RESUMO
In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.
Assuntos
Antibacterianos , Infecções por Bacteroides , Humanos , Antibacterianos/farmacologia , Bacteroides , Bacteroides fragilis/genética , Equador , beta-Lactamases/genética , Infecções por Bacteroides/tratamento farmacológico , Infecções por Bacteroides/microbiologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Proteínas de Bactérias/genéticaRESUMO
Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.
Assuntos
Infecções por Bacteroides/imunologia , Bacteroides fragilis/imunologia , Fibras na Dieta/efeitos adversos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Peritonite/imunologia , Animais , Infecções por Bacteroides/microbiologia , Dieta , Fibras na Dieta/administração & dosagem , Modelos Animais de Doenças , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Peritonite/microbiologia , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.
Assuntos
Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/genética , Infecções por Bacteroides/microbiologia , Proteínas Repressoras/genética , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/isolamento & purificação , Bacteroides fragilis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Testes de Sensibilidade Microbiana , Proteínas Repressoras/metabolismoRESUMO
Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/genética , Proteínas Repressoras/genética , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/isolamento & purificação , Bacteroides fragilis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Testes de Sensibilidade Microbiana , Proteínas Repressoras/metabolismoRESUMO
CfiA (CcrA) metallo-ß-lactamase is the main carbapenem resistance mechanism in B. fragilis. From cfiA positive isolates detected in a previous surveillance study, 3 displayed resistance to imipenem while the remaining were susceptible. The aim of this study was to identify the cfiA alleles and to analyze the presence of IS elements in their upstream regions. CfiA-1, CfiA-4, CfiA-13, CfiA-19 and CfiA-22 were detected. IS elements belonging to IS21 family and IS942 group were identified upstream to cfiA in the 3 imipenem resistant isolates. We present an exhaustive analysis of cfiA/CfiA registers in databases, illustrating the inconsistencies in both organization and nomenclature. According to this analysis CfiA family comprises nowadays 15 different CfiA variants coded by 24 cfiA sequences. Curation of CfiA database is mandatory, if not new cfiA admission at GenBank will contribute to make this classification more complex.
Assuntos
Proteínas de Bactérias/genética , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/classificação , Bacteroides fragilis/genética , Fases de Leitura Aberta , beta-Lactamases/genética , Alelos , Antibacterianos/farmacologia , Argentina/epidemiologia , Infecções por Bacteroides/epidemiologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Vigilância em Saúde PúblicaRESUMO
Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.
Assuntos
Toxinas Bacterianas/genética , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/genética , Bacteroides fragilis/isolamento & purificação , Fezes/microbiologia , Genótipo , Metaloendopeptidases/genética , Animais , Infecções por Bacteroides/epidemiologia , Bacteroides fragilis/classificação , Brasil/epidemiologia , Criança , Pré-Escolar , DNA Bacteriano/genética , Feminino , Humanos , Incidência , Masculino , Tipagem Molecular , Reação em Cadeia da PolimeraseRESUMO
Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.
Assuntos
Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Toxinas Bacterianas/genética , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/genética , Bacteroides fragilis/isolamento & purificação , Fezes/microbiologia , Genótipo , Metaloendopeptidases/genética , Infecções por Bacteroides/epidemiologia , Bacteroides fragilis/classificação , Brasil/epidemiologia , DNA Bacteriano/genética , Incidência , Tipagem Molecular , Reação em Cadeia da PolimeraseRESUMO
As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.
