RESUMO
Background: Clostridium difficile strain DH/NAP11/106, a relatively antibiotic-susceptible strain, is now the most common cause of C. difficile infection (CDI) among adults in the USA. Objectives: To identify mechanisms underlying the evolution and transmission of an MDR DH/NAP11/106 clone. Methods: WGS (Illumina MiSeq), restriction endonuclease analysis (REA) and antibiotic susceptibility testing were performed on 134 C. difficile isolates collected from paediatric patients with CDI over a 2 year period. Results: Thirty-one of 134 (23%) isolates were REA group DH. Pairwise single-nucleotide variant (SNV) analyses identified a DH clone causing seven instances of CDI in two patients. During the 337 days between the first and second CDI, Patient 1 (P1) received 313 days of antibiotic therapy. Clindamycin and rifaximin resistance, and reduced vancomycin susceptibility (MIC 0.5-2 mg/L), were newly identified in the relapsed isolate. This MDR clone was transmitted to Patient 2 (P2) while P1 and P2 received care in adjacent private rooms. P1 and P2 each developed two additional CDI relapses. Comparative genomics analyses demonstrated SNVs in multiple antibiotic resistance genes, including rpoB (rifaximin resistance), gyrB and a gene encoding PBP; gyrB and PBP mutations did not consistently confer a resistance phenotype. The clone also acquired a 46â¯000 bp genomic element, likely a conjugative plasmid, which contained ermB (clindamycin resistance). The element shared 99% identity with the genomic sequence of Faecalibacterium prausnitzii, an enteric commensal. Conclusions: These data highlight the emergence of MDR in C. difficile strain DH/NAP11/106 through multiple independent mechanisms probably as a consequence of profound antibiotic pressure.
Assuntos
Clostridioides difficile/classificação , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Transmissão de Doença Infecciosa , Evolução Molecular , Genótipo , Sequenciamento Completo do Genoma , Adolescente , Criança , Pré-Escolar , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/transmissão , DNA Girase/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla , Feminino , Hospitais Pediátricos , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Proteínas de Ligação às Penicilinas/genética , Proibitinas , Mapeamento por Restrição , Estados Unidos/epidemiologiaRESUMO
Antibiotics have been shown to influence the risk of infection with specific Clostridium difficile strains as well as the risk of C. difficile infection (CDI). We performed a retrospective case-control study of patients infected with the epidemic BI/NAP1/027 strain in a U.S. hospital following recognition of increased CDI severity and culture of stools positive by C. difficile toxin immunoassay. Between 2005 and 2007, 72% (103/143) of patients with first-episode CDIs were infected with the BI strain by restriction endonuclease analysis (REA) typing. Most patients received multiple antibiotics within 6 weeks of CDI onset (median of 3 antibiotic classes). By multivariate analysis, fluoroquinolone and macrolide exposure was more frequent among BI cases than among non-BI-infected controls (odds ratio [OR] for fluoroquinolones, 3.2; 95% confidence interval [CI], 1.3 to 7.5; (P < 0.001; OR for macrolides, 5.2; 95% CI, 1.1 to 24.0; P = 0.04)). In contrast, clindamycin use was less frequent among the BI cases than among the controls (OR, 0.1; 95% CI, 0.03 to 0.4; P = 0.001). High-level resistance to moxifloxacin and azithromycin was more frequent among BI strains (moxifloxacin, 49/102 [48%] BI versus 0/40 non-BI, P = 0.0001; azithromycin, 100/102 [98%] BI versus 22/40 [55%] non-BI, P = 0.0001). High-level resistance to clindamycin was more frequent among non-BI strains (22/40 [55%] non-BI versus 7/102 [7%] BI, P = 0.0001). Fluoroquinolone use, macrolide use, and C. difficile resistance to these antibiotic classes were associated with infection by the epidemic BI strain of C. difficile in a U.S. hospital during a time when CDI rates were increasing nationally due to the highly fluoroquinolone-resistant BI/NAP1/027 strain.
