsing were determined from direct counts with a cell detachment procedure and from scintillation counts of cells, which were radiolabeled with tritiated leucine . RESULTS: With both methods, amebae showed significantly increased binding to hydrogel lenses with attached P aeruginosa . The numbers of amebae that were retained on lenses with attached S epidermidis were not significantly different from those that were retained on lenses without bacteria . The binding of amebae to unworn hydrogel lenses, in contrast to the irreversible adherence of P aeruginosa, was tenuous . CONCLUSIONS: The binding of Acanthamoeba species to unworn hydrogel lenses was tenuous and appeared to be related to water content, surface tensions, and ionic charge . The presence of adhered P aeruginosa on the hydrogel lenses facilitated the binding of Acanthamoeba species . The cocontamination of lens systems with bacteria (eg, P aeruginosa) may be a prime factor in the development of amebic keratitis.

Infect Immun, 1996 May, 64(5), 1819 - 25
Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids; Panjwani N et al.; Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium . Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3 . The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively . By use of a TLC-bacterial overlay procedure, 35S-labeled P . aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3 . P . aeruginosa binding to PL1 was concentration dependent . Alkaline methanolysis abolished the binding . PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate-treated TLC plates . Both PL1-I and PL1-S contained binding sites for P . aeruginosa . Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively . Radiolabeled P . aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC . A control Escherichia coli strain did not bind to either PS or PI . Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P . aeruginosa to PS or PI . P . aeruginosa bound to the monolayers of corneal epithelial cells . P . aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone . The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P . aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization.

Infect Immun, 1996 May, 64(5), 1659 - 65
A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge; Fattom AI et al.; The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models . A dose of 2 x 10(5) CFU of S . aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days . Mice passively immunized i.p . 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge . Animals actively immunized with the monovalent type 5 CP-P . aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals . The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively) . The IgG was further evaluated in mice challenged i.p . with a sublethal dose of 5 x 10(4) CFU per mouse . Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h . Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids . Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05) . These data suggest that S . aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S . aureus infections.

Infect Immun, 1996 May, 64(5), 1582 - 8
Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells; de Bentzmann S et al.; We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients . Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model . At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury . P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001) . In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P . aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001) . Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05) . Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1 . These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P . aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium . These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P . aeruginosa adherence.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4409 - 14
Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes; Ochsner UA et al.; The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur) . Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification . DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro . Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis . In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs) . While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes . Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function . Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors . Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified . The putative gene products of seven additional PIGs did not show significant homologies to any known proteins . The PIGs were mapped on the P.aeruginosa chromosome . Their possible role in iron metabolism and virulence of P . aeruginosa is discussed.

Carbohydr Res, 1996 Apr 18, 284(1), 85 - 99
Rheological studies of the interaction of mucins with alginate and polyacrylate; Fuongfuchat A et al.; The associative interaction of purified ovine and porcine submaxillary mucins (OSM and PSM) with sodium alginate was evaluated by comparing the rheological properties of mixtures against those of pure alginate and mucin in dilute, semi-dilute, and concentrated solutions . These systems were investigated as models for the interaction of mucin with the extracellular alginate produced by Pseudomonas aeruginosa . In dilute solution, evidence for such interaction cannot be obtained because aggregate species exist both in the OSM-alginate mixtures as well as in pure OSM . However, in the semi-dilute regime, mixtures containing a higher proportion of mucin show systematically higher viscosities than those predicted by simple additivity . In concentrated solutions containing higher proportions of mucin, an enhanced elastic response is observed . These results demonstrate a substantial binding interaction of mucins with alginate . This property is not observed in mixtures containing a high proportion of alginate, suggesting that mucins possess relatively low numbers of interacting sites . Introduction of 3 mM Ca2+ ions to all mucin-alginate mixtures enhances the elasticity due to gelation of alginate . Finally, comparison of the rheological properties of PSM-alginate mixtures with those of PSM-polyacrylate mixtures indicates that the binding strength of alginate to mucin is significantly weaker than that of polyacrylate.

Mol Gen Genet, 1996 Apr 10, 250(6), 692 - 8
Molecular analysis of the phosphate-specific transport (pst) operon of Pseudomonas aeruginosa; Nikata T et al.; The organization of the phosphate-specific transport (pst) operon in Pseudomonas aeruginosa has been determined . The gene order of the pst operon is pstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region . The most striking difference from the known Escherichia coli pst operon is the lack of the pstS gene encoding a periplasmic phosphate (Pi)-binding protein . Even though the three pst genes were absolutely required for P(i)-specific transport, expression of the pst operon at high levels did not increase P(i) uptake in P . aeruginosa . DNA sequences for the pstB and phoU genes have been determined previously . The newly identified pstC and pstA genes encode possible integral membrane proteins of 677 amino acids (M(r) 73,844) and 513 amino acids (M(r) 56,394) respectively . The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in their E . coli counterparts . A chromosomal deletion of the entire pst operon rendered P . aeruginosa unable to repress P(i) taxis under conditions of P(i) excess . The phoU and pstB genes are essential for repressing P(i) taxis . However, mutants lacking either PstC or PstA alone were able to repress P(i) taxis under conditions of P(i) excess.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 221 - 5
Desulfonation of aliphatic sulfonates by Pseudomonas aeruginosa PAO; Kertesz MA; Pseudomonas aeruginosa PAO1 used a broad range of alkanesulfonic acids as sole sulfur source for growth, with molar growth yields of 2.2 to 2.9 kg protein per mol sulfur . 4-Phenylbutane-1-sulfonate was desulfonated in vivo to yield 4-phenyl-1-butyric acid quantitatively as the sole product, suggesting that the desulfonation mechanism is the same as when alkanesulfonates serve as a carbon source for growth . This contrasts with aromatic sulfonate utilization in other organisms, where different desulfonation reactions are used to provide carbon and sulfur . Desulfonation of alkanesulfonates to provide sulfur was repressed by sulfate or thiocyanate, and derepressed in their absence . The alkanesulfonatase system is hence controlled as part of the sulfate starvation-induced stimulon.

Microbiology, 1996 Apr, 142 ( Pt 4), 1033 - 40
recA-dependence of the response of Pseudomonas aeruginosa to UVA and UVB irradiation; Kidambi SP et al.; The responses of the autochthonous soil and aquatic organism, Pseudomonas aeruginosa to UV radiation wavelengths (UVA, 320-400 nm, and UVB, 280-320 nm) has been investigated in this study . P . aeruginosa recA mutants were found to be more sensitive to both UVA and UVB radiation than were their isogenic RecA+ parents . Introduction of a low-copy-number plasmid containing the cloned wild-type P . aeruginosa recA gene restored UVA and UVB resistance to recA mutants . The concentration of RecA protein increased twofold 120 min after exposure to either UVA or UVB radiation, suggesting induction of expression of the recA gene by these wavelengths . In this study, we found that a functional RecA protein is required for activation of D3 prophage in lysogenic cells following exposure to UVB radiation . Prophage were not induced by exposure of their hosts to UVA radiation . Induction of damage-inducible (din) genes in response to UVA or UVB irradiation was also shown to be RecA dependent . These data indicate that the recA gene plays a role in the response of P . aeruginosa to exposure to wavelengths of UV radiation found in the solar spectrum.

Microbiology, 1996 Apr, 142 ( Pt 4), 881 - 8
Physiological and biochemical changes accompanying the loss of mucoidy by Pseudomonas aeruginosa; Williams SG et al.; Pseudomonas aeruginosa M60, a mucoid strain, was grown in continuous culture (D 0.05 h-1) under ammonia limitation with glucose as the carbon source . Steady-state alginate production occurred for only 1-2 d under these conditions {qalginate 0.097 g alginate h-1 (g dry wt cells)-1}, after which time the percentage of mucoid cells and the alginate concentration in the culture decreased in parallel and approached zero after approximately 10 d . These changes were accompanied by similar decreases in the activities of the alginate biosynthetic enzymes (represented by phosphomannomutase and GDP-mannose dehydrogenase) and by a large increase in the activity of the first enzyme of the 'external' non-phosphorylative pathway of glucose metabolism, glucose dehydrogenase . In contrast, the activities of other enzymes associated with this pathway (gluconate dehydrogenase, 2-ketogluconate kinase plus 2-ketogluconate-6-phosphate reductase) or with the 'internal' phosphorylative pathway of glucose metabolism (glucokinase and glucose-6-phosphate dehydrogenase) remained essentially unchanged . The loss of mucoidy and alginate production was accompanied by the appearance of low concentrations of intracellular polyhydroxyalkanoate (PHA) and of extracellular gluconate and 2-ketogluconate (partly at the expense of alginate production and partly as a result of increased glucose consumption) . It is suggested that ammonia-limited, glucose-excess cultures of P . aeruginosa growing at low dilution rate are unable fully to regulate the rate at which glucose and/or its 'external' pathway metabolites are taken up by the cell, and therefore form copious amounts of alginate in order both to overcome the potentially deleterious osmotic effects of accumulating surplus intracellular metabolites and to consume the surplus ATP generated by the further oxidation of these metabolites . The loss of mucoidy invokes the use of an alternative, but analogous, strategy via which non-mucoid cells produce an osmotically inactive intracellular product (PHA) plus increased amounts of the extracellular metabolites gluconate and 2-ketogluconate via the low-energy-yielding and, under these conditions, largely dead-end 'external' metabolic pathway.

Microbiology, 1996 Apr, 142 ( Pt 4), 873 - 80
The Azotobacter vinelandii gene algJ encodes an outer-membrane protein presumably involved in export of alginate; Rehm BH; The algJ gene from Azotobacter vinelandii was cloned using a labelled RNA probe representing the coding region of the algE gene from Pseudomonas aeruginosa . DNA sequencing revealed an ORF of 1452 bp encoding a protein of 484 amino acid residues with a calculated molecular mass of 54611 Da . An RNA probe corresponding to algE was also used for Southern hybridization of chromosomal DNA, which showed that algE-related DNA sequences are also present in the alginate-producing phytopathogen species Pseudomonas marginalis and Pseudomonas syringae pv . glycinea . The coding region of algJ was subcloned in the expression vector pT7-7, leading to a corresponding gene product with an apparent molecular mass of 54 kDa which could be identified in the outer membrane (OM) of Escherichia coli BL21(DE3) . Additionally, a cross-reacting protein with the same molecular mass was also found in the OM of A . vinelandii using an anti-AlgE antiserum . The derived amino acid sequence of AlgJ shared approximately 52% identity with AlgE from P . aeruginosa . The hydrophilicity profile as well as the amphipathicity of regions in the amino acid sequence of AlgJ showed significant similarities to AlgE . Based on these data, a topological model of AlgJ was created with the aid of known structures of outer-membrane proteins . This model presents AlgJ as a beta-barrel containing 18 beta-strands inserted in the OM.

Can J Microbiol, 1996 Apr, 42(4), 326 - 34
Physical mapping of several heat-shock genes in Pseudomonas aeruginosa and the cloning of the mopA (GroEL) gene; Farinha MA et al.; Using a series of oligonucleotides synthesized on the basis of conserved nucleotide or amino acid motifs in heat-shock genes/proteins, we have physically mapped the dnaK, lon, and hptG genes of Pseudomonas aeruginosa . Hybridization data suggest that there is a single copy of the mopBA (GroES/GroEL) operon but several additional copies of mopA . In addition, the map coordinates for the rpoD, rpoS, and rpoH genes were determined . The mopA gene from the mopBA operon was cloned and sequenced . The protein product of this gene showed 79% amino acid identity to the Escherichia coli GroEL and 98% identity to the GroEL sequence from P . aeruginosa ATCC 27853 . A number of discrepancies were found with the latter sequence.

Antimicrob Agents Chemother, 1996 Apr, 40(4), 909 - 13
Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa; Masuda N et al.; Various Pseudomonas aeruginosa PAO1 NfxB mutants were isolated on agar plates containing cefpirome and ofloxacin . They were classified into type A and type B, based on the degrees of changes in their susceptibilities . Type A mutants were four to eight times more resistant to ofloxacin, erythromycin, and new zwitterionic cephems, i.e., cefpirome, cefclidin, cefozopran, and cefoselis, than was the parent strain, PAO1 . In contrast, type B mutants were more resistant to tetracycline and chloramphenicol, as well as ofloxacin, erythromycin, and the new zwitterionic cephems, than was PAO1, and they were four to eight times more susceptible to carbenicillin, sulbenicillin, imipenem, panipenem, biapenem, moxalactam, aztreonam, gentamicin, and kanamycin that was PAO1 . The changes in susceptibilities of type B mutants were greater than those of type A mutants . The susceptibilities of both type A and type B mutants were restored to the level of PAO1 by transformation with plasmid pNF111, which contained the wild-type nfxB gene, demonstrating that they are NfxB mutants . Immunoblot analysis with a monoclonal antibody to OprJ revealed that type B mutants produced larger amounts of outer membrane protein OprJ than did type A mutants and that PAO1 produced an undetectable amount of it . Moreover, transconjugants obtained with the different types of NfxB mutants as the donor strains showed almost the same phenotypes as the corresponding donor strains . These results suggest that there are at least two nfxB mutations that show different phenotypes and that production of OprJ is associated with changes in susceptibilities of NfxB mutants.

Antimicrob Agents Chemother, 1996 Apr, 40(4), 904 - 8
Comparative in vitro pharmacodynamics of imipenem and meropenem against Pseudomonas aeruginosa; White R et al.; MICs are commonly used to assess the in vitro activities of antimicrobial agents; however, they provide minimal information on the pattern of bacterial activities . Time-kill studies with extensive sampling allow assessment of both the rate and extent of bacterial killing and regrowth . We compared imipenem and meropenem by both MIC-MBC testing and a time-kill study with P . aeruginosa 27853 . In the time-kill study, concentration/MIC ratios ranging from 0.0625 to 32 times the MIC were studied . The kill rate, time to 99.9% kill, doubling time of regrowth, and area under the bacterial killing curve (AUKC) were evaluated . Degradation during the testing procedure was accounted for by assessing actual drug exposure as determined by the area under the concentration-time curve . Pharmacodynamic parameters were compared by using the Wilcoxon signed-rank test . The modal MIC and MBC for imipenem were 2 and 4 micrograms/ml, respectively, and those for meropenem were 0.25 and 0.5 microgram/ml, respectively . In the time-kill study, both agents displayed concentration-dependent activity over a range of 0.25 to 4 times the MIC . Initial killing (0 to 1 h) was faster with imipenem at the same concentration/MIC ratios (P = 0.0506) . The time to 99.9% kill was approximately 5 h for both agents . When regrowth occurred, the doubling rate for imipenem, which was the same as that for the growth control, was twice as rapid as that for meropenem . At the same concentrations, the AUKCs over 24 h were lower for meropenem than for imipenem (P = 0.0280); however, when normalized by MIC, imipenem resulted in smaller AUKCs . Comparison of plots of area under the concentration-time curve versus AUKC, which accounted for drug degradation and actual drug exposure, revealed that meropenem was three times more active than imipenem, rather than the eightfold difference suggested by MICs . Time-kill curves with extensive sampling and measurement of actual drug exposure, rather than traditional MIC testing, may more accurately assess differences in the in vitro activities of antimicrobial agents.

Diagn Microbiol Infect Dis, 1996 Apr, 24(4), 179 - 90
Comparative usefulness of ribotyping, exotoxin A genotyping, and SalI restriction fragment length polymorphism analysis for Pseudomonas aeruginosa lineage assessment; Nociari MM et al.; Ribotyping, exotoxin A genotyping (EAGP), and restriction fragment length polymorphism (RFLP) analysis of total DNA with SalI (SalI RFLP) were compared for intraspecies discrimination of 93 Pseudomonas aeruginosa isolates . Type-ability of all methods was 100% and the results of typing with each method remained unchanged during laboratory manipulation . Clonal groups defined with each molecular method were largely coincident and, in those cases where inconsistencies were detected, isolates were analyzed by transverse alternating field gel electrophoresis (TAFE) and arbitrarily primed polymerase chain reaction (AP-PCR) . SalI RFLP analysis was highly discriminative so as to distinguish unrelated isolates of close lineage . However, it was not a good method to identify isolates of unrelated lineage because SalI RFLP appeared to be subjected to convergent evolution . The index of discrimination suggested by Hunter and Gaston was determined to assess the discriminatory power of the molecular methods utilized either alone or in several combinations . Combined use of ribotyping and SalI RFLP analysis reached the highest index of discrimination (0.982) and proved to be a very valuable tool for epidemiological differentiation of P . aeruginosa isolates.

Eur J Epidemiol, 1996 Apr, 12(2), 123 - 9
Pseudomonas aeruginosa clinical isolates: serotypes, resistance phenotypes and plasmid profiles; Millesimo M et al.; 78 Pseudomonas aeruginosa strains were isolated from the respiratory tract of 56 patients, 15 of which were affected by cystic fibrosis (CF) . The epidemiological typing scheme was based on serotyping, antibiotic resistance pattern and plasmid DNA profile . All strains (except 2 mucoid strains) were typed using a rapid slide O-agglutination technique . Most common serotypes in both group were 0:1, 0:10 and 0:6 . Moreover we observed a correlation among 0:12 serotype and CF patients . Plasmid DNA analysis showed that 45.2% (on average) of strains isolated from patients with and w/o CF harboured 1-3 plasmids ranging in size from 1 to 15 Md . Plasmid prevalence was higher in strains isolated from CF patients in specimens collected after antibiotic therapy . A correlation was found between 1 and 1.9 Md plasmids and resistance to aminoglycosides . Our results indicate that the analysis of antibiotic resistance phenotypes combined with plasmid analysis may be useful, in association to serotyping, to characterize the circulation of P . aeruginosa strains and the spread of resistance in these bacteria.

