Protein alterations in isolated outer membranes of polymyxin-resistantPseudomonas aeruginosa isolates

Isolated outer membranes fromPseudomonas aeruginosa strains resistant to polymyxin were compared with isolated outer membranes from polymyxin-sensitive strains as to their protein composition as determined by slab polyacrylamide gel electrophoresis. Both the porin protein and the H1 protein were decreased greatly in concentration in the outer membranes from the polymyxin-resistant strains. This confirms previous findings reduced concentrations of these proteins in cell envelopes of these strains. No evidence was found for these decreases being the result of an artifactual loss of these proteins from the outer membrane due to conditions used for growth or for preparation of cell envelopes. Nevertheless, the role of these outer membrane proteins in mediating polymyxin resistance is uncertain.     

Phospholipid fatty acid ester composition ofPasteurella multocida andActinobacillus lignieresii

Cell surface hydrophobicity properties vary dramatically, whereas cell envelope phospholipid composition is essentially identical among strains ofPasteurella multocida andActinobacillus lignieresii. Fatty acid ester composition of the major phospholipid fractions from cell surface hydrophobicity variants was examined to determine whether hydrophobic properties are influenced by cell envelope fatty acid content. Individual phospholipids were resolved by preparative thin-layer chromatography, and methanolysis was performed with boron trifluoride-methanol. Gas-liquid chromatographic analysis revealed the organisms to be similar qualitatively, whereas hydrophobic variants exhibited consistently, greater and more disparate C_16:0+C_16:1/C_14:0 ratios in all fractions. Fatty acid composition of phospholipids may be related to surface hydrophobicity properties ofP. multocida variants. However, comparative data obtained forA. lignieresii revealed a degree of similarity withP. multocida that precludes use of this parameter as a means for differentiation of thesePasteurellaceae type species, thereby supporting their taxonomic relatedness.     

Susceptibility to Polymyxin B of Penicillin G-induced Proteus mirabilis L Forms and Spheroplasts

A polymyxin B-resistant strain of Proteus mirabilis was converted into L forms and spheroplasts in the presence of penicillin G. This treatment caused a 400-fold increase in polymyxin B susceptibility. The acquired susceptibility was in the range of the natural susceptibility reported for susceptible gram-negative bacteria ( approximately 1 mug/ml). The high susceptibility to polymyxin B was lost as soon as the spheroplasts and L forms were allowed to reconvert into the bacillary form in penicillin-free media. This behavior is strong evidence that the natural resistance of Proteus strains to polymyxins is due to the impermeability of the outer cell wall structures to these antibiotic substances.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

ThesacB gene cannot be used as a counter-selectable marker inPasteurella multocida

The use of theBacillus subtilis sacB gene as a counter-selectable marker was assessed in serogroup A and B strains ofPasteurella multocida. Expression ofsacB failed to render any of the strains sensitive to sucrose, indicating that thesacB gene can not be used as a positive selection system inP. multocida.     

Potent In Vitro Bactericidal Activity of Polymyxin B against Methicillin-Resistant Staphylococcus aureus (MRSA)

Eradication of methicillin-resistant Staphylococcus aureus (MRSA) carried by inpatients or healthy hospital personnel by topical use of antibiotics is an important step for preventing outbreak of MRSA nosocomial infection. In the screening of the antibiotic best suited for this purpose, we have found that polymyxin B, a commonly used antibiotic for gram-negative infection, had an unexpected strong cytokilling activity towards MRSA clinical strains, which was more potent than that of vancomycin or gentamicin. The data suggested that polymyxin B could be an antibiotic of choice in the treatment of topical carriage of or infection caused by MRSA.     