Assuntos
Antibacterianos/efeitos adversos , DNA Bacteriano/genética , Enterocolite Pseudomembranosa/etiologia , Fluoroquinolonas/efeitos adversos , Macrolídeos/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Estudos de Casos e Controles , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Farmacorresistência Bacteriana Múltipla , Enterocolite Pseudomembranosa/tratamento farmacológico , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterotoxinas/imunologia , Enterotoxinas/isolamento & purificação , Fezes/microbiologia , Feminino , Fluoroquinolonas/administração & dosagem , Humanos , Macrolídeos/administração & dosagem , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Proibitinas , Estudos Retrospectivos , Análise de Sobrevida , Estados UnidosRESUMO
The rising incidence of Clostridium difficile infections (CDIs) in adults is partly related to the global spread of fluoroquinolone-resistant strains, namely, BI/NAP1/027. Although CDIs are also increasingly diagnosed in children, BI/NAP1/027 is relatively uncommon in children. Little is known about the antibiotic susceptibility of pediatric CDI isolates. C. difficile was cultured from tcdB-positive stools collected from children diagnosed with CDI between December 2012 and December 2013 at an academic children's hospital. CDI isolates were grouped by restriction endonuclease analysis (REA). MICs were measured by agar dilution method for 7 antibiotics. Susceptibility breakpoints were based on guidelines from CLSI and/or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MICs and REA groupings of C. difficile isolates from 74 adult patients (29 isolates underwent REA) from a temporally and geographically similar adult cohort were compared to those of pediatric isolates. Among 122 pediatric and 74 adult isolates, respectively, the rates of resistance were as follows: metronidazole, 0% and 0%; vancomycin, 0% and 8% (P = 0.003); rifaximin, 1.6% and 6.7% (P = 0.11); clindamycin, 18.9% and 25.3% (P = 0.29); and moxifloxacin, 2.5% and 36% (P = <0.0001). Only 1 of 122 (0.8%) BI/NAP1/027 isolates was identified among the children, compared to 9 of 29 (31%) isolates identified among the adults (P = <0.0001). The 3 moxifloxacin-resistant pediatric isolates were of REA groups BI and CF and a nonspecific group. The 2 rifaximin-resistant pediatric isolates were of REA groups DH and Y. The 21 clindamycin-resistant pediatric isolates were distributed among 9 REA groups (groups A, CF, DH, G, L, M, and Y and 2 unique nonspecific REA groups). These data suggest that a diverse array of relatively antibiotic-susceptible C. difficile strains predominate in a cohort of children with CDI compared to adults.
Assuntos
Antibacterianos/uso terapêutico , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Adulto , Técnicas de Tipagem Bacteriana/métodos , Farmacorresistência Bacteriana/efeitos dos fármacos , Fezes/microbiologia , Humanos , Lactente , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular/métodos , Proibitinas , Estudos RetrospectivosRESUMO
OBJECTIVES: The aim of this study was to examine the antibiotic resistance profiles, antibiotic resistance mechanisms and possible 'clonal' nature of some MDR Bacteroides fragilis strains that simultaneously harboured cfiA, nimB, IS1186 and IS4351. METHODS: Antibiotic susceptibilities were determined by Etests and antibiotic resistance genes and different genetic elements were detected by applying PCR methods. The environments of the cfiA and nimB genes were also determined by sequencing. The transferability of the cfiA, nimB and tet(Q) genes was tested by conjugation. The genetic relatedness of the test strains was tested by ERIC-PCR or PFGE. The complete genome sequences of two strains (B. fragilis BF8 and O:21) were determined by next-generation sequencing. RESULTS: Most of the seven B. fragilis strains tested displayed multidrug resistance phenotypes; five strains were resistant to at least five types of antibiotics. Besides the common genetic constitution, ERIC-PCR implied high genetic relatedness. Similarities in some of the antibiotic resistance mechanisms [carbapenems (cfiA) and metronidazole (nimB)] also confirmed their common origin, but some other resistance mechanisms {MLSB [erm(F)] and tetracycline [tet(Q)]} and PFGE typing revealed differences. In B. fragilis BF8 and O:21, erm(F) and tet(X) genes were found with IS4351 borders, thus constituting Tn4351. All the strains were tet(Q) positive and transferred this gene in conjugation experiments, but not the cfiA and nimB genes. CONCLUSIONS: An international cluster of MDR B. fragilis strains has been identified and characterized. This 'clone' may have emerged early in the evolution of division II B. fragilis strains, which was suggested by the low-complexity ERIC profiles and differences in the PFGE patterns.
