Influence of methylation on the antibacterial properties of triclosan in Pasteurella multocida and Pseudomonas aeruginosa variant strains.

The opportunistic bacterium Pasteurella multocida is extremely susceptible to the hydrophobic biocide triclosan by virtue of its markedly permeable outer membrane, while the nosocomial pathogen Pseudomonas aeruginosa is intrinsically resistant to levels far exceeding the triclosan aqueous solubility limit. Widespread incorporation of triclosan in health and personal care products has resulted in its concomitant accumulation with metabolites such as methyl triclosan in environmental and biological systems. The present study was undertaken to investigate the possibility that methylation of triclosan may mitigate its antiseptic efficacy in healthcare settings, as well as represent a potential resistance mechanism. Comparative standardised disc agar diffusion and batch cultural turbidimetric bioassays were employed to assess the relationship between triclosan-susceptible or -resistant bacteria and methyl triclosan. A wild-type P. aeruginosa parental strain and a mutant exhibiting a permeable outer cell envelope phenotype were examined in concert with a refractory wild-type strain sensitised to triclosan susceptibility using outer membrane permeabiliser compound 48/80. All organisms examined were resistant to methyl triclosan, and all organisms excluding P. aeruginosa were susceptible to triclosan over a wide concentration range. The permeable outer membrane phenotype in both mutant and chemically sensitised wild-type strains rendered P. aeruginosa susceptible to triclosan, but not to methyl triclosan. These data support the notion that methylation of triclosan renders the compound unable to inhibit the growth of disparate bacterial pathogens in a manner independent of an intact outer membrane. It can also be concluded that biocide modification may contribute to the intrinsic resistance of P. aeruginosa to triclosan.

Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements.

Surface interfacial physiology is particularly important to unicellular organisms with regard to maintenance of optimal cell function. Bacterial cell surfaces possess net negative electrostatic charge by virtue of ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules which are exposed to the extracellular environment. The degree of peripheral electronegativity influences overall cell surface polarity and can be assessed on the basis of zeta potential which is most often determined by estimating the electrophoretic mobility of cells in an electric field. The purpose of this review is to provide bacteriologists with assistance as they seek to better understand available instrumentation and fundamental principles concerning the estimation of zeta potential as it relates to bacterial surface physiology.

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 (P1p-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 P1p-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 P1p-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 P1p-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis.

Adaptive acquisition of novobiocin resistance in Pasteurella multocida strains of avian origin.

Naturally occurring strains of Pasteurella multocida are atypically susceptible to hydrophobic antibiotics such as novobiocin, despite their Gram-negative cell envelope ultrastructure. Four strains adaptively resistant to 1000 micrograms/ml of novobiocin were obtained by sequentially subculturing cell surface hydrophobic variants of avian origin in the presence of increasing antibiotic concentrations. Adaptive novobiocin resistance was accompanied in all cases by the concomitant acquisition of resistance to coumermycin, a hydrophobic antibiotic possessing the same mechanism of action, but not to the functionally disparate hydrophobic antibiotic rifamycin. The acquisition of resistance was not accompanied by alterations in the lipid composition of the cell envelope. Subsequent growth of adaptively resistant strains in the absence of novobiocin did not result in the restoration of susceptibility to either novobiocin or coumermycin. Acquisition of adaptive resistance in encapsulated parental strains resulted in an inability to synthesize capsular material and enhanced cell surface hydrophobicity; however, parental encapsulation and decreased cell surface hydrophobicity were restored upon removal of novobiocin. These data suggest that acquisition of adaptive resistance to novobiocin conferred in this manner is the result of a stable genetic event affecting the mechanistic target of both novobiocin and coumermycin rather than a physiological adaptation involving outer membrane impermeability.

Relationship between serotype A encapsulation and a 40-kDa lipoprotein in Pasteurella multocida.

Eleven serotype A encapsulated and nonencapsulated strains of Pasteurella multocida were examined with regard to lipoprotein content. Relative amounts of an approximately 40-kDa lipoprotein (Plp-40) were found to correlate directly with the degree of encapsulation in that heavily encapsulated strains exhibited the greatest amounts, while nonencapsulated strains possessed little or no Plp-40.