J Clin Microbiol, 1996 Apr, 34(4), 924 - 7
Selective isolation of vancomycin-resistant enterococci; van Horn KG et al.; Broth formulations of two media selective for enterococci, Enterococcel, M-Enterococcosel broths were supplemented with 6 micrograms of vancomycin per ml and evaluated for isolation of vancomycin-resistant enterococci (VRE) . Each broth was challenged with various concentrations of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and vancomycin-susceptible and vancomycin-resistant enterococci and with 193 perianal specimens obtained from patients at risk in our institution for VRE colonization . Both the Enterococcosel and M-Enterococcus broths with vancomycin detected as few as 1 to 9 CFU of VRE while inhibiting growth of the other organisms tested . Enterococcus faecium organisms (MIC, > 256 micrograms/ml) were recovered from 66 perianal swab cultures in the enterococcosel-vancomycin broth, and VRE were recovered from 62 perianal swab cultures in the M-Enterococcus-vancomycin broth . Enterococcosel-vancomycin broth detected VRE in perianal specimens 48 h earlier than did M-Enterococcus-vancomycin broth . Enterococcosel broth with 6 micrograms of vancomycin per ml can be used for the rapid and selective isolation of VRE from surveillance specimens.

Lab Anim, 1996 Apr, 30(2), 102 - 7
Isolation of Enterococcus durans and Pseudomonas aeruginosa in a scid mouse colony; Dietrich HM et al.; In a colony of severe combined immunodeficiency (SCID) mice conspicuously altered behavioural characteristics were observed: hunched position, apathy, dullness, short breath, bristled fur, emaciation, circling movements around their longitudinal axis and oblique head posture . This was most common in pregnant and lactating animals and also observed in 4 mice after experimental treatment . Pseudomonas aeruginosa, serotype P1 and Enterococcus durans, serotype D were isolated from various organs and from the middle ear . On autopsy, the mice showed signs of focal pericarditis and thickened liver capsules . The histological examination of the liver revealed mild, focal accumulations of mononuclear cells . In addition, it was observed that SCID mice with signs of this disease did not allow human peripheral blood mononuclear cells to engraft.

Eur J Clin Microbiol Infect Dis, 1996 Apr, 15(4), 309 - 12
Precipitating Pseudomonas aeruginosa antibodies and antimicrobial therapy in cystic fibrosis patients; Trancassini M et al.; Forty patients with cystic fibrosis were studied bacteriologically and serologically . Precipitating Pseudomonas aeruginosa antibodies were monitored by crossed-immunoelectrophoresis (CIE) in order to evaluate the possibility of preventing chronic colonization by Pseudomonas aeruginosa by cycles of antimicrobial therapy . Sputum or pharyngeal aspirate and serum samples from all patients were analyzed by means of spread on selective media and CIE, respectively . Significant differences in the number of precipitins were obtained: noncolonized and intermittently colonized patients had no precipitins, whereas the number of precipitins in the chronically colonized patients varied from 11 to 44 . An increase in the number of precipitins could be a good marker for initiation of therapy with antimicrobial agents that are either active against Pseudomonas aeruginosa or able to inhibit the release of virulence factors.

Tuber Lung Dis, 1996 Apr, 77(2), 168 - 72
Surgery for unilateral bronchiectasis: results and prognostic factors; Ashour M et al.; SETTING: King Khalid University Hospital referral centre for thoracic surgery, Riyadh, Saudi Arabia . OBJECTIVE: To assess the results of surgery and factors influencing its outcome in patients with unilateral bronchiectasis . DESIGN: A retrospective analysis of 40 patients with unilateral bronchiectasis who were operated upon consecutively at King Khalid Hospital, between July 1987 and May 1993 . RESULTS: Left-sided disease was seen in 60% (n = 24) and right-sided in 40% (n = 16) of the patients . The entire lung was involved in 30% of cases (n = 12) . Of these, the left lung was totally involved in 22.5% (n = 9) and the right in 7.5% (n = 3) . A lobectomy was performed on 21 patients, basal segmentectomy with preservation of apical segment on 7, and pneumonectomy on 12 . There was no operative mortality in this series . Six patients (15%) developed postoperative complications, bleeding (n = 4) and prolonged air leak (n = 2) . During an average follow-up period of 30.7 months (+/- 15.4 months), 29 patients (72.5%) were cured and the remaining 11 (27.5%) improved . No patients with Pseudomonas aeruginosa infection (n = 3) or obstructive airway disease (n = 5) were cured (P = 0.02 and P = 0.002 respectively) . CONCLUSION: Curative resection for selected patients with unilateral bronchiectasis can be performed safely with good results and low morbidity . Pseudomonas aeruginosa infection and obstructive airway disease have an adverse effect on postoperative cure.

Arzneimittelforschung, 1996 Apr, 46(4), 429 - 32
Influence of temperature on mutational resistance to 4-quinolones; Parte AC et al.; Minimum inhibitory concentration (MIC) values of ciprofloxacin (CAS 85721-33-1), ofloxacin (CAS 82419-36-1) or levofloxacin (CAS 100986-85-4) against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis were determined at 37, 30 and 25 degrees C on nutrient agar . With E . coli and S . aureus, reducing the incubation temperature from 37 to 30 to 25 degrees C decreased the MIC values for each drug . With P . aeruginosa, temperature reduction also decreased the MIC values for ciprofloxacin but increased the MICs for ofloxacin or levofloxacin . With S . epidermidis, temperature reduction increased the MICs for ciprofloxacin and ofloxacin but with levofloxacin the MICs were identical at 37 or 25 degrees C but higher at 30 degrees C . Mutants of these bacterial species were selected on nutrient agar containing five times their respective MICs of the 4-quinolones at 37, 30 and 25 degrees C . with S . epidermidis, temperature reduction always reduced its mutation frequency to resist 4-quinolones, whereas with the other three species the effect of temperature was more variable, and in some instances even elevated their mutation rates.

FEMS Immunol Med Microbiol, 1996 Apr, 13(4), 287 - 92
Ribotyping of Pseudomonas aeruginosa isolates from patients and water springs and genome fingerprinting of variants concerning mucoidy; Leitao JH et al.; Thirty-one strains of Pseudomonas aeruginosa, isolated from water springs, clinical isolates (some of which were from cystic fibrosis (CF) patients), and two type cultures, were characterized by ribotyping . After restriction of chromosomal DNA of the different isolates with EcoRI and hybridization of Southern transfer blots with 2-acetylaminofluorene labelled Escherichia coli 16S + 23S rRNA probe, eleven different ribopatterns were obtained, representing variations of a dominant profile . This largely predominant pattern included both type cultures, all six isolates from water springs, 33% of the nine CF isolates and 43% of fourteen other clinical isolates most of them from nosocomial infections . When the genomic macrorestriction fingerprints of three mucoid CF isolates, with Asel, Drai or BfrI were compared with those of their spontaneous variants, concerning mucoidy, no differences were detected.

Thorax, 1996 Apr, 51(4), 364 - 8
Nebulised antipseudomonal antibiotic therapy in cystic fibrosis: a meta-analysis of benefits and risks; Mukhopadhyay S et al.; BACKGROUND: To establish the benefits and risks of nebulised antipseudomonal therapy in cystic fibrosis the results of relevant randomised controlled trials were combined . METHODS: The therapeutic end points compared were (a) number of pulmonary exacerbations requiring treatment with systemic antibiotics, (b) measurable alteration in respiratory tract pseudomonal load, (c) alteration in lung function on spirometric assessment, (d) development of resistance in respiratory tract Pseudomonas strains to the nebulised antipseudomonal used in each randomised controlled trial, and (e) renal and auditory impairment . RESULTS: Five studies were suitable for meta-analysis, eight others could not be included because of inadequate outcome description or the lack of appropriate randomisation . Meta-analysis shows benefit for nebulised antipseudomonal antibiotic therapy with no demonstrable adverse effect other than a possible increase in in vitro antibiotic resistance of Pseudomonas aeruginosa of the respiratory tract . CONCLUSIONS: Although inferences drawn from individual randomised controlled trials concerning the benefits and risks of this form of therapy are conflicting, pooled effect size establishes benefit with nebulised antipseudomonal antibiotic therapy and emphasises its relevance to the integration of information in other areas of controversy relating to the treatment of this disease.

J Laryngol Otol, 1996 Apr, 110(4), 315 - 8
Effect of aerobic bacteriology on the clinical presentation and treatment results of chronic suppurative otitis media; Vartiainen E et al.; The effect of aerobic bacteriology on the clinical presentation, complications of the disease and long-term results of surgical treatment was assessed in a cohort of 368 patients with chronic suppurative otitis media . Bacteriological findings showed no significant difference between child and adult patients . Staphylococcus aureus was isolated in cholesteatoma ears more frequently than Pseudomonas aeruginosa, in chronic ears without cholesteatoma the situation was reversed . Bacteriological findings had no significant effect on the incidence of complications caused by the disease . Failures after surgical treatment were most common in Pseudomonas ears . The bacteriology had no significant effect on pre-operative hearing levels nor post-operative hearing results . It was concluded that, in order to improve results of chronic ear surgery, more attention should be paid to pre-operative conservative treatment of chronically discharging ears, especially those infected by P . aeruginosa.

CLAO J, 1996 Apr, 22(2), 118 - 21
Comparative disinfectant efficacy of two disinfecting solutions against Pseudomonas aeruginosa; Key JE et al.; PURPOSE . We compared the disinfecting efficacy of Pure Eyes Disinfectant and Opti-Free Rinsing, Disinfecting and Storage Solution, without recommended daily cleaning steps, against Pseudomonas aeruginosa . METHODS . Vifilcon A soft contact lenses were inoculated with the challenge organism, P . aeruginosa ATCC 15442, at a concentration of 1 x 10(8) colony forming units (CFU/mL) and disinfected with each solution for four and six hours, respectively . RESULTS . Pure Eyes eradicated P . aeruginosa after both four and six hour soak times . With all lots of Opti-Free, colony forming units were too numerous to count (> 200) for both soak times . CONCLUSIONS . We concluded that, because Pure Eyes disinfecting solutions can eradicate P . aeruginosa without the benefit of a daily cleaning step, this disinfecting process may offer protective benefits to noncompliant contact lens wearers.

CLAO J, 1996 Apr, 22(2), 114 - 7
The use of synthetic Cecropin (D5C) in disinfecting contact lens solutions; Sousa LB et al.; PURPOSE . Microbial keratitis caused by Pseudomonas aeruginosa is the most common contact lens associated corneal infection . Cecropins are microbicidal peptides isolated from the hemolymph of the Cecropia moth . Previous in vitro studies have demonstrated their efficacy against a broad spectrum of ocular pathogens . This study was designed: a) to evaluate the antimicrobial potency of three different contact lens solutions (Renu, Complete, and Opti-Free) against P . aeruginosa, and b) to evaluate the activity of the same contact lens solutions in combination with a synthetic cecropin analog, D5C, against the challenge organism in the presence of a soft contact lens . METHODS . A virulent strain of P.aeruginosa isolated from a case of ulcerative keratitis was used in the study . Three different concentrations of bacteria (10(3), 10(5) and 10(7) CFU/mL) were inoculated into the contact lens solutions and into buffered saline, which was employed as a control . The samples were incubated at 27 degrees C, and at time 0, 30, and 90 minutes, 24, 48, and 72 hours, and aliquots of the test solutions were plated and subcultured on nutrient agar . After 24 hours of incubation at 37 degrees C, colonies observed on the nutrient agar plates were counted . To study the antimicrobial efficacy of D5C (100 micrograms/mL), we used the identical test series and assay, adding a soft contact lens to the solutions and a larger inoculum of bacteria (10(9) CFU/mL) . RESULTS . After 72 hours, all of the contact lens solutions tested sterilized 10(3) CFU/mL of P . aeruginosa . At 10(7) CFU/mL, they yielded greater than 2 logs of killing of the bacteria, but the solutions were not sterilized . The addition of D5C (100 micrograms/mL) to the contact lens solutions yielded greater than 3 logs of killing with a larger inoculum of bacteria in the presence of the soft contact lens . CONCLUSION . The contact lens solutions tested were effective against P . aeruginosa at 27 degrees C for up to 72 hours with an inoculum of 10(3) CFU/mL . The addition of D5C augmented their antimicrobial activity in the presence of the contact lens.

J Antimicrob Chemother, 1996 Apr, 37(4), 809 - 13
Comparative distribution of resistance patterns and serotypes in Pseudomonas aeruginosa isolates from intensive care units and other wards; Bert F et al.; The resistance patterns and O-serotypes of 2952 Pseudomonas aeruginosa isolates were studied and the relationship between patterns and serotypes was investigated . The penicillinase-producing and cephalosporinase-overproducing phenotypes were significantly more frequent in intensive care units than other wards, but there was no difference for the intrinsic resistance phenotype . The predominant serotypes were O6, O11 and O1 . The incidence of serotype O12 was low . O11 isolates were more common in intensive care units and more resistant to all antibiotics than other isolates . Most O12 isolates had a penicillinase-producing phenotype and were resistant to aminoglycosides and ciprofloxacin, but susceptible to fosfomycin.

J Antimicrob Chemother, 1996 Apr, 37(4), 703 - 10
A pharmacodynamic evaluation of ciprofloxacin and ofloxacin against two strains of Pseudomonas aeruginosa; Madaras-Kelly KJ et al.; The greater potency of ciprofloxacin in vitro to that of ofloxacin against Pseudomonas aeruginosa may be potentially offset by the more favorable pharmacokinetic profile of the latter drug . In order to test this hypothesis, we generated time concentration kill curves for P . aeruginosa ATCC 27853 and a clinical isolate P . aeruginosa PSA 9258 using an in-vitro model to simulate the pharmacokinetic characteristics found in vivo for ciprofloxacin at a peak concentration (CPmax) of 5 mg/L and an elimination T1/2 of 4.5 h, and ofloxacin at a CPmax of 5 mg/L and a T1/2 of both 4.5 h and 6 h, and at a CPmax of 8.0 mg/L and a T1/2 of 6 h . A 3 log10 kill (T3 kill) of P . aeruginosa ATCC 27853 was achieved in 0.15 h by ciprofloxacin and of P . aeruginosa PSA 9258 in 0.09 h . Ofloxacin at a CPmax of 8 mg/L and T1/2 of 6 h achieved a T3 kill of P . aeruginosa ATCC 27853 in 0.74 h and of P . aeruginosa PSA 9258 in 0.16 h . The area under the kill curve (AUKC) was 1.10 x 10(4)and 1.96 x 10(3) mL-h/cfu for ciprofloxacin against P . aeruginosa ATCC 27853 and P . aeruginosa PSA 9258, respectively and that of ofloxacin at CPmax 8 mg/L and a T1/2 of 6 h was 9.78 x 10(4)and 2.20 x 10(4) mL-h/cfu respectively . Significant differences (P > or = 0.05) were evident between ciprofloxacin and all ofloxacin regimens against P . aeruginosa ATCC 27853 but not against P . aeruginosa PSA 9258 . There was a poor correlation (r = 0.22) between the AUKC and area under the time concentration curve (AUC) for P . aeruginosa ATCC 27853 but a strong correlation (r = 0.96) between the AUKC and area under the inhibitory curve (AUIC) . Similar results were obtained for P . aeruginosa PSA 9258 for which the correlation between AUKC and AUC was weak (r = 0.10) whereas that between the AUKC and AUIC was strong (r = 0.93) . When the data for both P . aeruginosa were combined, a correlation coefficient of r = 0.04 for AUC and r = 0.80 for AUIC was found . These limited data suggest that fluoroquinolones can be compared using the AUIC for specific bacterial isolates . In addition, the larger AUC, higher CP, and longer T1/2 of ofloxacin in vivo did not fully compensate for the intrinsic differences in the antibiotic susceptibility against P . aeruginosa.

J Chemother, 1996 Apr, 8(2), 130 - 6
An open, multicenter clinical trial of piperacillin/tazobactam in the treatment of pediatric patients with intra-abdominal infections; Arguedas A et al.; A total of 60 children with secondary peritonitis were enrolled in an open, non-comparative multicenter study designed to evaluate the safety, tolerance and efficacy of parenteral piperacillin/tazobactam (80/10 mg/kg every 8 hours) in young children . The most common diagnosis was perforated appendicitis (90%) and the three most common pathogens, obtained from the peritoneal cavity, were Escherichia coli (52 isolates), Pseudomonas aeruginosa (16 isolates) and Bacteroides sp . (19 isolates) . Patients were examined daily during therapy, 4-14 days and 4-6 weeks post-therapy . Of the 60 patients, 43 were evaluable . The majority of patients had polymicrobial infections (36 patients) . All the aerobic isolates were susceptible to piperacillin/tazobactam while 19 were resistant to piperacillin alone . Four of 43 clinically evaluable patients were considered a clinical failure and 3 of 40 bacteriologically evaluable patients were considered to have an unfavorable microbiological response . There were 2 clinically adverse events considered related to the study drug and several possibly related, mild and transitory, abnormalities in eosinophil counts and liver function tests . Based on the safety and efficacy results from this study, the advantages of using a single agent for the treatment of mixed infections of the peritoneal cavity and its potential activity against resistant organisms, we believe that further comparative clinical trials in children with intra-abdominal infections are warranted.

Planta Med, 1996 Apr, 62(2), 122 - 5
Mode of antibacterial action of totarol, a diterpene from Podocarpus nagi; Haraguchi H et al.; The antimicrobial mechanism of totarol was studied using Pseudomonas aeruginosa IFO 3080 . This diterpene inhibited oxygen consumption and respiratory-driven proton translocation in whole cells, and oxidation of NADH in membrane preparation . NADH-cytochrome c reductase was inhibited by totarol while cytochrome c oxidase was not . NADH-DPIP reductase and NADH-CoQ reductase were also inhibited . The site of respiratory inhibition of totarol was thought to be near CoQ in the bacterial electron transport chain.

