RESUMO
Triclosan is a hydrophobic antimicrobial agent commonly employed in health care settings. While it exhibits broad-spectrum antibacterial properties, the gram-negative nosocomial opportunists Pseudomonas aeruginosa and Serratia marcescens are atypically refractory. Intrinsic resistance to triclosan in P. aeruginosa is largely due to its outer membrane impermeability properties for hydrophobic and bulky substances. The present study was undertaken to determine the relationship between triclosan and the outer cell envelopes of thirteen strains of ten Serratia species reported to be opportunistic pathogens in humans. General intrinsic resistance to hydrophobic and other outer membrane impermeant compounds was assessed using cultural selection, disk agar diffusion, and macrobroth dilution bioassays. Uptake of the hydrophobic fluorescent probe 1-N-phenylnapthylamine was assessed in four disparate strains of S. marcescens. Batch culture kinetics in the presence of combinations of triclosan and outer membrane permeabilizer compound 48/80 allowed analysis of outer membrane involvement in intrinsic resistance. Aggregate results revealed that individual species ranged in response to hydrophobic and bulky molecules from generally refractory to extremely susceptible. Moreover, susceptivity to triclosan sensitization by chemical disruption of outer membrane exclusionary properties differed markedly among species which exhibited intrinsic resistance to triclosan. These data suggest that disparate opportunistic pathogens within the genus Serratia differ phenotypically regarding the degree to which outer membrane exclusion contributes to intrinsic resistance for impermeant molecules in general, and triclosan specifically. Ancillary resistance mechanisms appear to contribute in some species and may involve constitutive multi-drug efflux systems. Importance A paucity of knowledge exists regarding the cellular and molecular mechanisms by which opportunistically pathogenic members of the genus Serratia are able to infect immunocompromised and otherwise susceptible individuals, and then evade chemotherapy. This is especially true for species other than Serratia marcescens and Serratia liquefaciens, although much remains to be learned with regard to the nature of key virulence factors and infection mechanisms which allow for the typically nosocomial acquisition of even these species. The research described in the present study will provide a better understanding of the contribution of outer cell envelope permeability properties to the pathogenicity of these opportunistic species in an ever-increasing susceptible patient population. It is our hope that greater knowledge of the basic biology of these organisms will contribute to the mitigation of suffering they cause in patients with underlying diseases.
RESUMO
Burkholderia multivorans causes opportunistic pulmonary infections and is intrinsically resistant to many antibacterial compounds including the hydrophobic biocide triclosan. Chemical permeabilization of the Pseudomonas aeruginosa outer membrane affects sensitization to hydrophobic substances. The purpose of the present study was to determine if B. multivorans is similarly susceptive suggesting that outer membrane impermeability properties underlie triclosan resistance. Antibiograms and conventional macrobroth dilution bioassays were employed to establish baseline susceptibility levels to hydrophobic antibacterial compounds. Outer membrane permeabilizers compound 48/80, polymyxin B, polymyxin B-nonapeptide, and ethylenediaminetetraacetic acid were used in attempts to sensitize disparate B. multivorans isolates to the hydrophobic agents novobiocin and triclosan, and to potentiate partitioning of the hydrophobic fluorescent probe 1-N-phenylnapthylamine (NPN). The lipophilic agent resistance profiles for all B. multivorans strains were essentially the same as that of P. aeruginosa except that they were resistant to polymyxin B. Moreover, they resisted sensitization to hydrophobic compounds and remained inaccessible to NPN when treated with outer membrane permeabilizers. These data support the notion that while both phylogenetically-related organisms exhibit general intrinsic resistance properties to hydrophobic substances, the outer membrane of B. multivorans either resists permeabilization by chemical modification or sensitization is mitigated by a supplemental mechanism not present in P. aeruginosa.
Assuntos
Complexo Burkholderia cepacia , Triclosan , Triclosan/farmacologia , Polimixina B/farmacologia , Pseudomonas aeruginosa , Novobiocina/farmacologia , Antibacterianos/farmacologiaRESUMO
Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.