Comparative polyacrylamide electrophoresis of periplasmic proteins released from gram-negative bacteria by polymyxin B

Summary Several cell-wall and membrane affecting agents were tested for causing release of periplasmic proteins of E. coli B as compared by gel electrophoresis. Osmotic shock and polymyxin-treatment yielded the best differentiated protein patterns. The periplasmic proteins derived from different E. coli strains and other gram-negative bacteria by polymyxin-treatment were compared. Whereas related strains showed similarities in the protein positions, unrelated gram-negative bacteria showed great differences of the protein bands. The polymyxin-induced liberation of periplasmic proteins was dependent upon the growth phase and growth media of the bacteria and was severely inhibited by 10^-2 M magnesium chloride.Abbreviations PX polymyxin B - CTABr cetyltrimethylammonium-bromide - tris trishydroxymethylaminomethane - EDTA ethylenediaminetetraacetate - LPS lipopolysaccharide     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa pvdQ</Emphasis> Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl₂. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.     

Recombinant outer membrane protein F ofPseudomonas aeruginosa elicits antibodies that mediate opsonophagocytic killing,but not complement-mediated bacteriolysis,of various strains ofP. aeruginosa

Recombinant outer membrane protein F ofPseudomonas aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Rats were immunized intramuscularly with 25 g of recombinant protein F adsorbed to aluminum hydroxide adjuvant on days 1, 14, and 28 and then challenged on day 42 via intratracheal inoculation of agar beads containing cells of a clinical isolate ofP. aeruginosa. On day 49 the lungs were examined macroscopically for the presence and severity of lesions and submitted for quantitation of the bacteria present. The recombinant protein F vaccine afforded significant protection against subsequent challenge withP. aeruginosa in the immunized rats, as compared with control rats immunized with bovine serum albumin. Antisera from the recombinant protein F-immunized rats mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells ofP. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant ofP. aeruginosa. The antisera to recombinant protein F did not promote complement-mediated bacteriolysis ofP. aeruginosa. These data demonstrate that recombinantP. aeruginosa protein F has efficacy as a protective vaccine in a rat model of chronic pulmonary infection.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Effect of saline on the releasability of alkaline phosphatase from cells of Serratia marcescens.

The effect of 0.9% sodium chloride solution on the release of alkaline phosphatases from cells of four strains of Serratia marcescens was studied. Saline had a greater action in the releasability of the enzyme on cells of the polymyxin B sensitive strains than those of the polymyxin B resistant strains. SDS-polyacrylamide gel electrophoresis of the released materials showed the presence of proteins and lipopolysaccharide components of the outer membrane as well as enzyme activity in all four strains. Cells from strains harvested under higher temperatures contained more releasable activity in the salin wash fraction than those harvested under refrigerated condition. Active components with molecular weights of 190,000 and 110,000 daltons were either absent or present to a lesser degree in the extracts released by the polymyxin B treatment of the washed cells. However, active components not released by saline were found in the polymyxin B extracts. Contrary to other reports, results of this study clearly showed the ubiquitous nature of alkaline phosphatase in S. marcescens. It appears that their releasability is related to the polymyxin B susceptibility as well as the instability of the outer membrane of the cell envelope.     

Derivation of Extracellular Polysaccharide-Deficient Variants from a Serotype A Strain of Pasteurella multocida

The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (Plp-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [³H]-palmitate revealed capsular loss occurred with a concomitant diminution of Plp-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in Plp-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that Plp-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis. Received: 30 October 1998 / Accepted: 4 December 1998     

Response of RP1+ and RP1− strains ofEscherichia coli to antibacterial agents and transfer of resistance toPseudomonas aeruginosa

The sensitivity of strains ofEscherichia coli, with and without the RP1 R-factor, to antibiotics and other antibacterial agents has been studied. RP1⁺ strains ofE. coli were resistant to kanamycin, carbenicillin, and tetracycline, resistance to the first two antibiotics being produced by destruction of the drugs. This resistance could be transferred to two strains ofPseudomonas aeruginosa. The parent strain ofE. coli UB 1005, its two mutant strains (DC2 and DC3), and two of the strains with the RP1 R-factor showed a similar order of sensitivity to phenylmercuric nitrate, chlorhexidine, thiomersal, and mercuric chloride.E. coli strains DC2 and DC2 (RP1⁺) were the most sensitive to benzalkonium chloride and cetrimide. RP1⁺ strains were more resistant than RP1⁻ strains to lysozyme-ethylenediaminetetraacetic acid, but treatment of the former strains with acriflavine rendered the cells more sensitive to the lytic system. There was no evidence thatP. aeruginosa (RP1⁺) strains possessed increased resistance to polymyxin or to disinfectants, although they became somewhat less sensitive to lysozyme-ethylenediaminetetraacetic acid.     

