RESUMO
Pseudomonas aeruginosa forms chronic infections in the lungs of cystic fibrosis (CF) patients, and is the leading cause of morbidity and mortality in patients with CF. Understanding how this opportunistic pathogen adapts to the CF lung during chronic infections is important to increase the efficacy of treatment and is likely to increase insight into other long-term infections. Previous studies of P. aeruginosa adaptation and divergence in CF infections have focused on the genetic level, both identifying characteristic mutations and patterns of gene expression. However, these approaches are not sufficient to fully understand the metabolic changes that occur during long-term infection, as metabolic regulation is complex and takes place on different biological levels. We used untargeted metabolic profiling (metabolomics) of cell supernatants (exometabolome analysis, or metabolic footprinting) to compare 179 strains, collected over time periods ranging from 4 to 24 years for the individual patients, representing a series of mostly clonal lineages from 18 individual patients. There was clear evidence of metabolic adaptation to the CF lung environment: acetate production was highly significantly negatively associated with length of infection. For amino acids, which are available to the bacterium in the lung environment, the tendency of isolates to evolve more efficient uptake was related to the biosynthetic cost of producing each metabolite; conversely, for the non-mammalian metabolite trehalose, isolates had significantly reduced tendency to utilize this compound with length of infection. However, as well as adaptation across patients, there was also a striking degree of metabolic variation between the different clonal lineages: in fact, the patient the strains were isolated from was a greater source of variance than length of infection for all metabolites observed. Our data highlight the potential for metabolomic investigation of complex phenotypic adaptations during infection.
Assuntos
Adaptação Fisiológica , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Aminoácidos/metabolismo , Doença Crônica , Humanos , Pulmão/microbiologia , Metabolômica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Fatores de TempoRESUMO
The aim of this study was to compare two traditional pattern matching techniques, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), with the more reproducible technique of multilocus sequence typing (MLST) to genotype a blinded sample of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. A blinded sample of 48 well-characterized CF P. aeruginosa isolates was genotyped by PFGE, RAPD, and MLST, each performed in a different laboratory. The discriminatory power and congruence between the methods were compared using the Simpson's index, Rand index, and Wallace coefficient. PFGE and MLST had the greatest congruence with the highest Rand index (0.697). The discriminatory power of PFGE, RAPD, and MLST were comparable, with high Simpson's indices (range 0.973-0.980). MLST identified the most clonal relationships. When clonality was defined as agreement between two or more methods, MLST had the greatest predictive value (100 %) in labeling strains as unique, while PFGE had the greatest predictive value (96 %) in labeling strains as clonal. This study demonstrated the highest level of agreement between PFGE and MLST in genotyping P. aeruginosa isolates from CF patients. MLST had the greatest predictive value in identifying strains as unique and, thus, has the potential to be a cost-efficient, high-throughput, first-pass typing method.
Assuntos
Fibrose Cística/complicações , Impressões Digitais de DNA/métodos , Eletroforese em Gel de Campo Pulsado/métodos , Tipagem de Sequências Multilocus/métodos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Análise por Conglomerados , Genótipo , Humanos , Valor Preditivo dos Testes , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificaçãoRESUMO
Correlation of biofilm formation and presence of the icaADBC genes was studied in 161 clinical isolates of S. epidermidis from persistent and non-persistent neonatal infections. Biofilm formation was compared in trypticase soy broth with or without added glucose (0.5-9.23%), and total parenteral nutrition (TPN) containing 9.23% glucose. Detection of icaADBC genes was carried out using polymerase chain reaction and ica-specific primers. Quantitative biofilm formation for all isolates was highest in the presence of 1% glucose, followed by trypticase soy broth and TPN. There was no significant difference between the amount of biofilm formed by persistent and non-persistent isolates under different test conditions. In contrast, 70% of the persistent isolates produced biofilm in TPN compared to the 56.3% of the non-persistent group. Neither the persistent bacterial phenotype nor presence of the icaADBC operon was correlated with biofilm formation.
Assuntos
Biofilmes , Óperon/genética , Polissacarídeos Bacterianos/genética , Staphylococcus epidermidis/genética , Infecção Hospitalar/genética , Infecção Hospitalar/microbiologia , Glucose/metabolismo , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Nutrição Parenteral Total , Staphylococcus epidermidis/metabolismoRESUMO
In previous work (E. E. Smith, D. G. Buckley, Z. Wu, C. Saenphimmachack, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson, Proc. Natl. Acad. Sci. USA 103:8487-8492, 2006) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.