Am J Pathol, 1996 Apr, 148(4), 1297 - 305
Role of CD 11/CD 18 in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia in rabbits; Kumasaka T et al.; This study examined CD11/CD18-mediated adhesion in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia . Neutrophil emigration during acute pneumonia was studied in anti-CD18 antibody or murine-IgG-pretreated rabbits 4 hours after intrabronchial instillation of P . aeruginosa . To examine emigration in recurrent pneumonias, rabbits given P . aeruginosa on day 0 received anti-CD18 antibody or IgG on day 7 . A second instillate was placed either at the initial site or in a separate lobe, and emigration into alveolar spaces was quantitated morphometrically after 4 hours . The results show that CD11/CD18 was required for neutrophil emigration in acute pneumonias and in recurrent pneumonias that occurred at a site distant from the initial infection . However, when the recurrent pneumonia occurred in the previously inflamed site, CD11/CD18 was not required . When the same number of organisms were instilled on days 0 and 7, emigration was reduced to 15 to 20 percent of the number that migrated initially and only CD18-independent adhesion pathways were used . Increasing the concentration of organisms threefold increased emigration through both CD18-dependent and CD18-independent pathways . These data indicate that P . aeruginosa induces CD11/CD18-dependent emigration during acute pneumonia and recurrent pneumonia at previously uninflamed sites . However, adhesion pathways are altered in regions of chronic inflammation, and a greater proportion of neutrophil emigration occurs through CD11/CD18-independent pathways.

J Bacteriol, 1996 Apr, 178(8), 2299 - 313
Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity; Leoni L et al.; The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa . Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA . The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site . RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level . Primer extension and S1 mapping experiments revealed two 5'termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation . The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA . Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites . A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigmaE -like factor PvdS were identified 5' of the T1 start site . The minimum DNA region required for iron-regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA . We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Fur and PvdS in the iron-regulated expression of pvdA . Two fur mutants of P . aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression . Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P . aeruginosa . Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Fur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation . Conversely, the promoter region of pvdA did not interact with Fur . Our results support the hypothesis that the P . aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes.

J Bacteriol, 1996 Apr, 178(8), 2186 - 95
Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation; Franklin MJ et al.; Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate . The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions . Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation . Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF . An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans . This indicated that the algIJF gene products were not required for polymer biosynthesis . To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette . Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected . Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate . Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant . The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results . Strains missing only algJ or algI also produced nonacetylated alginates . Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation . Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate . Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.

Chest, 1996 Apr, 109(4), 1019 - 29
Ventilator-associated pneumonia due to Pseudomonas aeruginosa; Crouch Brewer S et al.; OBJECTIVE: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa has been associated with higher case fatality rates than VAP caused by other bacterial etiologies . The causes of this excess mortality are unclear . DESIGN: Retrospective review of 38 consecutive ventilated patients with Pseudomonas pneumonia, documented by highly reliable methods . Charts of five additional patients were unavailable for review . SETTING: Medical ICUs of a university-affiliated Veterans Affairs Medical Center and a university-affiliated municipal hospital . MEASUREMENTS: Prospectively collected hospital admission acute physiologic and chronic health examination (APACHE) II scores and cause of ICU admission . Retrospectively calculated organ failure and APACHE scores, VAP score . Clinical and microbiologic variables . Antibiotic treatment and outcome . Direct cause of death by standard definitions . RESULTS: Overall mortality was 69% (26/38), significantly higher than the APACHE II predicted mortality of 42.6% (p=0.037) . At least 38% (10/26) of deaths were directly attributable to Pseudomonas VAP . Multivariate analysis of factors associated with death found infectious cause for ICU admission (odds ratio {OR}=8.67; 95% confidence interval {CI}, 0.86 to 85.94) and number of organ dysfunctions on the day of diagnosis (OR=1.73, 95% CI, 1.02 to 2.92) were significant . Septic shock from Pseudomonas VAP, septic shock from subsequent infection, and multiple organ dysfunction syndrome were the most common immediate causes of death . Mortality increased linearly with increasing APACHE III score on the day of diagnosis . Of initial antibiotic regimens, 67% (26/36) were considered failures . Persistent pneumonia occurred in 35% of patients while recurrent pneumonia was unusual (1/38) . CONCLUSIONS: Development of Pseudomonas pneumonia results in a mortality rate in excess of that due to the presenting illness . The attributable mortality determined by several means appears to approach 40% . The excess mortality appears to be related to the host defense response to the pneumonia rather than any characteristic of the pneumonia . Even standard antibiotic regimens fail frequently and do not prevent the excess mortality . Since at least 38% of deaths can be directly attributable to the Pseudomonas pneumonia, improvement in therapy is needed.

J Leukoc Biol, 1996 Apr, 59(4), 539 - 44
In vitro culture of murine peritoneal and alveolar macrophages modulates phagocytosis of Pseudomonas aeruginosa and glucose transport; Everett KD et al.; Phagocytosis by murine peritoneal macrophages (PM phi) of unopsonized Pseudomonas aeruginosa is a novel, glucose-dependent process occurring in concert with glucose or mannose transport via the GLUT1 facilitative transporter . The mechanism by which this transport triggers phagocytosis is not understood . The purpose of these investigations was to improve our understanding of this mechanism by delivery of an alternative sugar (fructose) to PM phi and to murine alveolar macrophages (AM phi) . Fructose-cultured PM phi developed fructose-dependent phagocytosis of P . aeruginosa with increased glucose-dependent phagocytosis, GLUT1 expression, and {14C}glucose transport . Freshly explanted AM phi, which were unable to transport {14C}glucose or to ingest P . aeruginosa acquired the ability to transport glucose and to phagocytose P . aeruginosa with culture in either glucose or fructose . Both fructose- and glucose-cultured AM phi remained viable but incapable of measurable fructose transport or fructose-dependent phagocytosis . These studies suggest that an intracellular metabolite of fructose, glucose, and mannose is involved in triggering macrophage phagocytosis of P . aeruginosa . We demonstrate that delivery of appropriate substrates can substantially improve AM phi phagocytic function and may therefore possibly improve pulmonary host defense against P . aeruginosa.

J Bacteriol, 1996 Apr, 178(7), 1793 - 9
Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii; Campos M et al.; Azotobacter vinelandii presents a differentiation process leading to the formation of desiccation-resistant cysts . Alginate, the exopolysaccharide produced by this bacterium, has been postulated to have a role in cyst formation . Here, we report the cloning and characterization of the A . vinelandii gene coding for the enzyme GDP-mannose dehydrogenase (algD), which is the key enzyme for alginate synthesis in Pseudomonas aeruginosa . This gene has a high degree of similarity with the algD gene from P . aeruginosa, and similar proteins seem to be involved in algD regulation in both bacteria . We show the existence of two mRNA start sites; one of these sites corresponds to a promoter transcribed by RNA polymerase containing a sigma E subunit . An A . vinelandii algD mutant which is completely impaired in alginate production and which is unable to form desiccation-resistant cells was constructed . The effects of NH4, NO3, and NaCl concentrations on algD transcription for three A . vinelandii strains producing different alginate levels were evaluated . We found a strict correlation between alginate production and algD transcription for the three strains studied; however, the effects on algD transcription under the conditions studied were different for each strain . The nitrogen source regulates algD expression in the wild-type strain.

J Bacteriol, 1996 Apr, 178(7), 1777 - 81
Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme; Shankar S et al.; Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells . We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk . We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form . The 12-kDa form was found to be predominantly associated with the P . aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic . In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates . The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100 . The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture . The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P . aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 526 - 31
Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma; Miyanoshita A et al.; A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma . The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins . The new defensin was found to be inducible by bacterial injection . Analysis of the antibacterial spectrum of A . dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family . In addition, A . dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S . aureus (MRSA) isolated from a patient.

Am J Epidemiol, 1996 Mar 15, 143(6), 637 - 47
Application of exponential smoothing for nosocomial infection surveillance; Ngo L et al.; Detection of outbreaks of infection or increases in bacterial resistance to antimicrobial agents is an essential component of hospital infection control surveillance . The authors applied the method of exponential smoothing to microbiology data from 1987-1992 to investigate a suspected outbreak of gentamicin resistance among Pseudomonas aeruginosa bacteria at the Department of Veterans Affairs Medical Center, San Francisco, California, in 1991-1992 . The years 1987-1990 were used to develop the baseline for the forecast model . Application of the model indicated that two observed prominent peaks in the annual cumulative incidence of gentamicin-resistant P . aeruginosa were within the upper bounds of their respective 95% confidence intervals as estimated by the forecast model--i.e., that no epidemic was in progress . This prediction was supported by investigations by the hospital's infection control team which indicated that the apparent increases were due to readmission of patients previously known to harbor these organisms . In contrast, application of a typically employed method that ignores the time series data structure indicated that there were 6 months in which incidence rates exceeded the upper bounds of their respective 95% confidence intervals, thereby erroneously suggesting that an epidemic was in progress . Recursive algorithms and some simplifying assumptions that do not affect the validity of inferences make the application of this method practical for nosocomial infection control programs.

J Antimicrob Chemother, 1996 Mar, 37(3), 599 - 604
Effects of synthetic form of tracheal antimicrobial peptide on respiratory pathogens; Lawyer C et al.; We have synthesized a C-terminal portion of tracheal antimicrobial peptide (TAP) with 38 amino acids and tested it for efficacy on various clinical isolates of Pseudomonas aeruginosa strains from patients with cystic fibrosis and also on Aspergillus fumigatus . Our results indicate that the synthetic TAP has both potent bactericidal and fungicidal activities and that a combination of TAP and amphotericin B showed strong additive effects of growth inhibition on A fumigatus . These results suggest that TAP is potentially an effective therapy for Aspergillus and multi-drug-resistant Pseudomonas, pathogens that are often a serious threat to patients with cystic fibrosis.

Eur J Pediatr, 1996 Mar, 155(3), 216 - 8
Necrotizing bowel lesions complicated by Pseudomonas septicaemia in previously healthy infants; Tsai MJ et al.; Two previously healthy infants with Pseudomonas septicaemia presented with necrotizing bowel lesions . Necrotizing bowel lesions should be suspected when infants presenting with a history of diarrhoea, develop abdominal distension and toxic signs . Pseudomonas aeruginosa should be regarded as one of the important aetiologies in such disorders, especially if there is associated neutropenia and ecthyma gangrenosum-like lesions . Antibiotics must be able to cover this pathogen to avert a catastrophic outcome . CONCLUSION: The intestine should be considered a possible site of involvement in Pseudomonas sepsis and special attention should be paid to examination of the abdomen.

J Clin Microbiol, 1996 Mar, 34(3), 575 - 8
Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis; Hla SW et al.; The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy . The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods . We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year . A high microbial load of P . aeruginosa was found in 70% of sputum samples collected . Of these, 55 sequential P . aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P . aeruginosa strains during chemotherapy . Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease . Of the eight patients, six harbored a single dominant strain of P . aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100% . The other two patients were infected with mixed bacterial isolates including P . aeruginosa . However, diversity was observed in the P . aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65% . The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P . aeruginosa strains in bronchiectasis patients without cystic fibrosis.

J Nat Prod, 1996 Mar, 59(3), 293 - 6
Metabolites from an Antarctic sponge-associated bacterium, Pseudomonas aeruginosa; Jayatilake GS et al.; In an ongoing survey of the bioactive potential of microorganisms associated with marine invertebrates, the culture media of a sponge-associated bacterial strain of Pseudomonas aeruginosa was found to contain metabolites which inhibit the growth of several Gram-positive microorganisms . A series of diketopiperazines (1-6) including a new natural product (6) and two known phenazine alkaloid antibiotics (7 and 8) were isolated from the culture broth of this bacterium.

Zentralbl Bakteriol, 1996 Mar, 283(3), 322 - 7
Postantibiotic effect and virulence factors depression induced by ciprofloxacin and by aminoglycosides in a clinical isolate of Pseudomonas aeruginosa; Hostacka A; The postantibiotic effect (PAE) was induced in Pseudomonas aeruginosa by ciprofloxacin, tobramycin and netilmicin (2x or 4x MIC) . A longer PAE was evoked by a higher concentration of antibiotics . The evaluation of Pseudomonas aeruginosa virulence factors after treatment with suprainhibitory concentrations of the antibiotics tested showed that a higher suppression of elastase activity was associated with a longer PAE . Elastase was suppressed more effectively than proteinase . The highest reduction of elastase and proteinase activity (to 24.9% and 76.4% of the control values) was observed after treatment with ciprofloxacin.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 784 - 6
Alteration of postantibiotic effect during one dosing interval of tobramycin, simulated in an in vitro pharmacokinetic model; den Hollander JG et al.; The kinetics of the postantibiotic effect (PAE) during one dosing interval of tobramycin against Staphylococcus aureus and Pseudomonas aeruginosa was investigated . We determined the PAE at different time points during this dosing interval of 12 h in an in vitro pharmacokinetic model simulating human pharmacokinetics in which the half-life of tobramycin was adjusted to 2.4 +/- 0.2 h . Using an enzymatic method to inactivate tobramycin, we determined PAEs in samples extracted from the model at 1, 5, 8, and 12 h, corresponding with tobramycin concentrations of 20, 5, 2, and 1 times the MIC for the test organism . The PAE decreased significantly from 2.5 h at 1 h to 0 h at 12 h . No change in MIC was observed for the strains during the experiments . We conclude that the PAE decreases with decreasing tobramycin concentrations during a 12-h dosing interval and completely disappears after the concentration has reached the MIC for the test organism . On the basis of these observations, the emphasis that is placed on the PAE in discussions about the optimal dosing interval in aminoglycoside therapy is questionable.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 763 - 6
Cerebrospinal fluid ceftazidime kinetics in patients with external ventriculostomies; Nau R et al.; Ceftazidime has proven to be effective for the treatment of bacterial meningitis caused by multiresistant gram-negative bacteria . Since nosocomial central nervous system infections are often accompanied by only a minor dysfunction of the blood-cerebrospinal fluid (CSF) barrier, patients with noninflammatory occlusive hydrocephalus who had undergone external ventriculostomy were studied (n = 8) . Serum and CSF were drawn repeatedly after the administration of the first dose of ceftazidime (3 g over 30 min intravenously), and concentrations were determined by high-performance liquid chromatography by using UV detection . The concentrations of ceftazidime in CSF were maximal at 1 to 13 h (median, 5.5 h) after the end of the infusion and ranged from 0.73 to 2.80 mg/liter (median, 1.56 mg/liter) . The elimination half-lives were 3.13 to 18.1 h (median, 10.7 h) in CSF compared with 2.02 to 5.24 h (median, 3.74 h) in serum . The ratios of the areas under the concentration-time curves in CSF and serum (AUCCSF/AUCS) ranged from 0.027 to 0.123 (median, 0.054) . After the administration of a single dose of 3 g, the maximum concentrations of ceftazidime in CSF were approximately four times higher than those after the administration of 2-g intravenous doses of cefotaxime (median, 0.44 mg/liter) and ceftriaxone (median, 0.43 mg/liter) (R . Nau, H . W . Prange, P . Muth, G . Mahr, S . Menck, H . Kolenda, and F . Sorgel, Antimicrob . Agents Chemother . 37:1518-1524, 1993) . The median AUCCSF/AUCS ratio of ceftazidime was slightly below that of cefotaxime (0.12), but it was 1 order of magnitude above the median AUCCSF/AUCS of ceftriaxone (0.007) (Nau et al., Antimicrob . Agents Chemother . 37:1518-1524, 1993) . The concentrations of ceftazidime observed in CSF were above the MICs for most Pseudomonas aeruginosa strains . However, they are probably not high enough to be rapidly bactericidal . For this reason, the daily dose should be increased to 12 g in cases of P . aeruginosa infections of the central nervous system when the blood-CSF barrier is minimally impaired.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 739 - 42
Uptake and intracellular activity of NM394, a new quinolone, in human polymorphonuclear leukocytes; Ozaki M et al.; The uptake of NM394, a new quinolone, by and its subsequent elution from human polymorphonuclear leukocytes were studied and compared with those of ofloxacin and ciprofloxacin . The kinetics of the uptake of NM394 was similar to that of ciprofloxacin . The maximum intracellular-to-extracellular concentration ratio was 12.3, compared with 8.6 for ciprofloxacin and 4.9 for ofloxacin at the extracellular concentration of 20 micrograms/ml . The elution of NM394 from human polymorphonuclear leukocytes occurs relatively slowly; 5 min after the removal of extracellular NM394, nearly 100% still remained in polymorphonuclear leukocytes, compared with ofloxacin, which was so rapidly eluted that only 12% remained . The uptake of NM394 was significantly decreased at 4 degrees C and by the presence of NaCN but was not affected by the presence of L-glycine, L-leucine, L-serine, adenosine, or NaF . NM394 showed intracellular activity at a concentration of 0.1 microgram/ml that significantly reduced the number of phagocytosed Pseudomonas aeruginosa cells with 2 h of incubation . These results suggest that uptake of NM394 by human polymorphonuclear leukocytes occurs via an active transport system differing from that of ofloxacin, whose uptake is affected by the presence of L-glycine and L-leucine, and that once accumulated, NM394 remains intracellularly active and participates in protection against bacterial infection.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 691 - 5
Continuous infusion versus intermittent administration of ceftazidime in critically ill patients with suspected gram-negative infections; Benko AS et al.; The pharmacodynamics and pharmacokinetics of ceftazidime administered by continuous infusion and intermittent bolus over a 4-day period were compared . We conducted a prospective, randomized, crossover study of 12 critically ill patients with suspected gram-negative infections . The patients were randomized to receive ceftazidime either as a 2-g intravenous (i.v.) loading dose followed by a 3-g continuous infusion (CI) over 24 h or as 2 g i.v . every 8 h (q8h), each for 2 days . After 2 days, the patients were crossed over and received the opposite regimen . Each regimen also included tobramycin (4 to 7 mg/kg of body weight, given i.v . q24h) . Eighteen blood samples were drawn on study days 2 and 4 to evaluate the pharmacokinetics of ceftazidime and its pharmacodynamics against a clinical isolate of Pseudomonas aeruginosa (R288) . The patient demographics (means +/- standard deviations) were as follows: age, 57 +/- 12 years; sex, nine males and three females; APACHE II score, 15 +/- 3; diagnosis, 9 of 12 patients with pneumonia . The mean pharmacokinetic parameters for ceftazidime given as an intermittent bolus (IB) (means +/- standard deviations) were as follows: maximum concentration of drug in serum, 124.4 +/- 52.6 micrograms/ml; minimum concentration in serum, 25.0 +/- 17.5 micrograms/ml; elimination constant, 0.268 +/- 0.205 h-1; half-life, 3.48 +/- 1.61 h; and volume of distribution, 18.9 +/- 9.0 liters . The steady-state ceftazidime concentration for CI was 29.7 +/- 17.4 micrograms/ml, which was not significantly different from the targeted concentrations . The range of mean steady-state ceftazidime concentrations for the 12 patients was 10.6 to 62.4 micrograms/ml . Tobramycin peak concentrations ranged between 7 and 20 micrograms/ml . As expected, the area under the curve for the 2-g q8h regimen was larger than that for CI (P = 0.003) . For IB and CI, the times that the serum drug concentration was greater than the MIC were 92 and 100%, respectively, for each regimen against the P . aeruginosa clinical isolate . The 24-h bactericidal titers in serum, at which the tobramycin concentrations were < 1.0 microgram/ml in all patients, were the same for CI and IB (1:4) . In the presence of tobramycin, the area under the bactericidal titer-time curve (AUBC) was significantly greater for IB than CI (P = 0.001) . After tobramycin was removed from the serum, no significant difference existed between the AUBCs for CI and IB . We conclude that CI of ceftazidime utilizing one-half the IB daily dose was equivalent to the IB treatment as judged by pharmacodynamic analysis of critically ill patients with suspected gram-negative infections . No evaluation comparing the clinical efficacies of these two dosage regimens was performed.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 677 - 83
Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa; Cappelletty DM et al.; The purpose of this study was to determine if synergism was maintained for various combinations of beta-lactams with an aminoglycoside against four clinical strains and one laboratory strain of Pseudomonas aeruginosa which were resistant, according to the MICs, to the beta-lactams and/or aminoglycoside . The results from both the checkerboard and killing curve methodologies were compared . The laboratory strain (ATCC 27853) was manipulated in vitro by serial passage onto agar containing increasing concentrations of each antibiotic to select for resistance . One clinical isolate (R61) was also serially passed to raise the MIC of piperacillin from 128 to 1,024 micrograms/ml . The fractional inhibitory concentration indices for all isolates indicated indifference for all combination therapies, with values ranging from 0.6 to 3 . In contrast, killing curve results for all isolates demonstrated synergism with drug concentrations at either one-fourth or one-half the MIC for each organism . The MIC of piperacillin for the laboratory-manipulated clinical isolate R61 was 1,024 micrograms/ml, and synergism was still observed with concentrations of one-half the MIC of piperacillin and amikacin . For clinical isolate R166, which was beta-lactam and tobramycin resistant, synergism continued to be demonstrated with concentrations of tobramycin (1/16 MIC) in combination with piperacillin and cefepime at 1/2 the MIC . The results of this study indicate that against P . aeruginosa, synergism is observed in spite of resistance to beta-lactams and/or aminoglycosides . Synergism appears to be maintained even at very high MICs (piperacillin, 1,024 micrograms/ml; tobramycin, 128 micrograms/ml) with drug concentrations within achievable therapeutic ranges . With current definitions of synergism there was a complete lack of correlation between the results obtained by the checkerboard and killing curve methodologies, with the fractional inhibitory concentration indices showing indifference and killing curves resulting in synergism . The methodologies and definitions of synergism or antagonism are variable and not standardized and should be reevaluated.