Assuntos
Infecções por Bacteroides/microbiologia , Bacteroides fragilis/classificação , Bacteroides fragilis/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Genótipo , Infecções por Bacteroides/epidemiologia , Bacteroides fragilis/genética , Bacteroides fragilis/isolamento & purificação , Análise por Conglomerados , Conjugação Genética , Elementos de DNA Transponíveis , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Eletroforese em Gel de Campo Pulsado , Ordem dos Genes , Transferência Genética Horizontal , Genes Bacterianos , Genoma Bacteriano , Saúde Global , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Tipagem Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes.
Assuntos
Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Conjugação Genética , Plasmídeos/genética , Animais , Sequência de Bases , Clostridium perfringens/classificação , Clostridium perfringens/isolamento & purificação , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos , Humanos , Tipagem de Sequências Multilocus , Plasmídeos/química , Prevalência , Microbiologia do SoloRESUMO
Antimicrobial susceptibility testing of anaerobic isolates was conducted at four independent sites from 2010 to 2012 and compared to results from three sites during the period of 2007-2009. This data comparison shows significant changes in antimicrobial resistance in some anaerobic groups. Therefore, we continue to recommend institutions regularly perform susceptibility testing when anaerobes are cultured from pertinent sites. Annual generation of an institutional-specific antibiogram is recommended for tracking of resistance trends over time.
Assuntos
Antibacterianos/farmacologia , Bactérias Anaeróbias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Farmacorresistência Bacteriana/fisiologia , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/fisiologia , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Estados Unidos/epidemiologiaRESUMO
Clostridium difficile is the causative agent of C. difficile-associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates (n=168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms (n=598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC90=0.25 µg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC50>32 µg/ml; n=270). Potent anti-C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 µM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti-C. difficile agent.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Animais , Antibacterianos/farmacocinética , Cricetinae , Masculino , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: The rationale and lessons learned through the evolution of the National Survey for the Susceptibility of Bacteroides fragilis Group from its initiation in 1981 through 2007 are reviewed here. The survey was conceived in 1980 to track emerging antimicrobial resistance in Bacteroides species. METHODS: Data from the last 11 years of the survey (1997-2007), including 6574 isolates from 13 medical centers, were analyzed for in vitro antimicrobial resistance to both frequently used and newly developed anti-anaerobic agents. The minimum inhibitory concentrations of the antibiotics were determined using agar dilution in accordance with Clinical and Laboratory Standards Institute recommendations. RESULTS: The analyses revealed that the carbapenems (imipenem, meropenem, ertapenem, and doripenem) and piperacillin-tazobactam were the most active agents against these pathogens, with resistance rates of 0.9%-2.3%. In the most recent 3 years of the survey (2005-2007), resistance to some agents was shown to depend on the species, such as ampicillin-sulbactam against Bacteroides distasonis (20.6%) and tigecycline against Bacteroides uniformis and Bacteroides eggerthii ( approximately 7%). Very high resistance rates (>50%) were noted for moxifloxacin and trovafloxacin, particularly against Bacteroides vulgatus. During that period of study, non-B. fragilis Bacteroides species had >40% resistance to clindamycin. Metronidazole-resistant Bacteroides strains were also first reported during that period. CONCLUSIONS: In summary, resistance to antibiotics was greater among non-B. fragilis Bacteroides species than among B. fragilis and was especially greater among species with a low frequency of isolation, such as Bacteroides caccae and B. uniformis. The emergence of resistance among the non-B. fragilis Bacteroides species underscores the need for speciation of B. fragilis group isolates and for clinicians to be aware of associations between species and drug resistance.
Assuntos
Antibacterianos/farmacologia , Bactérias Anaeróbias/efeitos dos fármacos , Bacteroides fragilis/efeitos dos fármacos , Bacteroides/efeitos dos fármacos , Farmacorresistência Bacteriana , Bacteriemia/microbiologia , Bacteroides/classificação , Bacteroides/isolamento & purificação , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/isolamento & purificação , Coleta de Dados , Humanos , Testes de Sensibilidade MicrobianaRESUMO
The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.
Assuntos
Antibacterianos/farmacologia , Clostridium perfringens/efeitos dos fármacos , Minociclina/farmacologia , Resistência a Tetraciclina/genética , Tetraciclina/farmacologia , Animais , Antiporters/genética , Proteínas de Bactérias/genética , Clostridium perfringens/genética , Genes Bacterianos , Humanos , Testes de Sensibilidade MicrobianaRESUMO
We report our continued experience with rifaximin as a post-vancomycin treatment strategy in six patients with multiple recurrences of C. difficile infection (CDI). Four of the six patients (67%) had no further diarrhea episodes, but two patients failed shortly after or during the rifaximin treatment. C. difficile isolates from one of the two patients who failed treatment had an MIC of >256 microg/ml to rifampin. Serial therapy with vancomycin, followed by rifaximin remains an option for some patients with multiple CDI recurrences.