Assuntos
Adaptação Biológica/genética , Fibrose Cística/microbiologia , Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Infecções Respiratórias/microbiologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Fibrose Cística/complicações , Reparo de Erro de Pareamento de DNA , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Evolução Molecular , Genes Bacterianos , Teste de Complementação Genética , Genoma Bacteriano , Humanos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/classificação , Infecções Respiratórias/complicaçõesRESUMO
Distinctive international clones of penicillin-nonsusceptible and multidrug-resistant Streptococcus pneumoniae are increasingly being reported. We investigated the spread of these clones in Canada through an active surveillance that was carried out at 11 Canadian pediatric tertiary care centers from 1991 to 1998. All penicillin-nonsusceptible isolates were serotyped, tested for antibiotic susceptibility, and genotyped by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Forty-five penicillin-nonsusceptible S. pneumoniae isolates were evaluated. Eleven serotype 9V isolates and six serotype 14 isolates displayed identical RAPD and PFGE fingerprint profiles. Twelve (70%) of these isolates were encountered in Quebec. The 9V/14 clone and the Spanish-French clone had similar PFGE fingerprint patterns. Eight isolates of serotype 23F and two isolates of serogroup 14 had the same fingerprint profiles and displayed resistance to three or more antibiotic drug classes. This clone was first detected in Calgary (Alberta) and in 1996 appeared simultaneously in various regions of Canada. This clone showed a PFGE fingerprint pattern similar to that of the Spanish-U.S. 23F clone. Our data show the emergence across Canada of two international clones of penicillin-nonsusceptible S. pneumoniae: (i) serotypes 9V and 14 related to the Spanish-French clone and (ii) the 23F Spanish-U.S. clone. The source of the first clone was in Quebec and the second international clone was probably originated from the United States. The exact reasons for the successful spread of these clones within Canada and their contribution to increased resistance to antibiotics have yet to be explored.
Assuntos
Resistência às Penicilinas , Infecções Pneumocócicas/epidemiologia , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/efeitos dos fármacos , Adolescente , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Canadá/epidemiologia , Criança , Pré-Escolar , Farmacorresistência Bacteriana Múltipla , Eletroforese em Gel de Campo Pulsado , Humanos , Lactente , Testes de Sensibilidade Microbiana , Infecções Pneumocócicas/microbiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidadeRESUMO
Infection with Burkholderia cepacia complex in patients with cystic fibrosis (CF) results in highly variable clinical outcomes. The purpose of this study was to determine if there are genomovar-specific disparities in transmission and disease severity. B. cepacia complex was recovered from 62 patients with CF on > or =1 occasions (genomovar III, 46 patients; genomovar II [B. multivorans], 19 patients; genomovar IV [B. stabilis], 1 patient; genomovar V [B. vietnamiensis], 1 patient; and an unclassified B. cepacia complex strain, 1 patient). Patient-to-patient spread was observed with B. cepacia genomovar III, but not with B. multivorans. Genomovar III strains replaced B. multivorans in 6 patients. Genomovar III strains were also associated with a poor clinical course and high mortality. Infection control practices should be designed with knowledge about B. cepacia complex genomovar status; patients infected with transmissible genomovar III strains should not be cohorted with patients infected with B. multivorans and other B. cepacia genomovars.
Assuntos
Infecções por Burkholderia/microbiologia , Burkholderia cepacia/genética , Burkholderia/genética , Fibrose Cística/microbiologia , Adolescente , Adulto , Colúmbia Britânica/epidemiologia , Burkholderia/classificação , Burkholderia/isolamento & purificação , Burkholderia/patogenicidade , Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/mortalidade , Infecções por Burkholderia/transmissão , Burkholderia cepacia/classificação , Burkholderia cepacia/isolamento & purificação , Burkholderia cepacia/patogenicidade , Criança , Fibrose Cística/complicações , Humanos , Prevalência , VirulênciaRESUMO
A polyphasic taxonomic study, including amplified fragment length polymorphism (AFLP) fingerprinting, DNA-DNA hybridizations, DNA base-ratio determinations, phylogenetic analysis, whole-cell fatty acid analyses and an extensive biochemical characterization, was performed on 19 Burkholderia cepacia-like isolates from the environment and cystic fibrosis (CF) patients. Several of the environmental isolates have attracted considerable interest due to their biocontrol properties. The polyphasic taxonomic data showed that the strains represent a new member of the B. cepacia complex, for which the name Burkholderia ambifaria sp. nov. is proposed. The type strain is strain LMG 19182T. B. ambifaria can be differentiated from the other members of the B. cepacia complex by means of AFLP fingerprinting, whole-cell fatty acid analysis, biochemical tests (including ornithine and lysine decarboxylase activity, acidification of sucrose and beta-haemolysis) and a newly developed recA gene-based PCR assay. 16S rDNA-based RFLP analysis and PCR tests allowed differentiation of B. ambifaria from Burkholderia multivorans, Burkholderia vietnamiensis and B. cepacia genomovar VI, but not from B. cepacia genomovars I and III and Burkholderia stabilis. The finding that this new taxon includes both strains isolated from CF patients and potentially useful biocontrol strains supports the general consensus that the large-scale use of biocontrol strains belonging to the B. cepacia complex would be ill-advised until more is known about their potential pathogenic mechanisms.