Biochemical characterization of lipopolysaccharides extracted from a hydrophobic strain of Pasteurella multocida.

Lipopolysaccharides were extracted from freeze-dried cells of Pasteurella multocida strain P-1581 (serotype 8) by the phenol-chloroform-petroleum ether method and biochemically analysed using standard procedures. The primary neutral sugars were glucose, galactose and heptose. No deoxy sugars were detected. Amino sugars included galactosamine, glucosamine and glucosamine-6-phosphate. 3-Deoxy-D-manno-2-octulosonic acid was present at a relatively low concentration. The analyses included identification and quantification of phosphate and alanine. The primary fatty acids and their approximate relative ratios were 3-hydroxytetradecanoate and tetradecanoate 2:1. Tetradecanoic acid was bound almost exclusively by ester linkages. 3-Hydroxytetradecanoic acid was bound primarily by amide linkages, although significant numbers of ester-bound residues were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses indicated that the lipopolysaccharides were of low molecular weight.

Effect of polymyxin B nonapeptide on daptomycin permeability and cell surface properties in Pseudomonas aeruginosa, Escherichia coli, and Pasteurella multocida.

The present study was carried out to determine if sensitization of Gram-negative bacteria to the polyanionic antibiotic daptomycin by cationic molecules can be explained on the basis of decreased cell surface charge in order to better understand intrinsic resistance. Turbidimetric assessments of batch cultural growth kinetics revealed the outer membrane permeabilizer polymyxin B nonapeptide sensitized Pseudomonas aeruginosa and Escherichia coli to the hydrophobic probe novobiocin, whereas little or no sensitization was observed for two surface hydrophobicity variants of Pasteurella multocida. Polymyxin B nonapeptide and daptomycin synergistically inhibited growth of P. aeruginosa only. A hydrocarbon adherence assay revealed permeabilizing concentrations of polymyxin B nonapeptide increased cell surface hydrophobicity of P. aeruginosa and the hydrophobic P. multocida variant, while E. coli and the hydrophilic P. multocida variant remained unaffected. Measurement of cellular electrophoretic mobility showed polymyxin B nonapeptide permeabilization of P. aeruginosa to daptomycin occurred concomitantly with a significant decrease in cell surface charge, while no such sensitization occurred in organisms which failed to undergo polymyxin B nonapeptide-induced surface charge decreases. These data suggest that sensitization of Gram-negative bacteria to polyanionic lipopeptides by growth in the presence of polycationic outer membrane permeabilizers such as polymyxin B nonapeptide is dependent on diminution of overall cell surface charge and polarity, thereby allowing outer cell envelope permeation.

Postnatal changes in selected bacterial groups of the pig colonic microflora.

The importance of the colonic microflora in health and nutrition is well known, but how they colonize and become established in the colon is not well understood. We therefore characterized the quantitative and qualitative changes of the colonic microflora during the first 120 days of postnatal development. Unlike previous studies, changes were defined for individual pigs using in situ samples collected anaerobically and aseptically from the distal colon. Although the colons were sterile at birth, they were rapidly colonized, and within 12 h bacterial densities had stabilized at 10(-9)-10(10) bacteria/g colonic content. Facultative anaerobes, notably coliforms, initially dominated the microflora, but were supplanted within 48 h after birth by obligate anaerobes, which constituted greater than 90% of the microflora thereafter. Bacteroides spp., the predominant anaerobes in the adult colon, did not markedly increase in abundance until after weaning and were still increasing by postnatal day 120. Shifts in the relative abundances of different bacterial populations throughout the first 120 days after birth confirm previous reports that the establishment of the adult colonic microflora is a gradual, sequential process, and highlight the need to focus research on anaerobic groups.

Adaptive resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa.