Assuntos
Antibacterianos , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/tratamento farmacológico , Enterocolite Pseudomembranosa/tratamento farmacológico , Fármacos Gastrointestinais , Rifamicinas , Vancomicina , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Clostridium/prevenção & controle , Quimioterapia Combinada , Enterocolite Pseudomembranosa/prevenção & controle , Feminino , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Humanos , Masculino , Testes de Sensibilidade Microbiana , Rifamicinas/administração & dosagem , Rifamicinas/farmacologia , Rifamicinas/uso terapêutico , Rifaximina , Prevenção Secundária , Resultado do Tratamento , Vancomicina/farmacologia , Vancomicina/uso terapêuticoRESUMO
Rifaximin, a poorly absorbed rifamycin derivative, is a promising alternative for the treatment of Clostridium difficile infections. Resistance to this agent has been reported, but no commercial test for rifaximin resistance exists and the molecular basis of this resistance has not been previously studied in C. difficile. To evaluate whether the rifampin Etest would be a suitable substitute for rifaximin susceptibility testing in the clinical setting, we analyzed the in vitro rifaximin susceptibilities of 80 clinical isolates from our collection by agar dilution and compared these results to rifampin susceptibility results obtained by agar dilution and Etest. We found rifaximin susceptibility data to agree with rifampin susceptibility; the MICs of both antimicrobials for all isolates were either very low or very high. Fourteen rifaximin-resistant (MIC, > or = 32 microg/ml) unique isolates from patients at diverse locations in three countries were identified. Molecular typing analysis showed that nine (64%) of these isolates belonged to the epidemic BI/NAP1/027 group that is responsible for multiple outbreaks and increased disease severity in the United Kingdom, Europe, and North America. The molecular basis of rifaximin and rifampin resistance in these isolates was investigated by sequence analysis of rpoB, which encodes the beta subunit of RNA polymerase, the target of rifamycins. Resistance-associated rpoB sequence differences that resulted in specific amino acid substitutions in an otherwise conserved region of RpoB were found in all resistant isolates. Seven different RpoB amino acid substitutions were identified in the resistant isolates, which were divided into five distinct groups by restriction endonuclease analysis typing. These results suggest that the amino acid substitutions associated with rifamycin resistance were independently derived rather than disseminated from specific rifamycin-resistant clones. We propose that rifaximin resistance in C. difficile results from mutations in RpoB and that rifampin resistance predicts rifaximin resistance for this organism.
Assuntos
Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Rifampina/farmacologia , Rifamicinas/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Enterocolite Pseudomembranosa/microbiologia , Humanos , Dados de Sequência Molecular , Rifaximina , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Clostridium difficile PCR ribotype 106 (also identified as restriction endonuclease analysis [REA] group DH) recently emerged as the most common strain causing C. difficile infection (CDI) among US adults. We previously identified this strain predominating our pediatric cohort. Pediatric clinical CDI isolates previously characterized by REA underwent antibiotic resistance testing and whole genome sequencing. Of 134 isolates collected from children, 31 (23%) were REA group DH. We performed a comparative genomics analysis to identify DH-associated accessory genes. We identified five DH-associated genes that are associated with virulence in other bacterial species but not previously known to contribute to CDI. These genes are associated with intestinal mucosal adhesion (collagen-binding surface protein), sporulation (sporulation integral membrane protein YtvI), and protection from oxidative stress and foreign DNA (DNA phosphorothioation-dependent restriction proteins, sulfurtransferase, and DNA sulfur modification proteins). The association of these genes was validated in a cohort of 623 publicly available C. difficile sequences, 10 (1.6%) of which were monophyletic to REA group DH through in silico multilocus sequence typing and core genome phylogenetic analysis. Further investigation is required to determine the contribution of these genes to the emergence and virulence of this epidemic strain.