Assuntos
Burkholderia cepacia/classificação , Burkholderia cepacia/isolamento & purificação , Burkholderia/classificação , Burkholderia/isolamento & purificação , Fibrose Cística/microbiologia , Composição de Bases , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia cepacia/genética , Burkholderia cepacia/metabolismo , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/genética , Microbiologia Ambiental , Ácidos Graxos/análise , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Filogenia , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
Pulmonary infections caused by Burkholderia cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy and a propensity to cause bacteraemic infections in patients with CF. A mouse respiratory challenge model was used to investigate the invasion phenotype of B. cepacia in vivo. After intratracheal inoculation, epidemic B. cepacia strains translocated from lung to liver and spleen; however, all bacteria were cleared from all organs within 7 days. B. cepacia strains, irrespective of cable piliation, were capable of attaching to and then invading murine respiratory tract epithelial cells. Histopathological examination of lungs showed interstitial infiltrates comprised mainly of polymorphonuclear leucocytes and were associated with widened alveolar septa. Electron microscopy demonstrated B. cepacia within epithelial cells and pulmonary macrophages. This study provides support for in-vitro observations that B. cepacia strains from patients with CF adhere to and then invade respiratory epithelial cells. The invasion phenotype in B. cepacia may be an important virulence factor in CF infections.
Assuntos
Burkholderia cepacia/patogenicidade , Sistema Respiratório/microbiologia , Animais , Infecções por Burkholderia/complicações , Infecções por Burkholderia/microbiologia , Fibrose Cística/complicações , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Feminino , Fímbrias Bacterianas/fisiologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia EletrônicaRESUMO
Children with leukocyte adhesion deficiency type I are at risk for overwhelming infection because their neutrophils lack surface beta 2 integrins (CD18/CD11) that normally interact with endothelial cell adhesion molecules and mediate migration to sites of bacterial invasion. In vitro studies of phagocytic cells from an infant with leukocyte adhesion deficiency type I demonstrated that complement receptor 3 (CD18/CD11b) mediates nonopsonic phagocytosis of some Pseudomonas aeruginosa strains and might play a control role in the control of Pseudomonas infections at sites where there are low levels of opsonins.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/complicações , Fagocitose/imunologia , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/imunologia , Antígenos CD18 , Humanos , Recém-Nascido , Síndrome da Aderência Leucocítica Deficitária/imunologia , Antígeno de Macrófago 1 , Pseudomonas aeruginosaRESUMO
A polyphasic taxonomic study was performed on 23 strains isolated from cystic fibrosis (CF) patients in the USA. These strains were tentatively identified as Burkholderia cepacia, Burkholderia vietnamiensis and Burkholderia or Ralstonia sp. using biochemical tests and 16S rDNA-based PCR assays. Visual comparison of protein profiles indicated that they belonged to a single new group ('group 13'). The polyphasic taxonomic data showed that 18 of these strains represent a new member of the B. cepacia complex, referred to in this report as B. cepacia genomovar VI, whereas the other five strains belonged to Burkholderia multivorans. By means of biochemical tests, B. cepacia genomovar VI strains can be separated from B. cepacia genomovars I and III, Burkholderia stabilis, B. vietnamiensis and Burkholderia gladioli, but not from B. multivorans. Separation of B. cepacia genomovar VI and B. multivorans is possible using AFLP (amplified fragment length polymorphism) fingerprinting and DNA-DNA hybridizations. Retrospective analysis of epidemiological and genotypic data suggests that strains of B. cepacia genomovar VI have been involved in chronic colonization of CF patients and have been spread from person to person.