Assuntos
Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Pseudomonas/efeitos dos fármacos , Triclosan/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Água Doce/microbiologia , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Oklahoma , Pseudomonas/genética , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S , Microbiologia da Água , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
INTRODUCTION: Skin and subcutaneous infections are dangerous sequelae of soft tissue injuries, especially in austere situations where medical technology is not available. Numerous plant species endemic to North America have been described as having antibacterial properties. Of these, St. John's wort (Hypericum perforatum), chamomile (Matricaria chamomilla), and white oak (Quercus alba) were selected for testing against Staphylococcus aureus. Our objective was to assess the suitability of all 3 plants as potential antiseptic agents using methods easily replicated in a resource-scarce environment. METHODS: Water-soluble natural products were extracted from different concentrations of each plant part using either mechanical agitation at ambient temperature or boiling in unsterilized tap water. Antibacterial activity of each extract against S aureus was assessed using a conventional agar well diffusion bioassay. Zones of inhibition were measured using electronic calipers and were compared to tap water as the negative control. RESULTS: Aqueous extracts of St. John's wort and white oak bark displayed antibacterial effects against S aureus, with St. John's wort being more potent. Chamomile displayed no inhibitory properties at the concentrations examined. CONCLUSIONS: These data suggest that both St. John's wort and white oak are potential candidates for infection prophylaxis and therapy in austere wilderness scenarios, with St. John's wort being the more potent agent. White oak may be more logistically feasible because the larger surface area of a white oak tree allows for harvesting a larger quantity of bark compared to the smaller surface area of the St. John's wort plant.
Assuntos
Hypericum/química , Matricaria/química , Extratos Vegetais/farmacologia , Quercus/química , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Humanos , América do Norte , Extratos Vegetais/química , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: The purpose of the present study was to obtain a better understanding of the relationship between cell surface physiology and outer cellular envelope permeability for hydrophobic substances in mucoid and non-mucoid B. multivorans strains, as well as in two capsule-deficient derivatives of a mucoid parental strain. METHODOLOGY: Cell surface hydrophobicity properties were determined using the hydrocarbon adherence method, while outer cell envelope accessibility and permeability for non-polar compounds were measured using hydrophobic antimicrobial agent susceptibility and fluorescent probe assays. Extracellular polysaccharide (EPS) production was assessed by cultivating strains of disparate origin on yeast extract agar (YEA) containing different sugars, while the resultant colonial and cellular morphological parameters were assessed macro- and microscopically, respectively.Results/Key findings. The cell surfaces of all the strains were hydrophilic, impermeable to mechanistically disparate hydrophobic antibacterial agents and inaccessible to the hydrophobic probe N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of basal YEA with eight different sugars enhanced macroscopic EPS expression for all but one non-mucoid strain, with mannose potentiating the greatest effect. Despite acquisition of the mucoid phenotype, non-mucoid strains remained non-capsulated and capsulation of a hyper-mucoid strain and its two non-mucoid derivative strains was unaffected, as judged by microscopic observation. CONCLUSION: These data support the conclusion that EPS expression and the consistent mucoid phenotype are not necessarily associated with the ability of the outer cell surface to associate with non-polar substances or cellular capsulation.
Assuntos
Burkholderia/química , Burkholderia/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Permeabilidade , Propriedades de Superfície , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Aderência Bacteriana , Corantes Fluorescentes/metabolismo , Hidrocarbonetos/metabolismoRESUMO
Enterobacter cloacae strain JD6301 was isolated from a mixed culture with wastewater collected from a municipal treatment facility and oleaginous microorganisms. A draft genome sequence of this organism indicates that it has a genome size of 4,772,910 bp, an average G+C content of 53%, and 4,509 protein-coding genes.
RESUMO
Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C(16:0) (palmitic acid), C(16:1) (palmitoleic acid), and C(18:1) (oleic acid), while C(14:0) (myristic acid), ΔC(17:0) (methylene hexadecanoic acid), C(18:0) (stearic acid), and ΔC(19:0) (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C(16:0), C(16:1), ΔC(17:0), C(18:1), and ΔC(19:0) with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.