The activity of some antibiotic combinations onSalmonella

Summary The effects of some antibiotics acting alone or in combination upon 72 strains of variousSalmonella species were investigated usingElek andHilson’s variant ofLederberg’s replica technique. Chloramphenicol and tetracycline were found to act mainly bacteriostatically; streptomycin and neomycin varied in their action from bacteriostatic for some strains to bactericidal for other ones. The action of polymyxin B appeared to be more especially bactericidal. But for a few exceptions no interaction was found between the combinations streptomycin-chloramphenicol and streptomycin-tetracycline. The pair neomycin-chloramphenicol was often antagonistic; neomycin-polymyxin B, on the other hand, prevailingly synergistic. Under the conditions of experimentation a pronounced synergism was noticed between polymyxin B and chloramphenicol. The prospects of the latter synergistic combination as a possibly more radical means of combatingSalmonella infections are shortly discussed. With the technical assistance of MissM. J. Wisse.     

Autolytic nature of iridescent lysis inPseudomonas aeruginosa

Attempts to demonstrate a filterable agent to be the cause of iridescent lysis inPseudomonas aeruginosa were uniformly negative. It was not possible to transmit the principle by needle transfer from iridescent plaques to non-iridescent cultures, and plaques produced byP. aeruginosa bacteriophages were never iridescent. Iridescent lysis and bacteriophage lysis were subjected to antibiotics, anti-metabolites, agar at different pH values, antisera to bacteriophage-lysed and to iridescent-lysed bacteria, different oxygen concentrations, and to different nutritional sources. Certain antibiotics, notably tetracycline, streptomycin and polymyxin inducedde novo or enhanced formation of metallic lysis in nutrient agar surface cultures ofP. aeruginosa. Bacteriophage was not induced. Antimetabolites of amino acids, carbohydrates and vitamins inhibited iridescence, or inhibited it at high concentrations and enhanced it at low concentrations. Bacteriophage action was unaffected. Metallic lysis was completely inhibited at pH 6.0; it was inhibited on media containing dye or bile salt and at lowered oxygen concentrations. Bacteriophage action was not affected under these conditions. Antisera to iridescent lysates and to bacteriophage lysates ofP. aeruginosa were tested. Phage antiserum strongly neutralized phage lysis but had no effect on iridescent lysis; antisera to iridescent lysates had no effect on either. No evidence for phage mediation of iridescent lysis was seen in any of the experiments. Iridescent lysis ofP. aeruginosa was demonstrated to be based on metabolic autolysis.     

The cell wall amidase AmiB is essential for Pseudomonas aeruginosa cell division,drug resistance and viability

The physiological function of cell wall amidases has been investigated in several proteobacterial species. In all cases, they have been implicated in the cleavage of cell wall material synthesized by the cytokinetic ring. Although typically non‐essential, this activity is critical for daughter cell separation and outer membrane invagination during division. In Escherichia coli, proteins with LytM domains also participate in cell separation by stimulating amidase activity. Here, we investigated the function of amidases and LytM proteins in the opportunistic pathogen Pseudomonas aeruginosa. In agreement with studies in other organisms, ^PaAmiB and three LytM proteins were found to play crucial roles in P. aeruginosa cell separation, envelope integrity and antibiotic resistance. Importantly, the phenotype of amidase‐defective P. aeruginosa cells also differed in informative ways from the E. coli paradigm; ^PaAmiB was found to be essential for viability and the successful completion of cell constriction. Our results thus reveal a key role for amidase activity in cytokinetic ring contraction. Furthermore, we show that the essential function of ^PaAmiB can be bypassed in mutants activated for a Cpx‐like envelope stress response, suggesting that this signaling system may elicit the repair of division machinery defects in addition to general envelope damage.     

Ultrastructural Study of Salmonella typhimurium Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components

Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of Salmonella typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B(+) and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B(+) was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.     

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.

It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.