Assuntos
Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Genoma Bacteriano/genética , Genômica , Adolescente , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Criança , Pré-Escolar , Clostridioides difficile/classificação , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/patogenicidade , Infecções por Clostridium/enzimologia , Infecções por Clostridium/epidemiologia , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Proibitinas , Estados Unidos/epidemiologia , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Clostridium perfringens is an important human and animal pathogen that causes a number of diseases that vary in their etiology and severity. Differences between strains regarding toxin gene composition and toxin production partly explain why some strains cause radically different diseases than others. However, they do not provide a complete explanation. The purpose of this study was to determine if there is a phylogenetic component that explains the variance in C. perfringens strain virulence by assessing patterns of genetic polymorphism in genes (colA gyrA, plc, pfoS, and rplL) that form part of the core genome in 248 type A strains. We found that purifying selection plays a central role in shaping the patterns of nucleotide substitution and polymorphism in both housekeeping and virulence genes. In contrast, recombination was found to be a significant factor only for the virulence genes plc and colA and the housekeeping gene gyrA. Finally, we found that the strains grouped into five distinct evolutionary lineages that show evidence of host adaptation and the early stages of speciation. The discovery of these previously unknown lineages and their association with distinct disease presentations carries important implications for human and veterinary clostridial disease epidemiology and provides important insights into the pathways through which virulence has evolved in C. perfringens.
Assuntos
Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Animais , Primers do DNA/química , Evolução Molecular , Variação Genética , Genoma Bacteriano , Humanos , Modelos Genéticos , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Recombinação Genética , Temperatura , VirulênciaRESUMO
We have previously described the identification of BTF-37, an autonomously transferable chromosomal element isolated from Bacteroides fragilis clinical isolate LV23. In this study, we determined that BTF-37 harbors a 16kb conjugal transfer-encoding region that contains an almost identical copy of a previously identified Bacteroides sp. conjugation-specific gene bctA. BctA has been shown to be required for conjugation in other Bacteroides sp. strains, but no information is available regarding its function. We now report strain distribution and gene expression profiles of bctA. The bctA gene was present in conjugative transposon-harboring B. fragilis strains, but not on a non-transferable B. fragilis plasmid. We also showed that recombinant BctA predominantly localized to the bacterial membrane, and that its N-terminal 32 amino acids were cleaved in an Escherichia coli protein expression system, indicating the presence of a signal sequence. Expression of bctA consistently increased ~3-fold upon pre-exposure of conjugating B. fragilis LV23 to subinhibitory concentrations of tetracycline. Maximum expression occurred 60min post-tetracycline induction, which also coincided with the time at which highest conjugation frequencies were seen for strain LV23. Based on localization, signal sequence and tetracycline inducibility, our results indicate that BctA is indeed an important member of the Bacteroides conjugal apparatus, since its gene is regulated by conditions that specifically control conjugation.
Assuntos
Proteínas de Bactérias/genética , Bacteroides fragilis/genética , Conjugação Genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/química , Membrana Celular/química , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Plasmídeos , Sinais Direcionadores de Proteínas , Tetraciclina/farmacologiaRESUMO
We report here the complete genome sequence of Clostridioides difficile strain DH/NAP11/106/ST-42, which is now the most common strain causing C. difficile infection among U.S. adults. This strain was isolated from the stool from a hospitalized pediatric patient with frequent relapses of C. difficile infection.