Assuntos
Burkholderia/química , Membrana Celular/química , Fosfolipídeos/análise , Burkholderia/isolamento & purificação , Infecções por Burkholderia/microbiologia , Cromatografia em Camada Fina , Fibrose Cística/complicações , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas , HumanosRESUMO
Triclosan is a broad-spectrum antimicrobial agent having low toxicity which facilitates its incorporation into numerous personal and health care products. Although triclosan acts against a wide range of gram-positive and gram-negative bacteria by affecting fatty acid biosynthesis, it is ineffective against the opportunistic pathogen Pseudomonas aeruginosa. Wild-type strain P. aeruginosa PAO1 was used as a model system to determine the effects of triclosan on fatty acid metabolism in resistant microorganisms. This was accomplished by cultivating P. aeruginosa PAO1 cultures in the presence of different concentrations of triclosan, monitoring growth rates turbidimetrically, and harvesting in stationary phase. Readily extractable lipids (RELs) were obtained from freeze-dried cells after washing and analyzed using gas chromatography coupled with mass spectrometry. Resultant data demonstrated that triclosan caused dose-dependent increases in the amounts of trans-C(16:1) and trans-C(18:1) fatty acids, with concomitant decreases in their respective cyclopropyl analogs. Triclosan did not affect the relative concentrations of saturated, cis unsaturated, or the overall ratios of combined C(16) to C(18) fatty acid species. The readily extractable lipid fractions contained triclosan proportional to triclosan concentrations in the growth media. The presence or absence of triclosan in either liquid or solid media did not affect the antimicrobial susceptibilities of P. aeruginosa PAO1 to a battery of unrelated antimicrobials. Triclosan decreased growth rate in a dose-dependent manner at soluble concentrations. Incorporation of triclosan into the REL fraction was accompanied by increased levels of trans unsaturated fatty acids, decreased levels of cyclopropyl fatty acids, and decrease in growth rate. These alterations may contribute to triclosan resistance in P. aeruginosa PAO1.
Assuntos
Anti-Infecciosos/farmacologia , Ácidos Graxos Insaturados/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Triclosan/farmacologia , Biomassa , Ácidos Graxos Insaturados/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Nefelometria e Turbidimetria , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
Pasteurella multocida exhibits nonspecific susceptibility to nonpolar antimicrobial agents such as triclosan, despite possessing an ultrastructurally typical gram-negative cell envelope. Capsulated and noncapsulated cell surface variants were examined to investigate the role outer membrane permeability plays in triclosan susceptibility. Test strains were unable to initiate growth in the presence of bile salts and were susceptible to triclosan with minimal inhibitory concentrations (MICs) ranging from 0.06 to 0.25 microg/ml. Disk agar diffusion bioassays revealed triclosan susceptibility to be dose dependent and all strains were susceptible to the hydrophobic antibiotics novobiocin, rifamycin SV, and chloramphenicol. Triclosan minimal bactericidal concentrations were greater than MICs, thereby suggesting that dose dependency reflected both bacteriostatic and bactericidal effects. Total and viable cell density growth kinetic determinations revealed a triclosan concentration of 2.0 microg/ml resulted in loss of batch culture viability within 4-24 h. Concentrations of 0.02 and 0.2 microg/ml exerted either a bacteriostatic or bactericidal effect depending on the strain. Uptake of the hydrophobic probe 1-N-phenylnaphthylamine was greater in P. multocida strains than refractory control organisms Pseudomonas aeruginosa and Escherichia coli thereby suggesting the presence of phospholipid bilayer regions in the outer membrane. Because triclosan inhibits a conserved enoyl-ACP reductase necessary for bacterial fatty acid biosynthesis, these data support the notion that extreme susceptibility in P. multocida is due to the general inability of the outer membrane to exclude nonpolar compounds. Moreover, susceptibility is independent of the presence of capsular material and the biocide is bactericidal in a concentration dependent manner.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores da Síntese de Ácidos Graxos/farmacologia , Pasteurella multocida/efeitos dos fármacos , Triclosan/farmacologia , 1-Naftilamina/análogos & derivados , 1-Naftilamina/química , Animais , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana/veterinária , Pasteurella multocida/crescimento & desenvolvimentoRESUMO
Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.
Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos Locais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Triclosan/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologia , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cloranfenicol/farmacologia , Meios de Cultura , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
A 40-kDa lipoprotein (Plp-40) is expressed by serotype A strains of Pasteurella multocida in amounts which correlate with the amount of capsular material present. We hypothesized that Plp-40 is exposed at the outer surface of the outer membrane (OM) of the cell and is associated with the serotype A exopolysaccharide material. The objectives of the present study were to confirm the lipoprotein nature of Plp-40 and to determine its subcellular location. Plp-40 maturation was shown to be sensitive to globomycin, thereby confirming it to be a bacterial lipoprotein. Plp-40 was shown to be present in the OM fractions of P. multocida obtained by both sarkosyl extraction and sucrose density gradient centrifugation, as well as in capsule fractions obtained by either hyaluronidase treatment or warm buffer extraction. [(3)H]palmitic acid-labeled Plp-40 could be removed from the surface of whole cells by exposure to proteinase K. Autoradiography of (125)I-labeled cell surface proteins exhibited a 40-kDa band that was prominent in capsulated strains and greatly diminished in a noncapsulated strain. These results support the hypothesis that Plp-40 is a lipid-modified OM protein, which is exposed on the outer cell surface and is likely associated with serotype A extracellular polysaccharide.
Assuntos
Lipoproteínas/metabolismo , Pasteurella multocida/metabolismo , Frações Subcelulares/metabolismo , Autorradiografia , Fracionamento Celular , Eletroforese em Gel de PoliacrilamidaRESUMO
The present study was undertaken to investigate the possibility that outer cell envelope impermeability might be involved in the intrinsic resistance of Pseudomonas aeruginosa to low levels of the hydrophobic biocide triclosan. Macrobroth dilution and batch cultural turbidimetric assays were employed to assess the ability of compounds that render the Gram-negative outer membrane permeable to non-polar molecules to sensitise cell envelope variants to triclosan. Pseudomonas aeruginosa strains possessing highly refractory (PAO1) and atypically permeable (Z61) outer cell envelopes as well as a PAO1 derivative lacking four multidrug efflux pumps (YM64) were examined. Whilst the triclosan minimal inhibitory concentrations (MICs) differed dramatically for both PAO1 and Z61, significant decreases were seen for both strains in the presence of the outer membrane permeabiliser polymyxin B-nonapeptide. Strain YM64 was as resistant to triclosan as strain PAO1. Turbidimetric assessments of batch cultural growth kinetics revealed that the three chemically unrelated outer membrane permeabilisers polymyxin B-nonapeptide, compound 48/80 and ethylenediaminetetraacetate (EDTA) sensitised all strains to a sub-MIC concentration of triclosan (2.0 microg/mL). These data support the notion that the outer membrane exclusionary properties of P. aeruginosa for non-polar molecules confer intrinsic resistance to low concentrations of triclosan such as might be expected to occur in environmental residues. Moreover, a role for outer cell envelope impermeability is suggested for resistance to high triclosan concentrations in vitro.