Assuntos
Infecções por Burkholderia/microbiologia , Burkholderia cepacia/classificação , Fibrose Cística/microbiologia , Proteínas de Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Composição de Bases , Infecções por Burkholderia/complicações , Burkholderia cepacia/genética , Classificação , Fibrose Cística/complicações , DNA Ribossômico/genética , Ácidos Graxos/isolamento & purificação , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Escarro/microbiologia , Estados UnidosRESUMO
Complement receptor 3 (CR3) mediates both opsonic and nonopsonic phagocytosis of bacteria. Leukocyte adhesion deficiency (LAD) allows for the study of CR3-dependent phagocyte-bacterial ingestion, since LAD phagocytes do not express this receptor. Phagocytes from an infant with LAD were unable to ingest 50% of the Pseudomonas aeruginosa strains studied, which indicates a requirement for CR3. However, the remaining strains were phagocytosed in the absence of CR3, and ingestion was blocked by monoclonal antibodies directed at CD14. This CR3/CD14 receptor bias was further confirmed by using thioglycollate-elicited murine peritoneal macrophages, which have nonfunctional CR3 before activation. Results indicate that either CR3 or CD14 is involved independently in nonopsonic phagocytosis of different P. aeruginosa strains. Clearance of P. aeruginosa from the endobronchial space may be facilitated by nonopsonic phagocytosis, since low levels of opsonins are present. The impact of lung infection with P. aeruginosa may be determined, in part, by the phagocytic receptor that mediates ingestion.
Assuntos
Receptores de Lipopolissacarídeos/sangue , Antígeno de Macrófago 1/sangue , Monócitos/microbiologia , Neutrófilos/microbiologia , Fagocitose/fisiologia , Pseudomonas aeruginosa , Adulto , Anticorpos Monoclonais/farmacologia , Antígenos CD/sangue , Fibrose Cística/microbiologia , Sangue Fetal , Citometria de Fluxo , Humanos , Recém-Nascido , Síndrome da Aderência Leucocítica Deficitária/sangue , Síndrome da Aderência Leucocítica Deficitária/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Neutrófilos/imunologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/isolamento & purificação , Valores de Referência , Especificidade da EspécieRESUMO
Recent taxonomic advances have demonstrated that Burkholderia cepacia is a cluster of at least seven closely related genomic species (or genomovars) collectively referred to as the B. cepacia complex, all of which may cause infections among cystic fibrosis patients and other vulnerable individuals. Thus, it is important for clinical microbiologists to be able to differentiate genomovars. Prior to this study, 361 B. cepacia complex isolates and 51 isolates easily confused with B. cepacia complex previously had been identified using a polyphasic approach, and in this study, a comparison of phenotypic and biochemical tests was carried out. It was determined that Burkholderia multivorans and Burkholderia stabilis could reliably be separated from other members of the B. cepacia complex by phenotypic methods. A combination of phenotypic and molecular tests such as recA PCR and 16S rRNA RFLP are recommended for differentiation among the genomovars of the B. cepacia complex. A biochemical reaction scheme for the identification of B. gladioli, Pandoraea species, and Ralstonia pickettii and the differentiation of these species from the B. cepacia complex is also presented.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções por Burkholderia/microbiologia , Burkholderia cepacia/classificação , Burkholderia cepacia/genética , Fibrose Cística/microbiologia , Burkholderia cepacia/metabolismo , DNA Ribossômico/análise , Genes de RNAr , Humanos , Fenótipo , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Recombinases Rec A/genéticaRESUMO
Pulmonary alveolar macrophages (AMphis) are incompetent to phagocytose unopsonized Pseudomonas aeruginosa, but ingestion by other macrophage phenotypes (i.e., peritoneal macrophages) occurs efficiently. The purpose of this study was to explore factors that might control such phenotypic differences. Our laboratory has demonstrated that AMphis exposed to sodium azide display enhanced phagocytosis of P. aeruginosa. Here we report that the phagocytic-enhancing effect of sodium azide was abrogated by inhibitors of protein kinase C (PKC). Furthermore, the addition of PKC agonists, such as phorbol myristate acetate (PMA), and tumor necrosis factor alpha (TNF-alpha), mimicked the phagocytic enhancing effect of sodium azide. We conclude that AM4phis are normally incompetent to phagocytose P. aeruginosa. Factors that up-regulate AMphi function (azide, PMA, TNF-alpha) can reverse the phagocytic incompetence in vitro. Although these compounds are not appropriate candidate therapeutic agents, their effects provide insights for understanding of the pathways responsible for regulation of P. aeruginosa phagocytosis.