Aminoglycoside-resistant variants of Pseudomonas aeruginosa strain PAO1 were readily selected by culturing the organism in medium containing increasing concentrations of gentamicin, tobramycin or amikacin until the strains were growing in a concentration of drug 128-fold greater than the minimal inhibitory concentration for the sensitive parent strain. These resistant strains exhibited characteristics previously associated with the impermeability type of resistance mechanism, i.e., they grew more slowly than the parent strain, the resistance was unstable in the absence of the antibiotic, and adaptation to one of the antibiotics conferred cross-resistance to other aminoglycosides. The adapted strains grew, with minimal morphological alterations, in concentrations of the various aminoglycosides that normally produced cell envelope damage, misshapen and filamentous cell formation, and cell lysis in the sensitive strain. Neither protein H1 nor phospholipid alterations appear to play a significant role in adaptive resistance to aminoglycoside antibiotics in this model system. The acquisition of adaptive resistance to the aminoglycoside antibiotics did not confer resistance to polymyxin B, another cationic antibiotic which is thought to share binding sites within the outer membrane with the aminoglycosides.

Susceptibility to hydrophobic molecules and phospholipid composition in Pasteurella multocida and Actinobacillus lignieresii.

Despite its typically gram-negative cell envelope ultrastructure, Pasteurella multocida is susceptible to the hydrophobic antibiotic novobiocin and is unable to initiate growth on MacConkey agar, a parameter often used to effect is differentiation from other members of the family Pasteurellaceae such as Actinobacillus lignieresii. However, growth on basal medium supplemented with individual selective factors and an agar diffusion assay revealed the bile salts contained in MacConkey agar to be toxic to both organisms. Four P. multocida surface hydrophobicity variants exhibited consistent in vitro susceptibility to the hydrophobic antibiotics novobiocin, rifamycin SV, and actinomycin D as determined by broth dilution. Readily extractable lipid fractions were obtained by chloroform-methanol extraction of freeze-dried whole cells from exponential-phase cultures. No major differences in total cellular readily extractable lipid content were observed among the P. multocida and A. lignieresii strains examined, although hydrophobic P. multocida strains appeared to contain slightly more than did hydrophilic strains. Analytical thin-layer chromatography and quantitation of resolved readily extractable lipid components revealed the major cell envelope phospholipids of both organisms to be phosphatidylethanolamine and phosphatidylglycerol in a molar ratio of approximately 4:1 regardless of cell surface hydrophobicity properties. Similar results were obtained for Pseudomonas aeruginosa, which is notably refractory to hydrophobic molecules. These data support the conclusion that the permeability of the P. multocida cell envelope to structurally unrelated, hydrophobic molecules is not dependent on cell surface hydrophobicity and cannot be explained on the basis of anomalous polar lipid composition.

Low propensity for poultry isolates of Pasteurella multocida to acquire adaptive resistance to oxytetracycline.

Thirty independently derived reference strains and clinical isolates of Pasteurella multocida were tested to determine their potential for acquiring adaptive resistance to oxytetracycline in an effort to better understand the prolonged high efficacy of the antibiotic for pasteurellosis in poultry. All reference strains and clinical isolates exhibited uniform susceptibility as measured with the broth dilution method. None of the strains or isolates readily acquired significant resistance when grown in subinhibitory oxytetracycline levels under the conditions employed. These data support the conclusions that spontaneous variation in P. multocida resulting in oxytetracycline resistance is uncommon in the field and that the organism possesses a very low propensity for acquiring adaptive resistance in response to growth in the presence of the antibiotic.

Variability of cell surface hydrophobicity among Pasteurella multocida somatic serotype and Actinobacillus lignieresii strains.