Assuntos
Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Triclosan/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/metabolismoRESUMO
The objective of this study was to determine the effects of sub-minimum inhibitory concentrations (sub-MICs) of 2 veterinary antibiotic preparations, chlortetracycline (CTC) and chlortetracycline-sulfamethazine (CTC + SMZ), on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus at normal and febrile body temperatures. Sub-minimum inhibitory concentrations of both antibiotics reduced the growth rates of M. haemolytica and H. somnus. Growth of both species was not inhibited when grown at 41 degrees C compared to 37 degrees C. There was no detectable consistent effect of antibiotic or temperature on outer membrane protein expression for either species. Our study indicates that sub-MIC levels of CTC and CTC + SMZ markedly impair growth of clinical M. haemolytica and H. somnus isolates, potentially allowing more effective host clearance during infection.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Clortetraciclina/farmacologia , Haemophilus somnus/efeitos dos fármacos , Mannheimia haemolytica/efeitos dos fármacos , Sulfametazina/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Quimioterapia Combinada , Eletroforese em Gel de Poliacrilamida/veterinária , Regulação Bacteriana da Expressão Gênica , Haemophilus somnus/crescimento & desenvolvimento , Cinética , Mannheimia haemolytica/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana/veterinária , TemperaturaRESUMO
The capsule of Pasteurella multocida serotype A strain ATCC 11039 is composed of hyaluronic acid and is an important virulence factor. Repeated subculturing of certain capsular serotype A strains results in dissociation from a capsulated to a noncapsulated phenotype with a concomitant loss of virulence. Although noncapsulated variants have been thought to arise as a result of mutation, the molecular mechanisms underlying this event are unknown. In this study, we demonstrate that restoration of the capsulated phenotype occurs in vivo subsequent to intraperitoneal inoculation of BALB/c mice with a noncapsulated variant. Moreover, reverse transcription polymerase chain reaction analysis revealed the capsule locus to be under transcriptional control. Cloning and sequencing of a 290-bp fragment within the promoter containing intergenic region of the capsule locus of 11039/iso revealed no significant alterations occurred subsequent to subculturing. These results demonstrate that serotype A P. multocida strain ATCC 11039 regulates capsule expression in response to an unidentified environmental factor(s), thereby providing insights into the molecular mechanisms underlying colonial dissociation.
Assuntos
Cápsulas Bacterianas/biossíntese , Pasteurella multocida/metabolismo , Animais , Cápsulas Bacterianas/genética , Sequência de Bases , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pasteurella multocida/classificação , Pasteurella multocida/genética , Pasteurella multocida/patogenicidade , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sorotipagem , VirulênciaRESUMO
Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017-1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.
Assuntos
Actinobacillus pleuropneumoniae/genética , Genes Bacterianos , Antígenos O/biossíntese , Antígenos O/genética , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , DNA Bacteriano/análise , Dados de Sequência Molecular , Mutagênese Insercional , Reação em Cadeia da Polimerase/veterinária , Sorotipagem/veterinária , SuínosRESUMO
Capsular serotype A strains of Pasteurella multocida of avian origin express a 40-kDa lipoprotein (Plp-40) thought to attach the extracellular polysaccharide to the cell surface. The objective of the present study was to assess the prevalence of Plp-40 in P. multocida strains of disparate serotypes and host origins, as well as other pathogenic members of the family Pasteurellaceae. Exponential-phase reference and clinical isolates were radiolabeled with [3H]-palmitate, lysed to obtain whole-cell protein fractions, and analyzed using SDS-PAGE and fluorography to assess lipoprotein content. The ability to produce Plp-40 was found to be conserved among certain P. multocida reference and clinical strains of different host origins including avian, human, porcine, bovine, feline, canine, ovine, and cervine, but not rabbit. Production of a 40-kDa lipoprotein was exhibited by all clinical isolates of Pasteurella aerogenes, Pasteurella pneumotropica, Actinobacillus suis, Actinobacillus suis-like organism, and Actinobacillus pleuropneumoniae examined, but not Pasteurella (Mannheimia) haemolytica, Actinobacillus lignieresii, or Haemophilus spp. These data suggest that, while not all Pasteurellaceae are able to produce a 40-kDa lipoprotein under the present experimental conditions, expression is somewhat conserved among diverse isolates of disparate host origins.
Assuntos
Cápsulas Bacterianas/química , Lipoproteínas/análise , Infecções por Pasteurella/veterinária , Pasteurellaceae/química , Animais , Aves , Gatos , Bovinos , Galinhas , Cervos , Cães , Eletroforese em Gel de Poliacrilamida , Furões , Humanos , Lipoproteínas/química , Peso Molecular , Pasteurella multocida/química , Pasteurellaceae/patogenicidade , Coelhos , Ratos , Sorotipagem , Ovinos , Suínos , PerusRESUMO
Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.