Assuntos
Macrófagos Alveolares/fisiologia , Fagocitose/fisiologia , Proteína Quinase C/fisiologia , Pseudomonas aeruginosa/fisiologia , Animais , Carcinógenos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Macrófagos Alveolares/microbiologia , Camundongos , Fagocitose/efeitos dos fármacos , Azida Sódica/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.
Assuntos
Técnicas de Tipagem Bacteriana , Pseudomonas aeruginosa/classificação , Técnica de Amplificação ao Acaso de DNA Polimórfico , Fibrose Cística/microbiologia , Humanos , Pseudomonas aeruginosa/genéticaRESUMO
Alveolar macrophages (AM) provide one of the first lines of defense against microbial invasion in the lower airways. The role of AM in the clearance of Pseudomonas aeruginosa in mice after intrapulmonary challenge was evaluated. AM were depleted by intranasal administration of liposome-encapsulated dichloromethylene diphosphonate. At 24 h following the instillation of liposomes, a sublethal dose of P. aeruginosa was inoculated intranasally. Spleen, liver, and lung tissue was then evaluated for viable bacteria and for histopathology. AM depletion of 78 to 88% did not affect the survival rate of infected mice or clearance of P. aeruginosa from the spleen, liver, or lung, compared to the control group, but the mice's susceptibility to Klebsiella pneumoniae was greatly enhanced. The recruitment of neutrophils to the lung was also not affected. Freshly explanted AM were not competent to phagocytose unopsonized P. aeruginosa but were able to phagocytose zymosan particles. Further studies were conducted to assess the in situ phagocytic activities of AM. Three hours after the intranasal instillation of P. aeruginosa or other particles, bronchoalveolar lavage was performed. AM phagocytosis of zymosan particles and latex beads exceeded that of P. aeruginosa. Neutrophils were recruited to the lung in response to a high-dose bacterial challenge. These results suggest that AM do not play an important role in defense of the lung against P. aeruginosa.
Assuntos
Pulmão/imunologia , Macrófagos Alveolares/imunologia , Infecções por Pseudomonas/imunologia , Animais , Linhagem Celular , Ácido Clodrônico/farmacologia , Klebsiella pneumoniae/imunologia , Lipossomos , Fígado/imunologia , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Baço/imunologiaRESUMO
Most Burkholderia cepacia strains are resistant to many, or all, of the antibacterial agents commonly used in cystic fibrosis (CF), and selection of appropriate antibiotics for treatment of pulmonary exacerbations is therefore difficult. We developed a technique for rapid in vitro testing of multiple antibiotic combinations for B. cepacia isolates. For each of 119 multi-drug-resistant isolates of B. cepacia, our multiple combination bactericidal test (MCBT) studied the bactericidal activity of 10 to 15 antimicrobial agents using 225 +/- 97 single, double, and triple antibiotic combinations. Of the 119 isolates, 50% were resistant to all single antibiotics tested, 8% were resistant to all two-drug antibiotic combinations, but all were inhibited by at least one bactericidal triple-drug combination. When used alone, meropenem, ceftazidime and high-dose tobramycin (200 microg/ml) were bactericidal against only 47, 15, and 14% of in vitro isolates, respectively. Using a double antibiotic combination improved bactericidal activity; meropenem-minocycline, meropenem-amikacin, and meropenem-ceftazidime combinations were bactericidal against 76, 73, and 73% of isolates, respectively. However, 47% of isolates demonstrated antagonism (growth of an organism when a second antibiotic was added to a bactericidal single antibiotic). Triple antibiotic combinations that contained tobramycin, meropenem, and an additional antibiotic were most effective, and were bactericidal against 81 to 93% of isolates. We conclude that triple-antibiotic combinations are more likely than double and single antibiotic combinations to be bactericidal against B. cepacia in vitro. MCBT testing is a useful technique to help clinicians decide on appropriate nonantagonistic combination antibiotic therapy for patients with CF infected with B. cepacia.