Assuntos
Antibacterianos/farmacologia , Infecções por Bacteroides/microbiologia , Bacteroides fragilis/efeitos dos fármacos , Bacteroides fragilis/crescimento & desenvolvimento , Animais , Infecções por Bacteroides/genética , Infecções por Bacteroides/metabolismo , Bacteroides fragilis/patogenicidade , Feminino , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Virulência/efeitos dos fármacosRESUMO
Multidrug resistance in Bacteroides spp. and related genera is uncommon and has not been described in Latin America until now. We studied phenotypically and genotypically the multidrug resistance of 10 clinical strains of Bacteroides, two of Parabacteroides distasonis, and one of Pseudoflavonifractor capillosus recovered in a national hospital between 2006 and 2010. To this end, we determined minimum inhibitory concentrations (MICs) of amoxicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, clindamycin, ciprofloxacin, tetracycline, and metronidazole using E-tests, evaluated the isolates for ß-lactamases with nitrocefin hydrolysis tests, performed a polymerase chain reaction (PCR)-based screening of erm, tet, and nim genes, obtained partial gyrA sequences, and studied the effect of tazobactam and efflux pump inhibitors (EPI) on the MIC of cefotaxime, clindamycin, and ciprofloxacin. Three isolates were resistant to four different classes of antibiotics and 10 were resistant to three. ß-lactam resistance was in most cases due to ß-lactamases susceptible of partial inhibition by tazobactam. Ten isolates were cfxA-positive and two isolates had cepA. Twelve isolates were highly resistant to clindamycin and nine were highly resistant to ciprofloxacin. However, these phenotypes were not linked to ermA, ermB, ermF, and ermG or mutations in gyrA. Addition of EPI lowered the MICs of clindamycin and ciprofloxacin of one and four isolates, respectively. Twelve isolates had tetQ and four were positive for tetM. In both cases, genes of the two-component system RteAB accompanied tet genes. Although metronidazole susceptibility was universal, nim genes were not present. To our knowledge, this is the first report of multidrug resistance due to less commonly identified or alternative mechanisms in strains of Bacteroides and related species from a developing country.
Assuntos
Antibacterianos/farmacologia , Infecções por Bacteroides/epidemiologia , Bacteroides/genética , Infecções por Clostridium/epidemiologia , Clostridium/genética , Genes Bacterianos , Bacteroides/efeitos dos fármacos , Bacteroides/enzimologia , Bacteroides/isolamento & purificação , Infecções por Bacteroides/tratamento farmacológico , Infecções por Bacteroides/microbiologia , Clindamicina/farmacologia , Clostridium/efeitos dos fármacos , Clostridium/enzimologia , Clostridium/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Costa Rica/epidemiologia , Farmacorresistência Bacteriana Múltipla , Genótipo , Humanos , Metronidazol/farmacologia , Mutação , Fenótipo , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologiaRESUMO
The frequency of finding of clindamycin-resistant anaerobic bacteria in clinical samples has doubled from 2008 to 2010 in Costa Rica. To determine whether this increase is due to dissemination of erm genes aided by tetQ elements, we analyzed 100 isolates of Bacteroides or Parabacteroides from a regional hospital, a national hospital, and the community. Antimicrobial susceptibilities were recorded with a broth micro-dilution method and erm genes were detected by PCR and Southern blotting. In addition, plasmid isolation and mating experiments were performed to clarify the location and mobility of the detected erm genes. Resistance to clindamycin was by far more frequent in the regional hospital (72%) than in the national hospital (29%) and the community (26%). Resistance to tetracycline was even more common, with the community (85%) outweighing the hospitals (71-72%). While MIC of clindamycin were higher in the hospitals than in the community (P < 0.05), the opposite was seen for tetracycline (P < 0.0001). Of the sought-after genes, only ermG (n = 2), ermA (n = 1), and ermF (n = 1) were detected in the hospitals and ermF in the community (n = 2). In opposition to the low frequency of finding of erm genes, 71% of the isolates were positive for tetQ. None of the detected genes were encoded on plasmids. Only three isolates from the hospitals transferred their erm genes laterally. By contrast, 13 hospital isolates and two community isolates transferred tetQ. Despite the widespread finding of tetracycline-resistant tetQ-positive bacteria, mobile erm genes were rare in our bacterial collection. We conclude that the detected erm genes are likely not included in typical conjugative transposons of Bacteroides and Parabacteroides.