Antimicrob Agents Chemother, 1996 Mar, 40(3), 665 - 9
Eradication of mucoid Pseudomonas aeruginosa with fluid liposome-encapsulated tobramycin in an animal model of chronic pulmonary infection; Beaulac C et al.; Despite controversies associated with forms and value of antibiotic therapy for cystic fibrosis patients, antibiotherapy remains a cornerstone in the management of those patients . Locally administered liposome-encapsulated antibiotics may offer advantages over free antibiotics, including sustained concentration of the antibiotic, minimal systemic absorption, reduced toxicity, and increased efficacy . We evaluated the efficacy of free and encapsulated tobramycin in fluid and rigid liposomal formulations administered to rats chronically infected with Pseudomonas aeruginosa . Chronic infection in lungs was established by intratracheal administration of 10(5) CFU of a mucoid variant of P . aeruginosa PA 508 prepared in agar beads . Antibiotic treatments were given intratracheally at time intervals of 16 h . After the last treatment, lung bacterial counts were determined and tobramycin levels in the lungs and kidneys were evaluated by high-performance liquid chromatographic analysis and microbiological assay . Two independent experiments showed that animals treated with encapsulated tobramycin in fluid liposomes had a number of CFU less than the minimal CFU number required to be statistically acceptable compared with > or = 10(6) CFU per pair of lungs for animals treated with encapsulated tobramycin in rigid liposomes, free antibiotic, or liposomes without tobramycin . Tobramycin measured in the lungs at 16 h after the last treatment following the administration of encapsulated antibiotic was still active, and its concentration was > or = 27 micrograms/mg of tissue . Low levels of tobramycin were detected in the kidneys (0.59 to 0.87 micrograms/mg of tissue) after the administration of encapsulated antibiotic, while 5.31 micrograms/mg of tissue was detected in the kidneys following the administration of free antibiotic . These results suggest that the local administration of fluid liposomes with encapsulated tobramycin could greatly improve the management of chronic pulmonary infection in cystic fibrosis patients.

Ann Pharmacother, 1996 Mar, 30(3), 246 - 8
Pseudomonas bacteremia precipitated by ticlopidine-induced neutropenia; Geletko SM et al.; OBJECTIVE: To report a case of ticlopidine-induced neutropenia resulting in Pseudomonas bacteremia . CASE SUMMARY: An 83-year-old white man developed febrile neutropenia 5 days after initiation of ticlopidine therapy . At presentation, the patient's white blood cell count was 1.1 x 10(9)/L with an absolute neutrophil count (ANC) of 0 . Ticlopidine was discontinued and the patient was treated empirically with ceftazidime, gentamicin, and filgrastim . The patient's blood cultures were positive for Pseudomonas aeruginosa . By day 6 of antibiotic and fllgrastim therapy, he was clinically improved and the ANC was 17 040 x 10(6) cells/L . The filgrastim and intravenous antibiotics were discontinued and oral ciprofloxacin was started . CONCLUSIONS: Ticlopidine-induced neutropenia can occur suddenly and may result in a serious infection, such as bacteremia.

An Esp Pediatr, 1996 Mar, 44(3), 239 - 41
{An increase in gamma-delta T-lymphocytes in the peripheral blood of cystic fibrosis patients}; Perez-Payarols J et al.; The function of the T gamma-delta cells of the human immune system is not well known at present . Only 3-10% of the T cells express the heterodimer composed of the gamma-delta chains . Recent studies have demonstrated a role of the T gamma-delta cells in the immunopathogenesis of autoimmune and infectious diseases . The present study was designed to evaluate the quantity of T gamma-delta cells in patients with cystic fibrosis with P . aeruginosa infections . These results were compared to blood levels of T cells found in patients with acute pulmonary infections, chronic pulmonary infections and healthy control patients . The cellular phenotype was determined by flow cytometry . Monoclonal antibodies against the different cell types studied were employed . The means of each group were compared by a Student's T test of Mann Whitney . We found that the percentage of T gamma-delta cells (TCR 1+) was significantly increased in patients with cystic fibrosis when compared to the pathological controls and healthy children . We conclude that our results demonstrate that children with cystic fibrosis infected with Pseudomonas aeruginosa demonstrate and increase in the subclass of T cells with the gamma-delta receptor.

C R Acad Sci III, 1996 Mar, 319(3), 153 - 60
{Alginates of Pseudomonas aeruginosa: a complex regulation of the pathway of biosynthesis}; Schmitt-Andrieu L et al.; Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections, especially in lungs of patients with cystic fibrosis . Environmental conditions induce the production by the bacteria of a viscous mucoid exopolysaccharide, called alginate, which is one of the most important factor of virulence of P . aeruginosa . Alginate is a linear polymer of beta-1, 4-linked L-guluronic acid and D-mannuronic acid . The alginate biosynthetic pathway involves genes called alg which are clustered at the 34 min region of chromosomal DNA of P . aeruginosa . The key enzyme of alginate biosynthesis, the GDP-mannose dehydrogenase is encoded by the gene algD . Expression of algD is positively controlled by several proteins, especially AlgU, a putative sigma factor homologous to sigma E of E . coli, AlgR and AlgP, a transactivator and an histone-like respectively . Here, a scheme of alginate biosynthetic pathway and a model for the alg genes regulation are described from results published in literature and from our own interpretation.

J Pediatr Orthop, 1996 Mar-Apr, 16(2), 224 - 30
Osteomyelitis of the calcaneus in children; Puffinbarger WR et al.; Eleven cases of calcaneal osteomyelitis in children are reported . Seven were hematogenous cases, and the remaining four were related to puncture wounds . The clinical presentation was less dramatic than that seen in typical long bone osteomyelitis . Laboratory findings were also less striking . A mixture of organisms was isolated from patients in the hematogenous group . In contrast, all puncture-related cases had cultures positive for Pseudomonas aeruginosa . Plain radiographic findings were noted at the time of presentation in 63% . Those findings were characteristically different in hematogenous and puncture-related cases . Oblique lateral radiographs can be important for diagnosis in puncture-related cases . Radionuclide bone scanning was an important diagnostic test in the absence of plain radiographic changes and in the very young patient . Surgery was performed in 82% of the cases . There were no recurrences or chronic infections . Two complications occurred in one patient, including residual scar sensitivity and early fusion of the calcaneal apophysis.

Virus Res, 1996 Mar, 41(1), 77 - 87
Plasmids containing cos ends inhibit the replication of phage phi CTX in Pseudomonas aeruginosa; Xiong G et al.; In bacteriophage phi CTX, the cohesive end sequences cos, the integrase gene int, the attachment site attP (the target site for int) and the gene ctx encoding a pore-forming cytotoxin CTX, are clustered . Phi CTX can infect some Pseudomonas aeruginosa strains with a subsequent induction of CTX expression . The 41 and 477 bp fragments containing cos ends of phi CTX DNA were cloned into the high copy number plasmid pHA10 . After pretransformation with the cos ends containing plasmids, plaque formation of phi CTX and cytotoxin production in phi CTX-infected Pseudomonas aeruginosa cells decreased by 100- and 10-fold respectively . Twelve hours after phi CTX infection proteins binding with cooperativity to cos sequence containing cleavable ends and the 10 bp flanking sequences were detected by gel electrophoretic mobility retardation of {32P}cos DNA . The results suggest that the cos binding proteins of phi CTX are involved in phi CTX replication.

Am J Otol, 1996 Mar, 17(2), 207 - 9
In vitro susceptibility of aural isolates of Pseudomonas aeruginosa to commonly used ototopical antibiotics; Dohar JE et al.; The choice of antimicrobial agents used to treat Pseudomonas aeruginosa infections of the ear is quite empiric . Yet in spite of this, very little has been published examining susceptibility patterns of aural isolates of P . aeruginosa . Recently, increasing concern has emerged over the development of resistance to many of the commonly used ototopical preparations with activity against P . aeruginosa . This concern stems from the fact that these preparations have been in use for a long time, and P . aeruginosa is known to develop resistance fairly readily . We prospectively studied the susceptibilities of aural isolates of P . aeruginosa in 231 consecutive children who were seen in the outpatient Pediatric Otolaryngology Department at Children's Hospital of Pittsburgh during the years 1992 and 1993 . The agents tested included neomycin, polymyxin B, colistin, and norfloxacin . We found that only 17.8% of the isolates were sensitive to neomycin, as opposed to > 95% for each of the other agents tested (polymyxin B, 99.6%; colistin, 97.4%; and norfloxacin, 98.3%) . This difference proved to be statistically significant (p < 0.05) . Given the concern of aminoglycoside-induced ototoxicity and the high rate of neomycin resistance, we believe that further investigation of other alternative ototopic agents with activity against P . aeruginosa is warranted.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Mar-Apr, (2), 7 - 9
{An immunological study of the cell wall proteins of Pseudomonas aeruginosa}; Makarenko TA et al.; A preparation containing P . aeruginosa cell-wall proteins with a mol . wt . of 9-55 kD has been obtained from P . aeruginosa by the method of extraction with the use of tris-EDTA buffer . In experiments on mice this protein preparation has shown pronounced protective properties, but, according to the data of Shwartzman's local reaction, proved to be toxic, perhaps due to the admixture of LPS . The purification of the protein preparation from the admixture of LPS will make it possible to obtain an effective vaccine against P . aeruginosa infection.

J Biomed Mater Res, 1996 Mar, 30(3), 403 - 10
Bacterial adhesion to orthopedic implant polymers; Barton AJ et al.; The degradable polymers poly(orthoester) (POE), poly(L-lactic acid) (PLA), and the nondegradable polymers polysulfone (PSF), polyethylene (PE), and poly(ether ether ketone) (PEEK) were exposed to cultures of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Escherichia coli . Bacteria washed and resuspended in phosphate buffered saline (PBS) adhered to polymers in amounts nearly twice those of bacteria that were left in their growth medium, tryptic soy broth (TSB) . In TSB, there was variation in adhesion from species to species, but no significant variation from polymer to polymer within one species . In PBS there were significant differences in the amounts of bacteria adhering to the various polymers with the exception, of S . epidermidis, which had similar adhesion to all polymers . As a whole, P . aeruginosa was the most adherent while S . epidermidis was the least adherent . The estimated values of the free energy of adhesion (delta Fadh) correlated with the amount of adherent P . aeruginosa . When POE, PLA, and PSF were exposed to hyaluronic acid (HA) before exposure to the bacteria, there was 50% more adhesion of E . coli and P . aeruginosa on POE and PLA . With respect to bacterial adhesion, the biodegradable polymers (POE and PLA) in general were not significantly different from the nondegradable polymers.

J Orthop Res, 1996 Mar, 14(2), 251 - 4
Removal of surface bacteria by irrigation; Anglen J et al.; We examined the efficacy of various irrigation solutions delivered through a power irrigator to remove bacteria from three different surfaces . Titanium, stainless-steel, and cortical bone surfaces were coated with three different bacterial species: Staphylococcus aureus, Pseudomonas aeruginosa, and Staphylococcus epidermidis . They were then irrigated with 1 L of fluid delivered by jet lavage . The fluids tested were normal saline and solutions of bacitracin, neomycin, and soap . One set of specimens was not irrigated, as a control . After irrigation, the specimens were sonicated to remove residual bacteria, and the sonicate was quantitatively cultured to allow evaluation of the amount of residual bacteria on the surface . The results showed that removal of bacteria reflects an interaction between bacterial species, surface characteristics, and irrigation solution . Fewer bacteria were present in all the irrigation groups than in the control . Soap solution was as good as or better than any other solution at removing all three types of bacteria from all three surfaces, although not all of the pairwise comparisons were statistically significant . There was a significant advantage to soap solution over antibiotic irrigant or saline alone in removing Staphylococcus epidermidis from metallic surfaces . The use of soap solution for irrigation seems to improve the removal of some bacteria from some surfaces in this experimental model and may represent a better type of irrigation additive.

J Med Microbiol, 1996 Mar, 44(3), 195 - 8
Assessment of clinical significance of positive blood cultures of relatively low-virulence isolates; Hirakata Y et al.; In Omori Hospital, Toho University School of Medicine, relatively low-virulence blood isolates, including coagulase-negative staphylococci (CNS), enterococci and nonfermentative gram-negative rods other than Pseudomonas aeruginosa comprised c . 60% of total blood isolates . A retrospective study was conducted to assess their clinical significance by reviewing a total of 91 hospital charts . The physicians' assessments of these positive blood cultures as recorded in the charts were classified into four categories--sepsis, possible sepsis, contamination and no comment . The episodes classified as sepsis accounted for 5.0-19.6% . These episodes were also evaluated by a graded clinical significance score based on multiple factors, including number of positive cultures and clinical signs . The scores for the 91 episodes covered a wide range from 1 to 9, indicating that both contaminants and causative organisms may have been involved . The episodes judged as sepsis or possible sepsis tended to have higher scores . The scores for the episodes associated with enterococci were also higher than those involving CNS or non-fermentative gram-negative rods . The scores for episodes associated with intravenous hyperalimentation catheters were higher than those not associated with the catheters.

J Bacteriol, 1996 Mar, 178(5), 1457 - 64
Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required; Sia EA et al.; Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient . Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer . Two distinct models have been generally considered for the mechanism of retrotransfer . In the two-way conduction model, no transfer of the conjugative plasmid is required . The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions . In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor . We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer . Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E . K.Ayres, V . J . Thomson, G . Merino, D . Balderes, and D . H . Figurski, J . Mol . Biol . 230:174-185, 1993) . The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type . Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer . Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient . The results prove that retrotransfer occurs by two sequential DNA transfer events.

J Bacteriol, 1996 Mar, 178(5), 1412 - 9
Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa; Yahr TL et al.; Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form . The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein . To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P . aeruginosa chromosomal digests . Cross-hybridizing bands were detected in chromosomal digests of a strain of P . aeruginosa in which exoS had been deleted by allelic exchange . A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS . Thirteen clones that cross-hybridized with the exoS probe were identified . One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS . The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S . A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS . Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS . These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes . In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.

Invest Ophthalmol Vis Sci, 1996 Mar, 37(4), 534 - 43
Pseudomonas keratitis . The role of an uncharacterized exoprotein, protease IV, in corneal virulence; O'Callaghan RJ et al.; PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV . METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent . Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria . RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate . Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02) . CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface . Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

Invest Ophthalmol Vis Sci, 1996 Mar, 37(4), 511 - 22
Effect of vitamin A deficiency on the early response to experimental Pseudomonas keratitis; Twining SS et al.; PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals . This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats . METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats . Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R) . Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P . aeruginosa organisms were applied to the corneal surface . The rats were killed using an overdose of sodium pentobarbital . Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination . Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea . Zymography was used to study caseinases, gelatinases, and plasminogen activators . Reverse zymography was used to detect proteinase inhibitors . Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency . RESULTS: Ulceration occurred within 24 hours when low numbers of P . aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas . When higher numbers of P . aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours . The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two . Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes . The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions . CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P . aeruginosa infection during the first 24 hours . The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.