Assuntos
Antibacterianos , Infecções por Burkholderia/complicações , Burkholderia cepacia/efeitos dos fármacos , Fibrose Cística/complicações , Quimioterapia Combinada/farmacologia , Adulto , Infecções por Burkholderia/tratamento farmacológico , Burkholderia cepacia/isolamento & purificação , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodosRESUMO
Pseudomonas aeruginosa causes severe respiratory tract infections in patients with cystic fibrosis (CF). We have been examining nonopsonic phagocytosis of P. aeruginosa by macrophages. To study the P. aeruginosa-macrophage interaction at the molecular level, we have constructed a transposon Tn5G bank in a clinical isolate of P. aeruginosa (strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocytosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of 900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85 putative mutants that were resistant to phagocytosis. In this study, we have characterized one of these transposon mutants, P. aeruginosa 4020 H27A, which was poorly ingested. H27A possessed a Tn5G insertion in a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have called this gene rpmA for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in P. aeruginosa. This rpmA::Tn5G mutant was motile both on agar plates and in visual examination of wet mounts. The phagocytosis defect was partially complemented by providing the rpmA gene in trans and fully complemented when both rpmA and rpmB were provided. A rpmA null mutant was ingested by macrophages similar to the H27A transposon mutant. These data suggest that the rpmA and rpmB gene products are required for the efficient ingestion of P. aeruginosa by macrophages.
Assuntos
Proteínas de Bactérias/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fagocitose/imunologia , Pseudomonas aeruginosa/imunologia , Proteínas de Bactérias/genética , Sequência de Bases , Criança , Fibrose Cística/microbiologia , Elementos de DNA Transponíveis , DNA Bacteriano , Teste de Complementação Genética , Humanos , Macrófagos/microbiologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Insercional , Pseudomonas aeruginosa/genéticaRESUMO
The Burkholderia cepacia complex currently comprises five genomic species, i.e., B. cepacia genomovar I, B. multivorans (formerly known as B. cepacia genomovar II), B. cepacia genomovar III, B. cepacia genomovar IV, and B. vietnamiensis (also known as B. cepacia genomovar V). In the absence of straightforward diagnostic tests for the identification of B. cepacia genomovars I, III, and IV, the last two genomic species were not formally classified as novel Burkholderia species (genomovar I contains the type strain and therefore retains the name B. cepacia). In the present study, we describe differential biochemical tests and a recA gene-based PCR assay for the routine identification of strains currently known as B. cepacia genomovar IV and propose formal classification of this organism as Burkholderia stabilis sp. nov. B. stabilis can indeed be differentiated from all other B. cepacia complex strains by the absence of beta-galactosidase activity, from strains of B. cepacia genomovars I and III and B. vietnamiensis by the inability to oxidize sucrose, and from B. multivorans by the lack of growth at 42 degrees C. In addition, analysis with the recA gene-derived primers BCRG41 (5'-ACCGGCGAGCAGGCGCTT-3') and BCRG42 (5'-ACGCCATCGGGCATGGCA-3') specifically allows the detection of B. stabilis strains in a conventional PCR assay. Examination of a set of 21 B. stabilis strains by means of random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis typing suggested that the genome of this organism is highly conserved, which is in sharp contrast to the generally accepted genomic diversity, variability, and plasticity among B. cepacia strains.
Assuntos
Burkholderia cepacia/classificação , Burkholderia cepacia/genética , Filogenia , Técnicas de Tipagem Bacteriana , Burkholderia cepacia/crescimento & desenvolvimento , Burkholderia cepacia/isolamento & purificação , Fibrose Cística/microbiologia , Impressões Digitais de DNA , Eletroforese em Gel de Campo Pulsado , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Recombinases Rec A/genética , Sacarose/metabolismo , beta-Galactosidase/análiseRESUMO
Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. A simple in vitro system to screen P. aeruginosa clinical isolates for their capacity to penetrate MDCK cell monolayers has been developed. By means of this system, P. aeruginosa clinical isolates, including 32 blood and 45 respiratory isolates, were examined. When monolayers were infected with 3.5x107 cfu of bacteria, significantly more blood (93.7%) than respiratory (54.4%) isolates (P<.001) were detected in the basolateral medium after 3 h. Penetration ability was usually independent of cytotoxicity. Only 8 (4 blood and 4 respiratory) isolates were cytotoxic, possessed exoU, and passed through the monolayer after epithelial cell death, associated with a marked drop in transepithelial electrical resistance. Conversely, noncytotoxic isolates with high penetration ability but without severe epithelial damage were invasive. This system is well suited for screening clinical isolates and their mutants for specific genes conferring the invasiveness phenotype.