Pasteurella multocida possesses a characteristically gram-negative ultrastructure, yet its inability to grow in the presence of hydrophobic compounds and the general penicillin susceptibility of genera making up the family Pasteurellaceae suggest a cell envelope having atypical permeability properties. The cell surface hydrophobicity properties of strains representing 15 of the 16 somatic serotypes of P. multocida and three strains of Actinobacillus lignieresii were assessed with hydrocarbon adherence and hydrophobic interaction chromatographic assays. These methods revealed surface hydrophobicity to vary dramatically among strains in both species. No direct correlation was observed with species, growth rate, or susceptibility to the antibiotics oxytetracycline (polar), polymyxin B (amphiphilic), or novobiocin (nonpolar) as measured with MIC determinations. All strains were susceptible to the antibiotics, although A. lignieresii was significantly less susceptible than P. multocida to novobiocin. These data suggest that cell surface hydrophobicity in P. multocida may be influenced by the type of lipopolysaccharide present but is not directly related to permeability of the antibiotics examined. The wide diversity of hydrophobic properties exhibited by strains of both P. multocida and A. lignieresii precludes the use of this parameter as a taxonomic acid.

Chemical alterations in cell envelopes of Pseudomonas aeruginosa upon exposure to polymyxin: a possible mechanism to explain adaptive resistance to polymyxin.

Polymyxin-susceptible cells of Pseudomonas aeruginosa were exposed for 10, 30, and 60 min to growth medium containing 6000 units of polymyxin per millilitre. Exposure for 10 min resulted in lipid alterations in the cell envelope. A large reduction in the content of both phosphatidylethanolamine and phosphatidylglycerol with a large increase in both diphosphatidylglycerol and free fatty acids was found upon analysis by thin-layer chromatography. The cellular percentage of readily extractable lipid (REL) was reduced, and the phospholipid proportion of the REL decreased with polymyxin exposure. Polymyxin exposure for 30 and 60 min only slightly enhanced these chemical alterations. The cell envelope alterations found were characteristic for strains which become adaptively resistant to polymyxin. Treatment of the cells with chloramphenicol or KCN prior to polymyxin exposure did not prevent the lipid alterations from occurring. These observations suggest that the polymyxin-susceptible cell population adapts to polymyxin resistance by the rapid alteration of the cell envelopes of the entire cell population. We propose the theory that cell envelope phospholipases and proteases play a major role in the adaptive response and that the cation content of the cell envelope may be a critical controlling factor in the process.

Conversion of phospholipids to free fatty acids in response to acquisition of polymyxin resistance in Pseudomonas aeruginosa.

The readily extractable lipids from a Pseudomonas aeruginosa isolate stepwise adapted to polymyxin resistance were compared with those of the susceptible parent and of a revertant strain which regained susceptibility. Significant qualitative and quantitative lipid alterations accompany the acquisition of resistance. Changes include the appearance of a major unidentified lipid (lipid X) unique to the readily extractable lipids of resistant cells. Comparative studies with parent and revertant strains indicated a significant decrease in the phospholipid content of resistant cells. Thin-layer chromatography of resistant-cell readily extractable lipids demonstrated: (i) the emergence of lipid X (36% of total readily extractable lipids), (ii) a decrease in phosphatidylethanolamine and phosphatidylglycerol, and (iii) an increase in diphosphatidylglycerol. Lipid X was purified by preparative silicic acid column chromatography and thin-layer chromatography and characterized by analytical thin-layer chromatography, column adsorption chromatography, and gas-liquid chromatography. Data from this study indicated that lipid X was a mixture of free fatty acids. The fatty acids present in lipid X were qualitatively and quantitatively the same as the fatty acids esterified to the phospholipids in the readily extractable lipids.

Noninvolvement of beauvericin in the entomopathogenicity of Beauveria bassiana.

Development of a microbiological autobiographic assay procedure permitted a detailed investigation of the possible role of beauvericin (a toxic ionophoric antibiotic produced by Beauveria bassiana) in the entomopathogenicity of B. bassiana against corn earworm (Heliothis zea) larvae. Analysis of spent media of B. bassiana and the hemolymph of infected and moribund larvae revealed that beauvericin was not present in a soluble form during the time that most (about 90%) larvae died of fungal infection (4 days). Intrahemocoelic injections of up to 6 micrograms of synthetic beauvericin failed to induce any deleterious effects. In addition, although methanol-soluble ionophores, such as valinomycin and bassianolide, were toxic to corn earworm larvae, no methanol-soluble toxin could be detected in the hemolymph of moribund larvae.