Carbohydr Res, 1996 Feb 28, 282(1), 81 - 99
Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates; Spiers ID et al.; The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described . The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite . The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported . myo-Inositol 1,2,3-trisphosphate, (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO . production in the iron-catalysed Haber-Weiss reaction . The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition, also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO . production . myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Biochemistry, 1996 Feb 27, 35(8), 2754 - 8
Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S; Liu S et al.; Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs) . Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381 . The specific activity of E381A mutant was 0.02% of wild-type delta N222 . Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity . In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222 . Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI) . The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222 . In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation . Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S.

FEBS Lett, 1996 Feb 26, 381(1-2), 140 - 2
The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa; Gorren AC et al.; The electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron acceptors in the conversion of 4-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH) . The reactivity of H117G-azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand . In the absence of imidazoles, H117G-azurin reacted directly with 4-ethylphenol; this reaction was abolished in the presence of imidazoles . The enzymatic reduction of H117G-azurin by 4-EPMH was 40 times slower than that of wild-type azurin . The rate of this reaction was enhanced by some imidazoles, diminished by others . In all cases the reduction of H117G-azurin was irreversible . These results demonstrate that His117 is vital for electron transfer and effectively protects the copper site against aspecific reactions.

Biochemistry, 1996 Feb 20, 35(7), 2429 - 36
Environment of copper in Pseudomonas aeruginosa azurin probed by binding of exogenous ligands to Met121X (X = Gly, Ala, Val, Leu, or Asp) mutants; Bonander N et al.; The binding of small exogenous ligands to mutants of the blue copper protein azurin from Pseudomonas aeruginosa, altered in the axial position, Met121X (X = Gly, Ala, Val, Leu, or Asp), has been studied with optical and electron paramagnetic resonance (EPR) spectroscopy . The results show that small molecules can enter the pocket left by the side chain of Met121 . For azide, the dissociation constants are Leu > Val > Ala, reflecting the increasing space available . The Gly and Asp mutants bind azide less strongly than the Ala mutant, due to competition with water (Gly) and the polar side chain (Asp) . Similar trends are found for thiocyanate . Cyanide binds equally well to the Ala and Val mutants . A number of other small potential ligands were tried . Alcohols do not affect room-temperature optical spectra, but at low temperatures, the EPR spectrum is stellacyanin-like, indicative of a weak axial interaction . Ligands binding with a carboxyl group or nitrogen (e.g . acetate or azide) convert the metal center to a form intermediate between regular types 1 and 2, presumably by pulling the copper ion out of the trigonal plane formed by Cys(S) and two His(N) . Cyanide interacts strongly as shown by the hyperfine coupling to the 13C nucleus . With increasing strength of the axial interaction, the two major bands in the visible region (600 and 400-500 nm) shift in parallel to higher energy, and at the same time, the strength of the latter transition increases at the expense of the former . This demonstrates that these transitions have a common origin, namely S-to-Cu charge transfer transition.

Eur J Biochem, 1996 Feb 15, 236(1), 283 - 93
Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa . Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity; Nguyen VT et al.; Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase) . Despite extensive sequence similarities, these enzymes function unidirectionally in vivo . In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity . When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed . This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly . To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E . coli argF gene to the P . aeruginosa arcB gene . A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity . Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E . coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity . Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly . Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity . Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly . These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions . Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154 . Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Experientia, 1996 Feb 15, 52(2), 167 - 74
Bactericidal activity against Pseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase; Mathy-Hartert M et al.; Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producing cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules . Pseudomonas aeruginosa (10(6) bacteria per 1ml) are killed within 1 h in vitro by a MPO/H2O2/C1- system (48mU=132ng of MPO) . The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme . Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages . These cells lost endogenous MPO within five days while H2O2 production in response to stimulation by phorbol myristate acetate (10(-6)M) decreased to 23% within ten days . On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten . Human macrophages cultured from eight days (when both H2O2 production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity against Pseudomonas aeruginosa . After a 1 h MPO loading period, macrophages (5X10(5) cells per ml) were incubated in the presence of bacteria (0.5 to 2X10(6) bacteria per ml) for 2 h at 37 degrees C . At a bacteria/macrophage ratio of 1, only 34.8+/-7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4+/-9.3% survived at a ratio of 4 . From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.

Biochemistry, 1996 Feb 6, 35(5), 1397 - 407
Spectroscopic and mechanistic studies of type-1 and type-2 copper sites in Pseudomonas aeruginosa azurin as obtained by addition of external ligands to mutant His46Gly; van Pouderoyen G et al.; The spectroscopic and mechanistic properties of the Cu-containing active site of azurin from Pseudomonas aeruginosa were investigated by the construction of a mutant in which one of the ligands of the metal, His46, was replaced by a glycine . Although the mutation creates a hole in the interior of the protein, the 3D structure of the protein does not change to any appreciable extent . However, the spectroscopic (optical, resonance Raman, EPR) properties of the mutant protein are strongly affected by the mutation . In the presence of external ligands, the properties of the original wild-type protein are restored to a smaller or larger extent, depending on the ligand . It is concluded that the hole created by the mutation, even though it is completely buried inside the protein, can be filled by external ligands, often resulting in the creation of a mixture of so-called type-1 and type-2 copper sites . Also, the redox properties (midpoint potential, kinetics of reduction/oxidation) appeared to be strongly affected by the mutation and the presence of external ligands . The results are compared with previous results obtained on the mutant His117Gly.

Acta Paediatr Jpn, 1996 Feb, 38(1), 12 - 6
Bacterial changes in neonatal intensive care unit; Endo A et al.; Organisms routinely cultured from throat swabs and infectious agents of sepsis and/or meningitis were reviewed . During the last 12 years, Klebsiella pneumoniae and Escherichia coli have been replaced by Staphylococcus aureus and Pseudomonas aeruginosa as the predominant isolates from throat swabs after admission . These change in the etiologic pattern of infectious agents of sepsis and/or meningitis, i.e., K . pneumoniae, E . coli, S . aureus, P . aeruginosa and staphylococcus epidermidis, were in agreement with the organisms isolated from the throat swabs after admission . The S . aureus isolated from throat swabs after admission showed a decrease in the bacterial activity of cloxacillin, cephazolin and cefotaxime since 1978.

Microbiology, 1996 Feb, 142 ( Pt 2), 299 - 307
The influence of A-band and B-band lipopolysaccharide on the surface characteristics and adhesion of Pseudomonas aeruginosa to surfaces; Makin SA et al.; Pseudomonas aeruginosa PAO1 possesses two distinct lipopolysaccharide (LPS) O-polysaccharide species, A- and B-band LPS, the relative expression of which appears to be under environmental control . In an attempt to identify the influence these LPS types have on surface characteristics and adhesion, we examined the surface hydrophobicity and surface charge of P . aeruginosa PAO1 (O5 serotype) and its isogenic LPS derivatives which possessed A+B-, A-B+ and A-B- LPS . The surface characteristics of the strains affected their ability to adhere to hydrophilic (glass) and hydrophobic (polystyrene) surfaces . Cells possessing only A-band LPS demonstrated the highest surface hydrophobicity, followed by the strain lacking both A- and B-band LPS . The presence of B-band LPS resulted in a more hydrophilic surface . Strains lacking B-band LPS (A+B- and A-B-) had more electronegative surfaces than those possessing B-band LPS (A+B+ and A-B+), with cells lacking both A- and B-band LPS showing the highest surface electronegativity . These data suggest that the main surface-charge-determining groups reside in the core region of the LPS molecule . Cells with the lowest surface hydrophobicity and lowest surface charge (A+B+, A-B+) adhered to glass the most efficiently, implying a role for electrostatic interaction, whereas adhesion to polystyrene mirrored the relative hydrophobicities of the strains (A+B- > A-B- > A+B+ > A-B+) . It is postulated that phenotypic variation in the relative expression of A- and B-band LPS may be a mechanism by which P . aeruginosa can alter its overall surface characteristics in such a way as to influence adhesion and favour survival.

Microbiology, 1996 Feb, 142 ( Pt 2), 289 - 97
Novel O-polysaccharide expression, as a lipid A-core-free form, in a lipopolysaccharide-core-defective mutant of Pseudomonas aeruginosa; Yokota S; Pseudomonas aeruginosa PML14e is a mutant strain, isolated from strain PML14 (Homma serotype I), that is resistant to all types of R-pyocins . PML14e completely lacked glucose and rhamnose as components of the lipopolysaccharide (LPS) outer core region . Whereas the O-polysaccharide attachment site on the LPS core was considered to be absent, PML14e was agglutinable with anti-serotype-I antibodies . The O-polysaccharide of PML14e was recovered in the supernatant after ultracentrifugation of the aqueous layer from a hot phenol/water extraction . Chromatographic behaviour and chemical analysis indicated that the PML14e O-polysaccharide was not linked to the lipid A . 1H-NMR spectroscopy indicated that the structure of the PML14e O-polysaccharide was the same as that of the O-polysaccharide from PML14 . The above evidence indicated that the O-polysaccharide is expressed on the cell surface of the mutant strain PML14e as the lipid A-free form . To examine the nature of the cell surface, the accessibility of monoclonal antibodies (mAbs) against cell surface antigens was tested by enzyme-linked immunosorbent assay . An anti-lipid A mAb and an anti-outer-membrane protein mAb, the epitopes for which are considered to be exposed on rough strains, bound to a greater extent to the PML14e cells than to two other LPS-core-defective rough mutants, PML14b and PML14d . Whereas these mutants appeared to have lesser defects in the LPS core, they expressed less O-polysaccharide than PML14e . The results indicated that the epitopes exposed on rough strains, such as lipid A and outer-membrane proteins, were mainly hindered by covalently linked core oligosaccharide rather than by the O-polysaccharide chain.

FEMS Microbiol Lett, 1996 Feb 1, 136(1), 85 - 90
Specific interaction of the protein-D2 porin of Pseudomonas aeruginosa with antibiotics; Ishii J et al.; The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics . We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel . Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.

Kansenshogaku Zasshi, 1996 Feb, 70(2), 123 - 31
{Development of multi-drug resistant mutants of clinical isolates of Pseudomonas aeruginosa after exposure to fluoroquinolone}; Hasegawa M et al.; Eight multi-drug resistant mutants (4.94%) were found in 162 clinical isolates of P . aeruginosa after exposure to norfloxacin at different concentrations (1/4, 1/2, 1, 2 and 4 MICs) and were investigated for the mechanisms of drug resistance . All the mutants were eight-times more resistant to norfloxacin than the respective parents, and showed the cross-resistance to the other fluoroquinolones . However, these mutants differed in the drug-resistant patterns; the 94-74 mutant was resistant to carbenicillin, ceftazidime and chloramphenicol, TA-15 mutant was resistant to imipenem, and the 93-183 was resistant to carbenicillin, ceftazidime and gentamicin . TA-16 mutant only showed a marked decrease in the bacterial uptake of norfloxacin . Profiles of the outer membrane proteins of the mutants were analyzed by SDS-PAGE method . The six mutants, except for TA-52 and 93-183 mutants, increased the intensity of bands in the 46 KD region . Three mutants (TA-15, TA-16 and 93-183) decreased the intensity of 44 KD (OMPE) and also the former two decreased the intensity of 22 KD (OMP G) . The gryA mutations associated with fluoroquinolone-resistance were investigated for the eight mutants, and the 93-183 mutant showed to have the gyrA mutation caused by the alteration in the amino acid sequence of gyrA; Thr-83 (ACC) to Ile (ATC) . The result of the present study indicate that multi-drug resistance of clinical isolates of P . aeruginosa develops through different types of mechanisms, and is not easily explained fully by the present results, and suggest that many factors attributed to the development of the resistances in those mutants remains to be clarified.

Kansenshogaku Zasshi, 1996 Feb, 70(2), 116 - 22
Sepsis associated with hematological malignancies: prophylaxis of Pseudomonas aeruginosa sepsis; Sakamoto M et al.; Underlying diseases, pathogenic bacteria, clinical background and outcome were studied during 91 febrile episodes complicated by sepsis in 55 patients with hematological malignancies, who had been admitted to our hospital (Jikei University Kashiwa Hospital) between January 1990 and December 1994 . Particularly in patients with P . aeruginosa sepsis, we compared the prophylactic effect of ciprofloxacin (CPFX) alone with that of the combination of polymyxin B (PL-B) plus kanamycin (KM) . The major underlying diseases were acute myelocytic leukemia and malignant lymphoma, followed by myelodysplastic syndrome, acute lymphocytic leukemia and chronic myelocytic leukemia . Nearly two-thirds of the pathogenic microorganisms isolated were gram-positive bacteria (including coagulase-negative staphylococci and Staphylococcus aureus); approximately one-quarter were gram-negative bacteria (such as Pseudomonas aeruginosa), and the remainder were fungi . These microorganisms usually induced sepsis when granulocyte counts were decreased . Sepsis was a direct cause of death in about 60% of the patients and P . aeruginosa sepsis had the worst outcome . Oral administration of CPFX was more effective than PL-B plus KM in preventing P . aeruginosa sepsis . The difference in effectiveness might depend on the absorption profile of the drugs.

Kansenshogaku Zasshi, 1996 Feb, 70(2), 108 - 15
{Septicemia associated with hematopoietic disorders and its features according to respective primary disorders}; Yokota T et al.; Two hundred eighty-seven episodes of septicemia which occurred in patients with hematological disorders between 1980 and 1993 were examined according to respective underlying diseases . The diagnosis of acute myelogenous leukemia (AML) was made in 155 patients, acute lymphocytic leukemia (ALL) in 45, chronic myelogenous leukemia (CML) in 29, malignant lymphoma in 36, adult T-cell leukemia (ATL) in 7, multiple myeloma (MM) in 8 and aplastic anemia (AA) in 7 . Three hundred and two strains were isolated from 287 patients . Fifty two point three percent of the total isolates were gram-negative bacilli, 26.8% were gram-positive cocci, 17.2% were fungi and 3.6% were anaerobic bacteria . In ALL patients gram-positive cocci accounted for 42.0% . This rate was significantly higher than in other disorder . Additionally, oral mucositis or gingivitis was evaluated as clinical background in 36.1% of ALL cases . Forty-seven point two percent of organisms which caused septicemia in ALL patients were isolated from surveillance cultures of the throat just before the onset of septicemia . These data suggested that in ALL cases microbiological organisms more frequently invaded through injuries of oral mucosa . In ATL, CML, MM and AA patients, fungi accounted for more than 25% of causative organisms . The most common organism of all of the strains was Pseudomonas aeruginosa (21.9%), but in ATL and MM patients Escherichia coli was more common than P . aeruginosa . At the onset of the septicemia, neutrophil counts were less than 100/mm3 in 76.6% of all patients, and more than 3,000/mm3 in only 5.0% . In contrast to this result, in 66.7% of ATL patients and 37.5% of MM patients, septicemia occurred even when neutrophil counts were more than 3,000/mm3 . Septicemia occurred in 28.2% of the total patients but died . The mortality rate in MM and AA patients (50.0% respectively) was higher than in other diseases . According to the mortality of each causative organisms, fungal septicemia had a terribly high mortality of 82.9% while other bacterial mortality was about 20%.

J Chemother, 1996 Feb, 8(1), 43 - 6
En bloc transduction of imipenem resistance with other resistance determinants from a clinical strain of Pseudomonas aeruginosa; Babalova M et al.; In this report we describe a lysogenic strain of Pseudomonas aeruginosa No . 229, from Frankfurt University Clinics, resistant to imipenem (IMI), cefotaxime (CTX), kanamycin (KAN), carbenicillin (CAR) as well as to other non-beta-lactam drugs, from which a wild-type phage could be isolated and used for transduction of an imipenem resistance determinant . All IMI-selected transductants were found to be co-resistant also to CTX, KAN and CAR . Conversely, transductants selected with CTX, KAN or CAR were also all resistant to IMI . It seems probable that IMI is hydrolyzed by the original donor strain as well as by transductant colonies by means of a clavulanate-inhibited beta-lactamase and not by a metallo-beta-lactamase.

J Chemother, 1996 Feb, 8(1), 25 - 32
Performance of the fractional maximal effect method: comparative interaction studies of ciprofloxacin and protein synthesis inhibitors; Li RC et al.; The performance of the recently developed Fractional Maximal Effect (FME) method was evaluated along with the conventional checkerboard technique and time-kill method . Ciprofloxacin in combination with tobramycin was tested against Escherichia coli and Pseudomonas aeruginosa and in combination with tetracycline, chloramphenicol, erythromycin against Escherichia coli . Two combinations, amoxicillin-tetracycline (antagonistic) and tobramycin-ticarcillin (synergistic), were included as reference interactions . The FME method unequivocally showed an antagonistic interaction between ciprofloxacin and all the protein synthesis inhibitors (PSIs) tested, while the other two methods yielded variable results . At a total FME (TFME) level of 1, the FME method demonstrated a similar degree of antagonism against ciprofloxacin by tetracycline, chloramphenicol, and erythromycin, and much lower by tobramycin . Internal consistency of the FME operation was demonstrated by the identical conclusions obtained at both TFME levels of 0.5 and 1 . The FME method appears to be a practical alternative for resolving the inconsistencies observed in conventional methods of antibiotic combination testing.

Antimicrob Agents Chemother, 1996 Feb, 40(2), 349 - 53
Multifocal outbreaks of metallo-beta-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including carbapenems; Senda K et al.; A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994 . Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (blaIMP)-specific probe . Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas . Three strains of P . aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between . These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems . In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P . aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid . Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital . These findings suggest that the metallo-beta-lactamase-producing P . aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds . Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.