RESUMO
Population pharmacokinetic modeling and Monte Carlo simulation (MCS) are approaches used to determine probability of target attainment (PTA) of antimicrobial therapy. The objectives of this study were 1) to determine a population pharmacokinetic model (PPM) using metronidazole and hydroxy-metronidazole concentrations from healthy subjects and critically ill patients, and 2) to determine the probability of attaining the pharmacodynamic target area under the plasma concentration (AUC)/MIC ratio >or=70 against 218 clinical isolates of Bacteroides fragilis using MCS. Eighteen healthy subjects were randomized to 3 dosages of intravenous metronidazole (500 mg every 8 h, 1000 mg day(-1), 1500 mg day(-1)) in an open-label 3-way crossover fashion. Serial blood samples were collected over 25.5 h on the 3rd day of each study period. An additional of 8 critically ill patients received intravenous metronidazole 500 mg every 8 h. Serial blood samples were collected over 8 h after the 2nd day of dosing. Plasma metronidazole and hydroxy-metronidazole concentrations were analyzed using a high-performance liquid chromatographic assay. The 834 plasma concentrations from 62 data sets were simultaneously modeled with Non-Parametric Adaptive Grid population modeling program. A 4-compartment model with a metabolite and zero-order infusion into the central compartment was used. The mean parameter vector and covariance matrix from PPM were inserted into the simulation module of ADAPT II. A 10,000-subject MCS was performed to determine the probability of PTA for a total drug AUC to MIC ratio >or=70 against 218 isolates of B. fragilis (MIC range, 0.125-2.0 mg L(-1)). Mean parameter values were CL(non-OH), 3.08 L h(-1); Vc, 35.4 L; K(OH), 0.04 h(-1); CL(OH), 2.78 L h(-1); and V(OH), 9.66 L. The regression values of the observed versus predicted concentrations (r2) of metronidazole and hydroxy-metronidazole were 0.972 and 0.980, respectively. The PTA for metronidazole 1500 mg day(-1) or 500 mg every 8 h (taken together) and 1000 mg day(-1) were 99.9% and 99.8%, respectively, over the reported MIC distribution range. For an MIC of 4 mg L(-1), the predicted PTA decreased to 80.0% and 28.5%, respectively. A PPM was determined by comodeling metronidazole and hydroxy-metronidazole concentrations from healthy subjects and critically ill patients. Based on this model, attainment of the target pharmacodynamic parameter (AUC/MIC ratio >or=70) against B. fragilis isolates is >99% when MICs are <2 mg L(-1), irrespective of the dosing interval of 24 h.
Assuntos
Anti-Infecciosos/farmacocinética , Bacteroides fragilis/efeitos dos fármacos , Metronidazol/farmacocinética , Administração Intravesical , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/sangue , Teorema de Bayes , Estudos Cross-Over , Esquema de Medicação , Quimioterapia Assistida por Computador/métodos , Humanos , Metronidazol/administração & dosagem , Metronidazol/sangue , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Método de Monte Carlo , Distribuição TecidualRESUMO
Hundreds of bacterial species make up human gut flora. Of these, 99% are anaerobic bacteria. Although anaerobes are part of the normal commensal flora, they can become opportunistic pathogens, causing serious, sometimes fatal infections if they escape from the colonic milieu. Most often, this escape occurs as a result of perforation, surgery, diverticulitis or cancer. Infections involving anaerobic bacteria are often difficult to treat because antibiotic resistance is increasing among the genera, mediated primarily through horizontal transfer of a plethora of mobile DNA transfer factors. Some of these transfer factors can also be transmitted to aerobic bacteria. It is becoming increasingly clear that antibiotic resistance trends have to be carefully monitored, and the transfer factors and mechanisms of transfer understood at a molecular level to avoid negative clinical outcomes when infections involve anaerobic bacteria.
Assuntos
Antibacterianos/uso terapêutico , Bactérias Anaeróbias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Intestinos/microbiologia , Farmacorresistência Bacteriana , HumanosRESUMO
Antibiotic-resistant anaerobic bacteria have become increasingly recognized as a confounding factor in the selection of therapeutic agents. The use of potent, broad-spectrum antibiotics as empirical therapy, along with appropriate adjunctive measures, has, in some ways, masked the magnitude of the antibiotic resistance problem that parallels that observed for nonanaerobic pathogens. The use of standardized testing methods that recognize resistance and an understanding of resistance mechanisms have become essential for the treatment of patients and the development of new agents.
Assuntos
Antibacterianos/farmacologia , Bactérias Anaeróbias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Resistência a Medicamentos/genética , Resistência a Medicamentos/fisiologia , Farmacorresistência Bacteriana/fisiologia , Frequência do Gene , Humanos , Testes de Sensibilidade Microbiana , Transdução GenéticaRESUMO
Susceptibility testing of anaerobes has been described for approximately 45 years, some 15 years fewer than descriptions for aerobic organisms. During that time period, >16 methods, >16 media, and a host of other variables have been described in the United States literature, culminating in the most recent standards published by the National Committee for Clinical Laboratory Standards (NCCLS) in 2001. These new guidelines include a single agar dilution reference method as well as an alternative minimal inhibitory concentration method validated by extensive multilaboratory collaborative trials. In addition, the Food and Drug Administration-approved E-test has proved valuable as a user-friendly alternative to NCCLS methods. Highlights of the "evolution" of various methods, their variations and their failures, factors that affected the development of the current standards, and the rationale for susceptibility testing of anaerobes are discussed herein.