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 enha