An Esp Pediatr, 1996 Feb, 44(2), 109 - 11
{An alpha 1-antitrypsin aerosol in the treatment of cystic fibrosis}; Ferrer Calvete J et al.; Alpha-1-antitrypsin (a-1AT) is a natural inhibitor of the elastase that is released physiologically by neutrophils in the lung . As a result of the increased neutrophil degranulation secondary to chronic epithelial inflammation in cystic fibrosis patients with chronic infections by Pseudomonas aeruginosa, there are larger amounts of elastase in airway secretions . This results in the a-1AT concentration being insufficient to inhibit the destructive proteolytic degradation, culminating in a chronic epithelial burden and a worsening of the cystic fibrosis pulmonary disease . In this preliminary study, we have evaluated the results obtained from the sputum of 4 cystic fibrosis patients treated with a-1AT (Prolastina, Bayer) in aerosol . The levels of a-1AT, neutrophil elastase, antineutrophil elastase activity, IgG, albumin and clinical parameters were measured . The concentration of sputum a-1AT was increased when compared to the same patient after 8 days with treatment (we compared means with Student's t-test and p < 0.05 was considered significant) . We did the same with the impairment of neutrophil elastase, although we found no significant results . Nevertheless, antineutrophil elastase activity increased (p < 0.05) . These results encourage us to continue the same treatment for a longer period of time to prevent pulmonary disease in CF subjects.

Mol Microbiol, 1996 Feb, 19(4), 857 - 69
Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion; Pepe CM et al.; Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin . These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria . In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene . PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin . An A . hydrophila genomic library was transferred into a P . aeruginosa pilD mutant that is defective for type IV pilus biogenesis . The A . hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P . aeruginosa . Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4 . Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P . aeruginosa type IV pilus biogenesis genes (pilABCD) . We showed that TapB and TapC are functionally homologous to P . aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili . In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P . aeruginosa prepilin as substrate . Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A . hydrophila.

Proteins, 1996 Feb, 24(2), 178 - 94
Comparison of low oxidoreduction potential cytochrome c553 from Desulfovibrio vulgaris with the class I cytochrome c family; Blackledge MJ et al.; The cytochrome c553 from Desulfovibrio vulgaris (DvH c553) is of importance in the understanding of the relationship of structure and function of cytochrome c due to its lack of sequence homology with other cytochromes, and its abnormally low oxido-reduction potential . In evolutionary terms, this protein also represents an important reference point for the understanding of both bacterial and mitochondrial cytochromes c . Using the recently determined nuclear magnetic resonance (NMR) structure of the reduced protein we compare the structural, dynamic, and functional characteristics of DvH c553 with members of both the mitochondrial and bacterial cytochromes c to characterize the protein in the context of the cytochrome c family, and to understand better the control of oxide-reduction potential in electron transfer proteins . Despite the low sequence homology, striking structural similarities between this protein and representatives of both eukaryotic {cytochrome c from tuna (tuna c)} and prokaryotic {Pseudomonas aeruginosa c551 (Psa c551)} cytochromes c have been recognized . The previously observed helical core is also found in the DvH c553 . The structural framework and hydrogen bonding network of the DvH c553 is most similar to that of the tuna c, with the exception of an insertion loop of 24 residues closing the heme pocket and protecting the propionates, which is absent in the DvH c553 . In contrast, the Psa c551 protects the propionates from the solvent principally by extending the methionine ligand arm . The electrostatic distribution at the recognized encounter surface around the heme in the mitochondrial cytochrome is reproduced in the DvH c553, and corresponding hydrogen bonding networks, particularly in the vicinity of the heme cleft, exist in both molecules . Thus, although the cytochrome DvH c553 exhibits higher primary sequence homology to other bacterial cytochromes c, the structural and physical homology is significantly greater with respect to the mitochondrial cytochrome c . The major structural and functional difference is the absence of solvent protection for the heme, differentiating this cytochrome from both reference cytochromes, which have evolved different mechanisms to cover the propionates . This suggests that the abnormal redox potential of the DvH c553 is linked to the raised accessibility of the heme and supports the theory that redox potential in cytochromes is controlled by heme propionate solvent accessibility.

Am J Physiol, 1996 Feb, 270(2 Pt 1), L232 - 41
Exotoxin A stimulates fluid reabsorption from distal airspaces of lung in anesthetized rats; Pittet JF et al.; To determine whether exotoxin A may affect the transport of fluid across the lung epithelium, two isogenic strains of Pseudomonas aeruginosa PA103 (10(8) colony-forming units), one (PA103 tox omega) with a structural gene mutation in exotoxin A, were instilled into the distal airspaces of anesthetized rats . PA103 parental strain, but not its mutant, stimulated the removal of fluid from the distal airspaces of the lung . Instillation of exotoxin A alone caused a dose-dependent increase in the fluid transport across the lung epithelium . Instillation of amiloride (10(-3) M) with exotoxin A demonstrated that this effect partially depended on increased uptake of sodium across the lung epithelium . The absence of stimulation after instillation of an exotoxin A mutant (PE delta Glu553) without ADP-ribosyltransferase activity demonstrated that the effect of exotoxin A depended on its ADP-ribosyltransferase activity . Finally, the instillation of exotoxin A in rats depleted of macrophages indicated that the effect of exotoxin A was not secondary to the activation of alveolar macrophages by this toxin . In conclusion, these results indicate that the in vivo release of exotoxin A by live airspace P . aeruginosa directly stimulates the fluid removal from the airspaces by the lung epithelium . This may alter the volume or composition of airway secretions, and may contribute to the lung disease in patients infected with P . aeruginosa.

Pathol Biol (Paris), 1996 Feb, 44(2), 125 - 31
Usefulness of a computerized expert system associated with systematic O-serotyping for the early detection of outbreaks of hospital acquired infections and for the presumptive antibiotic therapy of Pseudomonas aeruginosa infections; Watine J et al.; Over a four-year period, the systematic O-serotyping of all Pseudomonas aeruginosa isolated in Hopital de Rodez associated with the use of a computerized expert system, facilitated the early detection of two outbreaks of nosocomial infections with multiresistant serotype O:11 and multiresistant serotype O:12 P . aeruginosa respectively involving ten patients over 16 months and six patients over six months . Over this four-year period, serotype O:12 represented 14% of 404 clinical isolates of P . aeruginosa, and most isolates of this serotype were resistant to multiple antibiotics . Combination experiments showed that fosfomycin/amikacin together were active against 86% of O:12 isolates . Fosfomycin/amikacin might be considered as a therapeutic alternative to ceftazime/amikacin for the presumptive antipseudomonal therapy of serotype O:12 infections.

J Chemother, 1996 Feb, 8 Suppl 2, 48 - 56
Newer antipseudomonal cephalosporins; Watanabe NA; The in vitro antipseudomonal activity of eight cephalosporins with alpha-oxyimino-aminothiazolyl or aminothiadiazolyl group in the 7 beta-side chain and the quaternary ammonium moiety in the 3-position was evaluated . The order of antipseudomonal potency of these cephalosporins was as follows: cefaclidine > or = FK-518 > cefluprenam = cefozopran > or = ceftazidime > or = cefepime > cefpirome > or = cefoselis . Pseudomonas aeruginosa strains derepressed for the chromosomally mediated beta-lactamase (cephalosporinase) production were relatively susceptible to cefaclidine and FK-518, but were resistant to six other cephalosporins . P . aeruginosa strains producing the OXA-1-like penicillinase were resistant to cefaclidine, cefepime, and cefpirome, whose frequency was still lower . The higher antipseudomonal activity of these compounds than that of cefotaxime and some differences in activity among these compounds were suggested to result from cephalosporinase stability due to their low affinity for enzyme coupled with resistance to enzyme hydrolysis, assuming that the outer membrane permeation rates of compounds and their affinities for essential penicillin-binding proteins were almost the same . The aminothiadiazolyl cephalosporins showed higher antipseudomonal activity than the corresponding aminothiazolyl counterparts . This was also suggested to result from the same hypothesis as described above.

J Antimicrob Chemother, 1996 Feb, 37(2), 295 - 301
An 8 year Microbe Base survey of the epidemiology, frequency and antibiotic susceptibility of Pseudomonas aeruginosa hospital isolates in the United Kingdom; Spencer RC; Over the last 8 years selected United Kingdom hospitals have contributed antimicrobial susceptibility data to a central data base called Microbe Base . During that period, data on 1,000,067 isolates have been collected, including 29,425 isolates of Pseudomonas aeruginosa . The present study focused on the epidemiology, frequency and antibiotic susceptibility of P . aeruginosa isolates (17,440) from hospitalised patients . In such patients P . aeruginosa was predominant in general surgery (20%), care of the elderly (18%), general medicine (13%), and paediatrics and neonates (10%) . Ninety-five per cent of P . aeruginosa were susceptible in vitro to ceftazidime compared with 93% for piperacillin, 92% for gentamicin . 90% for ciprofloxacin, and 89% for imipenem . Excluding susceptibility data reported in 1991, there was no change in the susceptibility pattern of P . aeruginosa throughout the study period.

J Korean Med Sci, 1996 Feb, 11(1), 64 - 7
Ecthyma gangrenosum associated with aplastic anemia; Chun WH et al.; Ecthyma gangrenosum is a characteristic skin lesion of systemic infection due to Pseudomonas aeruginosa . It has a high incidence in patients with chronic disease and impaired defense mechanisms . Early diagnosis and appropriate systemic antibiotic therapy is crucial since its mortality rate is very high . We report a case of ecthyma gangrenosum in aplastic anemia.

J Hosp Infect, 1996 Feb, 32(2), 135 - 45
Discriminatory power and usefulness of pulsed-field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa; Talon D et al.; We assessed the discriminatory power of pulsed-field gel electrophoresis (PFGE) for the analysis of DNA restriction fragment length polymorphism (RFLP) in Pseudomonas aeruginosa . We determined DraI PFGE-RFLP of DNA of unrelated clinical and environmental strains, and clinical strains isolated from two intensive care units of the Besancon University Hospital . The typeability and reproducibility was 100% . The discriminatory index was 0.998, and the DNA patterns were stable in vitro and in vivo . There was a very low correlation between PFGE-RFLP and traditional typing methods . The typeability, reproducibility, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping . Further standardization and quantitative interpretation are possible and should lead to this technique becoming a library typing system.

Prenat Diagn, 1996 Feb, 16(2), 180 - 2
Blood contamination of amniotic fluid after amniocentesis in relation to placental location; Giorlandino C et al.; The aim of the present study was to evaluate blood contamination of the amniotic fluid collected in 20 patients undergoing a second amniocentesis performed 2 weeks after a first procedure that had failed due to Pseudomonas aeruginosa contamination of the cell cultures . Red blood cell and haemoglobin concentrations in the amniotic fluid were significantly higher in patients who had undergone a transplacental procedure compared with patients in whom the placenta was not traversed with the needle . For both groups, blood contamination of the amniotic fluid was significantly higher compared with a control group of 20 patients undergoing amniocentesis for the first time . Significant blood contamination of the amniotic fluid after amniocentesis occurs in every instance if evaluated at a "second-look' procedure; the blood contamination is higher when an anterior placenta is traversed with the needle . The clinical significance of these findings needs to be further evaluated.

J Photochem Photobiol B, 1996 Feb, 32(3), 159 - 64
Photoinactivation of bacteria . Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both gram-negative and gram-positive bacteria; Minnock A et al.; The photosensitization of microorganisms is potentially useful for sterilization and for the treatment of certain bacterial diseases . Until now, any broad spectrum approach has been inhibited because, although Gram-positive bacteria can be photoinactivated by a range of photosensitizers, Gram-negative bacteria have not usually been susceptible to photosensitized destruction . In the present work, it has been shown that the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Enterococcus seriolicida, can be photoinactivated when illuminated in the presence of a cationic water-soluble zinc pyridinium phthalocyanine (PPC) . The degree of photoinactivation is dependent on both the concentration of PPC and the illumination time . In contrast, the three bacteria are not photoinactivated by illumination in the presence of a neutral tetra-diethanolamine phthalocyanine (TDEPC) or negatively charged tetra-sulphonated phthalocyanine (TSPC) . Uptake studies have revealed that the lack of activity of TSPC is due to the fact that it has very little affinity for any of the organisms . However, the issue appears to be more complex than simply the gross levels of cellular uptake, since TDEPC and PPC are both taken up by the organisms but only PPC shows activity . This indicates that the localization and subcellular distribution of the phthalocyanines may be a crucial factor in determining their cell killing potential . Further analysis of the uptake data has revealed a cell-bound photosensitizer fraction, which remains tightly associated after several washings, and another weakly bound fraction, which is removed by successive washings . Analysis of the cell killing curves, carried out after successive washings of E . coli exposed to PPC, has revealed that it is the tightly associated fraction that is involved in the photosensitization . Taken together with other data, these results suggest that cationic photosensitizers may have a broader application in the photoinactivation of bacterial cells than the anionic or neutral photosensitizers commonly used in photodynamic therapy.

J Antibiot (Tokyo), 1996 Feb, 49(2), 199 - 209
A novel 1 beta-methylcarbapenem antibiotic, S-4661 . Synthesis and structure-activity relationships of 2-(5-substituted pyrrolidin-3-ylthio)-1 beta-methylcarbapenems; Iso Y et al.; The synthesis and biological activity of (1R,5S,6S)-2-{(3S,5S)-5-substituted pyrrolidin-3-ylthio}-6-{(1R)-1-hydroxyethyl}-1- methylcarbapen-2-em-3-carboxylic acids are described . These compounds exhibit potent antibacterial activity against a wide range of both Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa . Of these new carbapenems, (1R,5S,6S)-2-{(3S,5S)-5-sulfamoylaminomethyl pyrrolidin-3-ylthio}-6-{(1R)-1-hydroxyethyl}-1-methylcarb apen- 2-em-3-carboxyli c acid (S-4661) showed the most potent and well balanced activity and was selected as a candidate for further evaluation.

Transfusion, 1996 Feb, 36(2), 113 - 6
Differentiation between human red cells of Pk and p blood types using Pseudomonas aeruginosa PA-I lectin; Sudakevitz D et al.; BACKGROUND: The red cells of almost all human beings bear P antigen . Type P1 cells (around 75% of the population) contain P1 antigen in addition to P, and type P2 cells (around 25% of the population) contain only P . The red cells of only a few individuals are devoid of P: these cells may be of either Pk-positive (P1k and P2k{Pk}) or p type (the latter lack all the above-described antigens of the P system) . Differentiation between them is of clinical importance, but there is a shortage of specific reagents . This article offers reliable means for differentiation . STUDY DESIGN and METHODS: Agglutination of washed, papain-treated red cells of all the P types by PA-1 and soybean lectins and adsorption of the lectins onto the red cells were examined . RESULTS: PA-1 strongly agglutinated papain-treated red cells . Examination of its interactions with red cells having different P system antigens revealed that Pk (both P1k and P2k) red cells of O, A, and B blood types were agglutinated significantly faster than p red cells . The agglutination intensities of Pk red cells of types O and A (most people) was considerably stronger than those of p red cells . P1 and P2 type red cell agglutination was intermediate (P1>P2) . Adsorption tests with all the red cells, exhibited the same order of PA-1 affinities for the P system blood types: Pk>P1,>P2>p . The soybean lectin exhibited opposite behavior (p>P2>P1>Pl) . CONCLUSION: The galactose-binding lectins PA-1 and soybean may facilitate the determination of Pk and p red cells.

Biochem J, 1996 Feb 1, 313 ( Pt 3), 769 - 74
Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa; Mitchell CG; A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells . The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex . The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex . No complex can be reformed in the absence of CSI or CSII . Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared . More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase . The results support the idea of a 'metabolon' in this organism, with the composition of the CS component varying during the growth cycle.

Can J Surg, 1996 Feb, 39(1), 67 - 9
Infected internal iliac artery aneurysm: a case report; Hassan D et al.; Isolated internal iliac aneurysms are rare, and although most are of atherosclerotic origin the cause may also be congenital, traumatic, associated with pregnancy or infectious . A 56-year-old man presented with a swollen, painful left lower limb . Within a few days, weakness of the limb developed with fever and an acute abdomen with free air on x-ray . At emergency laparotomy a small perforation was found in the ascending colon . Examination of the left iliac fossa revealed a ruptured left internal iliac artery aneurysm . Extra-anatomic cross-femoral bypass grafting was done to revascularize the left lower extremity . The patient recovered without complication . At discharge the weakness had improved but knee flexion and extension were weak . Culture of the aneurysm contents grew Staphylococcus aureus and Pseudomonas aeruginosa . The authors discuss the presentation and management of infected internal iliac artery aneurysms.

Clin Exp Immunol, 1996 Feb, 103(2), 212 - 8
Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats; Johansen HK et al.; In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon-gamma (rrIFN-gamma) . Rats were treated either before or after intratracheal challenge with P . aeruginosa embedded in alginate beads . Rats treated after challenge had a significant reduction in the severity of macroscopic lung inflammation compared with rats treated before challenge (P = 0.004) and controls (P = 0.003) . The histopathology in controls was dominated by numerous polymorphonuclear leucocytes (PMN) (> or = 90%) surrounding the alginate beads like in CF . This could be caused by a Th2-like response . In contrast, a complete shift to a chronic-type inflammation dominated by mononuclear leucocytes (> or = 90% lymphocytes and plasma cells) and granulomas was observed in both rrIFN-gamma-treated groups of rats . This could be caused by a Th1-like response . There was no significant difference in lethality between the groups, and the antibody titres against P . aeruginosa sonicate and alginate were similar in the treated rats and controls . Since the ongoing lung tissue damage in CF patients has been shown to be caused by elastase secreted by PMN, which dominate the P . aeruginosa lung infection, our findings offer a possible new strategy of modifying the inflammatory response in CF patients.

J Bacteriol, 1996 Feb, 178(3), 831 - 9
Dual function of PilS during transcriptional activation of the Pseudomonas aeruginosa pilin subunit gene; Boyd JM et al.; The polar pili of Pseudomonas aeruginosa are composed of subunits encoded by the pilA gene . Expression of pilA requires the alternative sigma factor RpoN and a pair of regulatory elements, PilS and PilR . These two proteins are members of the two-component regulatory family, in which PilS is the sensory component and PilR is the response regulator . By using expression and localization analyses, in this work we show that PilS is synthesized as a 59-kDa polypeptide located in the P . aeruginosa cytoplasmic membrane . When the pilS gene is expressed in Escherichia coli, aberrant translational initiation results in a smaller, 40-kDa polypeptide . Unexpectedly, overexpression of pilS in P . aeruginosa results in decreased transcription of the pilA gene . Moreover, fully functional PilS was not required for this inhibitory effect . A mutation in the histidine residue essential for kinase activity resulted in a protein unable to activate transcription, yet when overexpressed in the presence of the wild-type PilS protein, this protein still repressed pilin synthesis . A shorter form of PilS, lacking its transmembrane segments, was active and fully capable of stimulating pilA transcription but when overexpressed did not show the inhibitory effect on pilin expression seen with full-length PilS . We also show that overexpression of pilR can activate transcription of pilA even in the absence of PilS . On the basis of our studies, we propose a complex mechanism of regulation of PilS function, involving other cellular factors that control PilS and its activities during the phosphorelay mechanism of signal transduction.

J Bacteriol, 1996 Feb, 178(3), 625 - 32
Alginate synthesis in Pseudomonas aeruginosa: the role of AlgL (alginate lyase) and AlgX; Monday SR et al.; Previous studies localized an alginate lyase gene (algL) within the alginate biosynthetic gene cluster at 34 min on the Pseudomonas aeruginosa chromosome . Insertion of a Tn501 polar transposon in a gene (algX) directly upstream of algL in mucoid P . aeruginosa FRD1 inactivated expression of algX, algL, and other downstream genes, including algA . This strain is phenotypically nonmucoid; however, alginate production could be restored by complementation in trans with a plasmid carrying all of the genes inactivated by the insertion, including algL and algX . Alginate production was also recovered when a merodiploid that generated a complete alginate gene cluster on the chromosome was constructed . However, alginate production by merodiploids formed in the algX::Tn501 mutant using an alginate cluster with an algL deletion was not restored to wild-type levels unless algL was provided on a plasmid in trans . In addition, complementation studies of Tn501 mutants using plasmids containing specific deletions in either algL or algX revealed that both genes were required to restore the mucoid phenotype . Escherichia coli strains which expressed algX produced a unique protein of approximately 53 kDa, consistent with the gene product predicted from the DNA sequencing data . These studies demonstrate that AlgX, whose biochemical function remains to be defined, and AlgL, which has alginate lyase activity, are both involved in alginate production by P . aeruginosa.

Infect Immun, 1996 Feb, 64(2), 600 - 5
Pseudomonas aeruginosa strains possess specific adhesins for laminin; Plotkowski MC et al.; Pseudomonas aeruginosa is a major human pathogen known to infect tissues that have been previously damaged in some way . In wounded human respiratory tissues, P . aeruginosa cells were found attached to exposed basement membranes following epithelial denudation, suggesting that the affinity for extracellular matrix proteins may account for the bacterium's opportunistic character . By using microtiter wells coated with different P . aeruginosa strains, we demonstrated that laminin binds to both colonizing bacterial strains, isolated from asymptomatic carriers, and strains isolated from infected patients . Binding of soluble laminin to piliated P . aeruginosa PAK and to the nonpiliated isogenic mutant PAK/p--was shown to be saturable . Binding of laminin to the piliated PAK strain was not different from binding to th nonpiliated PAK/p--strain but was significantly higher than binding to the avirulent, nonpiliated PAK-N1 rpoN mutant . By transmission electron microscopy, we localized the laminin-binding sites on a loose material in the outermost layer of the bacteria . Western immunoblotting results suggested that 57- and 59-kDa nonpilus adhesins from the microbial outer membranes account for the binding of P . aeruginosa to laminin . We speculate that bacterial affinity for laminin may be of biological significance in the pathogenesis of P . aeruginosa infection of injured tissues.

Infect Immun, 1996 Feb, 64(2), 518 - 23
Pyoverdin is essential for virulence of Pseudomonas aeruginosa; Meyer JM et al.; The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence . To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P . aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added . Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium . Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation . Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth . In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice . However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection . These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P . aeruginosa . Rapid removal of iron from {59Fe}ferritransferrin by pyoverdin in vitro supports this view.

Med Clin (Barc), 1996 Jan 27, 106(3), 91 - 4
{Risk factors for local infection in burns . Multivariate study}; Herruzo Cabrera R et al.; BACKGROUND: Infection, which is the most serious complication in burns has been little studied . There the aim of the present was to study this complication and its risk factors in patients admitted into a Burn Intensive Care Unit . METHODS: Patients admitted from 1988-1992 were prospectively studied . The characteristics of the patients were systematically collected on admission (i.e . age, body area burned {BAB}, previous disease) as were those of the patient's evolution in the Burn Unit (i.e . infection, manipulation, and therapy) . Prior to analysis the patients were randomly divided into two groups: a "study" group with 455 patients and a "validation" group of 163 patients . A uni and bivariate descriptive study was performed in addition to a multivariate study by unconditional multiple logistic regression obtaining one predictive and one explicative equation . The former was thereafter validated in a sample of 163 patients . RESULTS: No significant differences were observed between the two groups which were made up of severely ill patients (20% BAG 2/3 with dermodeep flame burns, mean age of 43 years) with a similar rate of infection 7.2% and 8.6%, respectively . The ethiologic agent was Pseudomonas aeruginosa in both groups . On applying logistic regression a predictive equation of burn infection and the main risk factors of infection were obtained: BAB (between 15-30%, odds ratio of 4, being 15 if BAB > 30%), duration of central catheterization prior to infection (1-15 days, odds ratio of 6, being 31 if it is longer than 15 days) and preinfection stay (between 15-30 days, odds ratio of 3, being 10 if the stay is longer than 30 days) . The validation was obtained on applying the predictive equation to the patients in both groups to calculate the number of infected patients . In both cases the prediction was correct . CONCLUSIONS: The main risk factors of burn infection are: body area burned, central catheterization and preinfection hospital stay . The equation which adequately predicts the probability of infection based on the characteristics of the burned patients is a good tool for reducing the frequency of infections in burned patients.

J Mol Biol, 1996 Jan 26, 255(3), 362 - 6
X-ray crystal structure of the two site-specific mutants Ile7Ser and Phe110Ser of azurin from Pseudomonas aeruginosa; Hammann C et al.; The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre . This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment . In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (17S and F110S) were introduced that were thought to increase the polarity and the mobility in its environment . The crystal structures of these mutants were solved at 2.2 A and 2.3 A resolution, respectively . These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein . We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues.

EMBO J, 1996 Jan 15, 15(2), 429 - 36
A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa; Lu HM et al.; A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium . The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated . A series of exotoxin A truncates, fused to beta-lactamase, was constructed . Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium . Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period . A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A . It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane . The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted . The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets . It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells . In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.

Science, 1996 Jan 5, 271(5245), 64 - 7
Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections; Pier GB et al.; Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections . Cultured human airway epithelial cells expressing the delta F508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P . aeruginosa compared with cells expressing the wild-type allele . Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs . CFTR may contribute to a host-defense mechanism that is important for clearance of P . aeruginosa from the respiratory tract.

J Med, 1996, 27(5-6), 293 - 302
Survival of nosocomial pathogenic bacteria at ambient temperature; Smith SM et al.; Staphylococcus aureus and many gram-negative rods are prevalent nosocomial pathogens . The mechanisms by which these organisms persist and spread within the hospital environment have not been clearly defined . We found that these bacteria have an extraordinary capability for survival in the environment . The viabilities of staphylococcal and gram-negative (Escherichia coli and Pseudomonas aeruginosa) isolates were assessed on three environmental surfaces: a non-nutrient surface, a woven cotton fiber, and a blood protein coagulum . The bacteria were dried on these surfaces and quantitatively subcultured over six months . The viability was consistently higher on dried blood surfaces . Viability was next highest on cotton strings . For both of these environments, staphylococci appeared to lose viability between three and six months, while E . coli and P . aeruginosa survived longer . Survival on a clean non-nutrient surface (tubes alone) for all organisms was much briefer and did not extend beyond four weeks . Such extended survival on blood and fiber surfaces, as observed in part, explains the difficulty in controlling colonization of patients by and spread of these nosocomial pathogens.

Acta Microbiol Immunol Hung, 1996, 43(4), 371 - 8
Effect of microwaves on survival of some bacterial strains; Atmaca S et al.; While the inhibitory effect of microwave radiation on microorganisms is being researched intensively, how microwave radiation brings about this effect has been a matter of discussion . Some researchers support that this effect is of a thermal character, whereas some others maintain a non-thermal effect . In this work, 1 ml suspensions of Pseudomonas aeruginosa, Pseudomonas acidovorans staphylococcus aureus and Staphylococcus epidermidis bacteria were subjected to microwave radiation at 2450 MHz and 550 Watts for periods of 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 25 and 30 seconds . When each result was compared with the CFU/ml results obtained from unradiated control group bacterial suspensions derived from stock cultures, significant conclusions were attained (P < 0.001) . The same experiments were repeated with the application of conventional heating . The difference between the CFU/ml values of similar bacterial suspensions subjected to microwave radiation and conventional heating was significant (P < 0.001) . Concurrently, the fact that the effect was exacerbated upon increasing of liquid volume during the application of microwave radiation was established via the results obtained through the application of microwave radiation to 1 ml and 5 ml bacterial suspensions (P < 0.001).

Acta Microbiol Immunol Hung, 1996, 43(4), 301 - 5
Incidence of wound infection in three different departments and the antibiotic sensitivity pattern of the isolates in a Saudi Arabian hospital; Abussaud MJ; The incidence of wound infection in three different departments: general surgery (GS), interdisciplinary intensive care (IIC) and pediatric intensive care (PIC) in King Khalid Hospital in Hail was studied during the hot months June-October . A total of 2,331 wounds were examined . One hundred and ninety-three of them were infected (8%) . The overall monthly infection rates in these departments were significantly different . They were 18, 9 and 4% in the IIC, GS, and PIC respectively . The monthly infection rates varied, the highest rates occurred in July . Two hundred and eighty three strains were isolated from the infected wounds . Seventy ones were isolated during June, and 69, 60, 42 and 41 strains were isolated during July, October, August and September, respectively- Thirty five per cent of these strains were identified as Staphylococcus aureus, 31% as Escherichia coli, 25% as Pseudomonas aeruginosa, and 10% as Klebsiella . S . aureus was the predominant organism during July; while E . coli predominated during August and October . S . aureus and P . aeruginosa strains were isolated in equal numbers during June . The sensitivity of these bacteria to ampicillin, cephalotin, cefotaxime, cefoxitin, tetracycline, chloramphenicol and gentamicin was tested by the standard disk diffusion technique . The most effective antibiotic against S . aureus was chloramphenicol (78%) followed by gentamicin (63%) and tetracycline (57%); against E . coli cefotaxime (82%) followed by cefoxitin (77%), cephalitin (62%) and chloramphenicol (53%); against Klebsiella cefotaxime (95%) followed by chloramphenicol (90%), tetracycline (70%), cephalotin and gentamicin (each 66%); and cefoxitin (60%); against P . aeruginosa cefotaxime (56%), gentamicin (33%) and chloramphenicol (29%).

Folia Microbiol (Praha), 1996, 41(2), 149 - 53
Pharmacodynamic parameters of aminoglycosides and their effect on exoenzymes of Pseudomonas aeruginosa; Hostacka A; Aminoglycosides at 2x or 4x minimum inhibitory concentration induced postantibiotic effects against Pseudomonas aeruginosa lasting 3.5-4.9 h (gentamicin) and 0.5-3.7 h (selemycin) . Postantibiotic effects of subinhibitory concentrations of the aminoglycosides tested were substantially longer . Some combinations of supra- and subinhibitory concentrations of antibiotics did not even allow any regrowth of the bacterial strain . The postantibiotic effects and postantibiotic effects of subinhibitory concentrations of gentamicin and selemycin were associated with changes of P . aeruginosa elastase and proteinase . Combinations of supra- and subinhibitory concentrations more pronouncedly suppressed enzymic activities than did suprainhibitory concentrations alone.

Folia Microbiol (Praha), 1996, 41(1), 61 - 4
Inhibition of Pseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics; Majtan V et al.; The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), beta-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by a P . aeruginosa strain were studied . Both beta-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC . Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate . Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.

Folia Microbiol (Praha), 1996, 41(1), 39 - 42
Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence on Pseudomonas aeruginosa enzymic activity; Hostacka A; The postantibiotic effects of subinhibitory concentrations (PA SMEs) and virulence factor alterations induced by ciprofloxacin, tobramycin and netilmicin in Pseudomonas aeruginosa were studied . After induction of the postantibiotic phase (PA) (2x or 4x MIC) the cultures were exposed to subinhibitory concentrations (0.1, 0.2 and 0.3x MIC) of the same antibiotic (PA SME) . The regrowth of treated as well as control cultures was followed for 24 or 45 h . In the sterile culture filtrates obtained from these bacterial cultures, elastase and proteinase were determined . Ciprofloxacin and aminoglycosides exhibited PA SMEs of 35-35 h for certain combinations of supra-subinhibitory antibiotic concentrations . Longer PA SMEs were observed after treatment with higher sub-MICs . Tobramycin at 0.2 and 0.3x MIC (postantibiotic phase induced by 2x MIC) and at alt sub-MICs added to the bacteria previously exposed to 4x MIC do not allow any regrowth of bacterial culture . PA SMEs of tested antibiotics affected virulence factors of P . aeruginosa . Elastase compared to proteinase was suppressed more effectively . Ciprofloxacin at 0.3x MIC reduced elastase and proteinase activity most significantly (to 14.2 and 60% of the control values).

Folia Microbiol (Praha), 1996, 41(1), 29 - 32
The influence of Pseudomonas aeruginosa on liposomes; Antos M et al.; The interaction of liposomes loaded with Ponceau red (used as a marker) with Pseudomonas aeruginosa cells was observed and it resulted in marker leakage . The marker leakage from liposomes was low in physiological fluids . The interaction was independent of secreted phospholipase C level and the serotype of the tested strain . Six of 37 examined isolates did not cause any release of the marker from the liposomes . Marker release of over 50% of total encapsulated material was observed only for ten of the strains tested.

Int J Clin Pharmacol Res, 1996, 16(2-3), 43 - 9
Imipenen-resistant Ps . aeruginosa bacteraemia in cancer patients: risk factors, clinical features and outcome; Krcmery V Jr et al.; Ninety-nine patients with 101 bacteraemic episodes due to Pseudomonas aeruginosa (PA) within 6 years were divided into two groups according to their resistance to imipenem; of these 91 episodes were due to imipenem-sensitive (ISPA) and 10 due to imipenem-resistant (IRPA) strains . Risk factors, clinical course and outcome were evaluated and compared in the two groups . Acute leukaemia, long-lasting neutropenia, previous therapy with amikacin, third-generation cephalosporins, imipenem and prophylaxis with quinolones were significantly more frequently associated with IRPA than with ISPA . Imipenem-resistant PA bactereamias were associated with a higher incidence of septic shock (40% vs 19.8%) p . 161 0.02) and death 33.3%) than were ISPA bacteraemias . Since 1992, when first IRPA appeared, the incidence of imipenem-resistance increased tenfold, and in 1994, up to 10% of the PA populations causing bloodstream infections in cancer patients in our centre were imipenem-resistant.

Microbios, 1996, 87(352), 141 - 7
Influence on Pseudomonas aeruginosa elastase, proteinase and alginate by supra-inhibitory and supra-subinhibitory concentrations of quinolones; Hostacka A et al.; Suppression of elastase, proteinase and alginate of Pseudomonas aeruginosa after the exposure of a bacterial suspension (30 min) to supra-inhibitory concentrations (2x or 4x MIC) of norfloxacin was found . The norfloxacin at 4x MIC was more effective, and this concentration depressed elastase to 13.0%, proteinase to 14.6% and alginate to 0% of the control values . Enoxacin at 2x MIC did not affect the virulence factors studied, and at a concentration of 4x MIC only elastase was effectively reduced to 61% of the control value . Supra-subinhibitory concentrations of the quinolones tested (2x or 4x MIC + sub-MICs) severely diminished all of the activities tested compared with supra-inhibitory concentrations alone . Combinations of 4x MIC + sub-MICs were more effective in the majority of cases.

J Drug Target, 1996, 4(4), 233 - 43
Distribution of DNA and alginate in purulent cystic fibrosis sputum: implications to pulmonary targeting strategies; Mrsny RJ et al.; Cystic fibrosis (CF) patients frequently experience recurring airway infections characterized by thick, viscous sputum . The consistency and nature of these purulent secretions may produce a significant barrier to the successful delivery of drugs and gene therapy vectors designed to treat CF . We have carried out a series of in vitro studies to determine the distribution of two macromolecular components typically present in purulent sputum, bacterial alginate and neutrophil-derived DNA . Sputum samples were obtained from hospitalized CF patients . DNA and alginate were disrupted, respectively, by the in vitro additions of human recombinant deoxyribonuclease I (rhDNase) or alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa . N-acetyl-L-cysteine (acetylcysteine) was similarly used to collapse the mucin matrix of these samples for comparison . Using a centrifugation-based rheological method known as the compaction assay, a greater maximal response was observed for rhDNase compared to alginate lyase treatment . A simultaneous addition of these enzymes to purulent sputum produced an additive compaction response . Electron microscopy was used to identify alginate and DNA components within the mucin matrix of sputa and to evaluate changes following treatment with high concentrations of alginate lyase or rhDNase . DNA was more widely distributed throughout purulent samples than alginate . Differences in the distribution of DNA and alginate may explain, at least in part, the larger compaction response to rhDNase versus alginate lyase treatment . An improved understanding of DNA and alginate distribution within purulent CF sputum may lead to improvements in drug and vector delivery to airway epithelial cells.

Antibiot Khimioter, 1996, 41(9), 53 - 6
{Clinical trials of ofloxacin in sequential use (intravenous and oral) in patients with serious hospital infection}; Iakovlev SV et al.; Fifteen patients with severe hospital infections such as postoperative pneumonia or intraabdominal sepsis were treated with ofloxacin in a dose of 400 mg once a day for 7 to 14 days (11 days at the average) . The drug was administered intravenously for the first 3-5 days and then orally till the end of the treatment course . The clinical effect was observed in 14 patients (93 per cent) and the positive bacteriological effect was stated in 11 out of 13 patients (85 per cent) . Before the treatment 18 microbial cultures were isolated from the patients . 94 per cent of them was susceptible to ofloxacin . The isolates of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were the most frequent . The treatment resulted in the eradication of 15 cultures (83 per cent) . The adverse reactions were observed in 3 patients but only in 1 of them they were for certain due to the drug use . All the adverse reactions were insignificant or moderate and did not require the treatment discontinuation . The trials showed that ofloxacin was a highly efficient agent useful in the empirical monotherapy of patients with severe hospital infection.

Antibiot Khimioter, 1996, 41(9), 43 - 6
{An attempt to use ofloxacin in patients with mucoviscidosis}; Chuchalin AG et al.; The literature data and the data of the authors on the pathogenesis and pathogenetic and etiotropic therapy of mucoviscidosis are presented . The use of ofloxacin as an antibacterial agent in the complex treatment of mucoviscidosis is considered expedient . The drug was administered intravenously in a dose of 400 mg twice a day for 5 days followed by the oral use of the drug in the form of tablets according to the same scheme . The microbiological investigation of the sputum specimens revealed diagnostically significant titers of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella spp . The isolates except for one case (Ps.aeruginosa) were susceptible to ofloxacin . The treatment with ofloxacin in accordance with the above scheme resulted in a rapid improvement of the patient state: the intoxication lowered, the expectoration and the sputum viscosity decreased, the body temperature normalized by the 5th day . The drug tolerance after the intravenous and enteral administration was good . The intravenous injections of ofloxacin induced a 1.5-fold increase in the intensity of the neutrophil oxygen burst . After the drug enteral administration there was observed a 2-fold increase the intensity of the neutrophil oxygen burst.

Pharmazie, 1996 Jan, 51(1), 54 - 6
Effect of organic ammonium salts on metabolic processes in Pseudomonas aeruginosa; Majtan V et al.; The effect of a homologous series of 2-(dodecanoylamino)-ethylalkyl-dimethylammonium bromides on the cell metabolism of a Pseudomonas aeruginosa strain isolated from a nosocomial infection was studied . The inhibition of the incorporation of radioactive precursors into macromolecules (DNA, RNA, proteins) was dependent on the length of the alkyl chain of the tested substances . Similar results were noted also by following the inhibition of the endogenous respiration of P . aeruginosa . The most effective substance at suppression of {14C} precursors incorporation was with hexyl, and in the case of respiration, the substance with octyl in the molecule . The total inhibition of the respiration in the presence of exogenous carbon substrates, i.e . glucose and glycerol, was found with the substance containing the octyl group (conc . 100 micrograms/ml) . We assumed that the studied substances interfere with cell energy-carbon metabolism of P . aeruginosa.

Microbiol Immunol, 1996, 40(5), 333 - 8
Reduced anti-Pseudomonas aeruginosa activity of a cephalosporin, cefodizime, in rats whose neutrophils were selectively depleted by a monoclonal antibody; Araki A et al.; In order to clarify the mechanisms of the in vivo antibacterial activity of a cephalosporin, cefodizime (CDZM), the effect of this antibiotic on Pseudomonas aeruginosa E7 infection was examined in rats whose neutrophils had been selectively depleted by monoclonal antibody RP-3 . CDZM was less effective in RP-3-treated rats than in untreated rats . However, treatment of rats with recombinant human granulocyte-colony stimulating factor (rhG-CSF) augmented the in vivo activity of this antibiotic . Furthermore, the in vivo antibacterial activity of two other cephalosporins, cefpimizole (CPIZ) and cefoperazone (CPZ), was bilaterally affected by a rise or fall in the neutrophil number, although to a lesser degree than was the case with CDZM . Taken together, neutrophils play an important role in the in vivo antibacterial activity of certain cephalosporins.

J Chemother, 1996 Jan, 8 Suppl 1, 3 - 30
{Tobramycin--clinical pharmacology and chemotherapy}; Periti P; Aminoglycosides are potent water-soluble antibiotics, with peak concentration-dependent bactericidal activity against many pathogenic aerobic Gram-negative bacilli and Staphylococcus aureus: they exhibit enduring antibacterial activity many hours after tissue concentrations become negligible and appreciation of this postantibiotic effect is leading to replacement of conventional multiple daily doses by large once-daily doses . Cotreatment with betalactams is commonly employed in order to exploit a synergism between these antimicrobial agents, particularly in severe Gram-negative sepsis . Resistance to aminoglycosides may be observed at several levels and is generally high when due to the acquisition of aminoglycoside modifying enzymes which may be plasmid-borne or transferred by transposable elements . Tobramycin is more effective than gentamicin and the other aminoglycosides against Pseudomonas aeruginosa and is less nephrotoxic than gentamicin . Higher serum tobramycin concentrations at the peak are associated with a longer postantibiotic effect and increased bactericidal activity . A longer dosage interval may decrease the risk of nephrotoxicity because higher transient serum aminoglycoside levels appear to be less nephrotoxic than lower but more persistent serum concentrations . Once-daily administration may also reduce the risk of ototoxicity through a similar mechanism . In a multicenter Italian study of 104 adult patients with severe bacterial lower respiratory tract infections, the safety and efficacy of a regimen of high dose, once-daily tobramycin alone or in combination with antipseudomonas betalactams was assessed . The overall bacteriological response was an elimination of the original pathogen in 70% of the patients while the clinical response mirrored the bacteriological results with a successful clinical outcome in 78% of patients . Adverse experiences were, in general, few and mild without oto- or nephrotoxicity . The once-daily, high dose regimen of tobramycin proved to be a safe and efficacious therapy for severe lower respiratory tract infections in adult patients.

Acta Otolaryngol Suppl, 1996, 521, 3 - 16
Malignant external otitis: review and personal experience; Amorosa L et al.; Malignant external otitis (MEO) is an infrequent but severe infective disorder, generally due to Pseudomonas aeruginosa, which most often affects elderly diabetics patients . The clinical features rarely permit exact diagnosis of MEO to be made promptly, and initially at least they are difficult to distinguish from those of external otitis . This explains the frequent delay in diagnosis with respect to the onset of symptoms . Physical examination almost always reveals the presence of aspecific granulation tissue in the external auditory canal, while the most common laboratory finding is raised erythrocyte sedimentation rate (ESR) . Imaging has great diagnostic relevance: CT and MRI are very useful for spatial resolution, while radionuclide scanning and, in our experience, SPECT are superior for detecting early osteitis and monitoring response to therapy . We present an extensive review of the literature and our personal experience . In particular, we stress the relevance of immunological study of MEO patients: all our 4 patients had defective immune defences . As regards therapy, like other authors we underline the fundamental importance of long-term antibiotic treatment . The availability of quinolones and latest generation cephalosporins has greatly simplified the choice of antibiotic treatment, although clinicians should be aware of the possibility of drug-resistant bacterial strains.

Scand J Infect Dis, 1996, 28(4), 417 - 8
X-linked agammaglobulinemia presenting as pseudomonas aeruginosa septicemia; Zenone T et al.; Usually, presenting infections in children with agammaglobulinemia include pneumonia and otitis media caused by pyogenic bacteria . We report here two cases of Pseudomonas aeruginosa septicemia with ecthyma gangrenosum, in previously healthy boys, leading to the diagnosis of X-linked agammaglobulinemia.

Jpn J Ophthalmol, 1996, 40(2), 212 - 9
The effectiveness of two ciprofloxacin formulations for experimental Pseudomonas and Staphylococcus keratitis; Engel LS et al.; Ciprofloxacin is a fluoroquinolone antibiotic with broad spectrum bactericidal activity . A commercially available form of ciprofloxacin contains benzalkonium chloride (BAC) (0.006%) and EDTA (0.05%) as preservatives . Since BAC has been shown to cause adverse changes in corneal epithelial cells, a formulation of ciprofloxacin devoid of BAC and EDTA but with the same effectiveness would be valuable . We present here the results of experiments designed to assess the efficacy of a BAC-free and EDTA-free formulation of ciprofloxacin, Ciprofloxacin-polystyrene sulfonate (PSS), in experimental models of Pseudomonas aeruginosa and Staphylococcus aureus keratitis . Both formulations of ciprofloxacin sterilized corneas infected with P aeruginosa, and both formulations showed equal bactericidal activity for S aureus . Normal eyes treated with either formulation showed mild conjunctival irritation compared to untreated normal eyes . The bactericidal activities of both formulations of ciprofloxacin were excellent . Therefore, the Ciprofloxacin-PSS formulation could serve as an effective single drug therapy for ocular infections.

Microbiol Immunol, 1996, 40(2), 177 - 82
Airway interleukin-8 in elderly patients with bacterial lower respiratory tract infections; Ponglertnapagorn P et al.; We investigated the interleukin-8 (IL-8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents . The IL-8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens . We also demonstrated that human recombinant IL-8 at a range of 6.25-25 ng/ml significantly enhanced opsonophagocytic killing of P . aeruginosa immunotype-1 strain by human neutrophils in the presence of a serotype-specific anti-lipopolysaccharide monoclonal antibody and fresh normal human serum . These data suggest that the level of IL-8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL-8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.

Microbiol Immunol, 1996, 40(2), 107 - 14
The trpF nucleotide sequence and its promoter analysis in Pseudomonas aeruginosa; Murata T; The nucleotide sequence of the gene for the phosphoribosyl anthranilate isomerase (PRAI) (trpF) in Pseudomonas aeruginosa was determined . The gene encoded a polypeptide of 211 amino acid residues and its initiation codon was UUG . Northern hybridization analysis showed only one transcript for trpF (about 700 nucleotides) . Primer extension analysis revealed three transcription start sites which were at positions 52, 55 and 57 bases upstream of the initiation codon . A highly GC-rich 9-base-pair inverted repeat with 5-base-pair spacing was found upstream of a putative Shine-Dalgarno sequence . This inverted repeat could form a 23-nucleotide stem-loop structure in the transcript . Deletion of the inverted repeat slightly decreased the promoter activity of trpF . The presence of tryptophan had no effect on transcription of trpF.

Microbios, 1996, 86(347), 77 - 83
A new gene, aadA2b, encoding an aminoglycoside adenylyltransferase, AAD(3")(9), isolated from integron InC in Pseudomonas aeruginosa; Kazama H et al.; A gene, designated aadA2 and encoding an aminoglycoside-adenylyltransferase, was located on integron InC, isolated from the R-plasmid of Pseudomonas aeruginosa, as a gene cassette . The aadA2 gene of InC was identical to those of pSa and R1033 with the exception of one base in each case . This single-base substitution did not influence the expression of streptomycin resistance . It is proposed that the aadA2 genes isolated from pSa and from R1033, and the aadA2 gene from InC, be designated aadA2a and aadA2b, respectively.

Infection, 1996 Jan-Feb, 24(1), 5 - 8
Adenovirus infection in cystic fibrosis patients: implications for the use of adenoviral vectors for gene transfer; Rosenecker J et al.; Clinical trials using replication-deficient adenovirus as vectors for gene transfer into the airways of cystic fibrosis (CF) patients are in progress . However, little is known about the prevalence of wild-type adenovirus infections in patients with cystic fibrosis and their effect on lung function . To answer these questions, serum IgG and IgM antibody titers against adenovirus type 5 were prospectively measured by an indirect immunofluorescence assay in 199 CF outpatients and in a control group of 45 healthy children and young adults . In addition, we performed pulmonary function tests when the patients were in stable clinical condition . IgM antibodies against adenovirus were present in 104 of the 199 cystic fibrosis patients (52.3%) . IgG antibodies against adenovirus were detected in 192 of the 199 cystic fibrosis patients (96.5%), and were significantly higher in cystic fibrosis patients older than 7 years than in younger patients and in age matched controls . IgG antibody titers measured a second time 11.8 months later in 143 of the 199 patients had increased in 48 (33.6%) patients . In 27 of these 48 patients, who had at least a 2-fold increase in antibody titer, FVC and FEV1 decreased by 9.8% (p < 0.05) and 8.3% (p = 0.05), respectively, over 45 months . In a comparison group matched for age, sex, and chronic Pseudomonas aeruginosa infection but no increase in antibody titers, FVC and FEV1 were unchanged . The results indicate that wild-type adenovirus infections are prevalent in cystic fibrosis patients and that wild-type adenovirus infections in cystic fibrosis patients seem to be associated with deterioration in lung function . These observations may have important implications for efficacy and safety considerations when using adenoviral vectors for gene therapy.

Jpn J Antibiot, 1996 Jan, 49(1), 71 - 82
{Antibacterial activities of combination uses of cefpirome with various antibiotics in vitro against clinically isolated glucose non-fermentative gram-negative rods: part 1 . the results against Pseudomonas aeruginosa}; Suzuki Y et al.; In order to evaluate antibacterial activities of combination uses of cefpirome (CPR) and various antibiotics in vitro, minimum inhibitory concentrations (MICs) of CPR alone and combinations of CPR+other drugs against freshly isolated clinical strains of Pseudomonas aeruginosa . The results are summarized as follows; 1 . Combined effects of CPR+beta-lactams, piperacillin (PIPC), aztreonam (AZT), imipenem (IPM) showed wider antibacterial spectra and stronger antibacterial activities than CPR alone with drugs concentrations of CPR and other drugs at sub-MIC levels . At concentrations of sub-MIC levels, antibacterial effects of CPR+PIPC and CPR+AZT combination were strong but CPR+IPM was weaker than those of the former two combinations . It appeared that stronger effects were demonstrated by some combinations against strains that were susceptible to both drugs of combination, but little additive effects were shown against strains that were resistant to both drugs . Antibacterial effect of CPR+fosfomycin combination was the same as those of CPR+PIPC and CPR+AZT combinations . 2 . Combined effects of CPR+aminoglycosides (AGs), gentamicin, tobramycin, amikacin showed wider antibacterial spectra and stronger antibacterial activities at sub-MIC levels of CPR or ATs . The effect against CPR-resistant strains was same . But the combined effect was weak against AGs-resistant strains . 3 . Effectiveness of combinations of CPR+beta-lactams and CPR+AGs depended on drug susceptibilities of strains tested . We cannot estimate effects of those combinations without investigating drug susceptibility of bacteria being tested . 4 . In none of the combinations tested, antagonism was observed.

Microbiol Immunol, 1996, 40(6), 415 - 23
Bradykinin generation triggered by Pseudomonas proteases facilitates invasion of the systemic circulation by Pseudomonas aeruginosa; Sakata Y et al.; To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation . P . aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease) . Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay . The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice . This enhancement was induced not only by pseudomonal proteases but also by bradykinin . More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination . Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin . Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p . simultaneously with PA 621 . On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo.

Mol Microbiol, 1996 Jan, 19(2), 297 - 306
Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa; Braun P et al.; Elastase of Pseudomonas aeruginosa is synthesized as a pre-proprotein . The propeptide has been shown to inhibit the enzymatic activity of elastase . In this study, we investigated a possible additional role of the propeptide in the folding and secretion of the enzyme . When elastase was expressed in Escherichia coli without its propeptide, no active elastase was produced . The enzyme was poorly released from the cytoplasmic membrane and, depending on the expression level, it was either degraded or it accumulated in an inactive form in the cell envelopes, probably as aggregates . Since proper folding is required for the release of translocated proteins from the cytoplasmic membrane and for the acquirement of a stable and active conformation, these results suggest that the propeptide is involved in the proper folding of the elastase and that it functions as an intramolecular chaperone . When mature elastase was expressed without its propeptide in P . aeruginosa, the enzyme was not secreted, and it was degraded . Therefore, proper folding of mature elastase appears to be required for secretion of the enzyme . Expression of the propeptide, as a separate polypeptide, in trans with mature elastase resulted in the formation of active elastase . This active enzyme was secreted in P . aeruginosa . Apparently, the propeptide can also function as an intermolecular chaperone.

Pharmacol Res, 1996 Jan, 33(1), 63 - 5
Anti-pseudomonal activity of collagen sponge with liposomal polymyxin B; Trafny EA et al.; An antibiotic delivery system has been developed using collagen sponge with liposome-encapsulated polymyxin B . Superficial, non-lethal infection was produced in mice by injecting Pseudomonas aeruginosa into the skin windows . Wounds were dressed with collagen sponge containing liposomal polymyxin B or containing empty liposomes (with PBS) as a control . Single dose of topically applied collagen sponge with encapsulated polymyxin B decreased bacterial cell number as compared to the control . Finally, after 8 days of experiment, the number of bacterial colonies dropped below 10(4) per biopsy . Presented polymyxin B delivery system offers potential clinical advantages.

Auris Nasus Larynx, 1996, 23, 13 - 9
Residual bacterial infection in the tympanic cavity following surgery for ears with chronic discharge; Gyo K et al.; In surgical treatment of ears with chronic discharge, pathogenic microorganisms may remain in the middle ear even after meticulous tympanomastoidectomy, and cause recurrent infection unless an appropriate antimicrobial agent is administered . The present study was conducted to determine the incidence of residual bacterial infection in the tympanic cavity by examining secretions from a drainage tube placed there via the mastoid cavity during surgery . Comparison of the bacterial flora before and after surgery demonstrated that some of the microorganisms continued to be present in the tympanic cavity for up to 2 weeks despite medication, and that the incidence of Pseudomonas aeruginosa and Staphylococcus epidermidis remained fairly high.

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