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
Bacteroides fragilis, an opportunistic pathogen and commensal bacterium in the gut, is one the most aerotolerant species among strict anaerobes. However, the mechanisms that control gene regulation in response to oxidative stress are not completely understood. In this study, we show that the MarR type regulator, BmoR, regulates the expression of genes involved in the homeostasis of intracellular redox state. Transcriptome analysis showed that absence of BmoR leads to altered expression in total of 167 genes. Sixteen of these genes had a 2-fold or greater change in their expression. Most of these genes are related to LPS biosynthesis and carbohydrates metabolism, but there was a significant increase in the expression of genes related to the redox balance inside the cell. A pyridine nucleotide-disulfide oxidoreductase located directly upstream of bmoR was shown to be repressed by direct binding of BmoR to the promoter region. The expression of two other genes, coding for a thiosulphate:quinone-oxidoreductase and a thioredoxin, are indirectly affected by bmoR mutation during oxygen exposure. Phenotypic assays showed that BmoR is important to maintain the thiol/disulfide balance in the cell, confirming its relevance to B. fragilis response to oxidative stress.
Assuntos
Bacteroides fragilis , Dissulfetos/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo/genética , Proteínas Repressoras , Compostos de Sulfidrila/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Perfilação da Expressão Gênica , Oxirredução , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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
Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.
Assuntos
Bacteroides fragilis/genética , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Animais , Bacteroides fragilis/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , RNA Mensageiro/isolamento & purificação , Análise de Sequência de RNARESUMO
Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.
Assuntos
Proteínas de Bactérias/genética , Bacteroides fragilis/genética , Bacteroides/genética , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/genética , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bacteroides/metabolismo , Bacteroides fragilis/metabolismo , Liases de Carbono-Enxofre/metabolismo , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Homosserina/biossíntese , Lactonas , Dados de Sequência Molecular , Percepção de Quorum , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
In the past few years, many studies revealed a remarkable genetic variability in Bacteroides fragilis species, and the existence of two divisions was proposed according to presence or absence of the cfiA (metallo-ß-lactamase/carbapenemase) gene. The aim of this study was to evaluate the use of DNA sequence analysis for glutamate dehydrogenase (gdh), phosphoglucomutase (pgm) and esterase (est) metabolic genes, in comparison to RNA polymerase ß subunit (rpoB) and 16S ribosomal RNA (rrs) gene sequencing, to identify the presence of these two groups in seventeen B. fragilis strains. Based on phylogenetic trees, only the est gene sequences generated a classification similar to rrs- and rpoB-genes. On the other hand, the genes pgm and gdh did not allow the discrimination of these divisions. The est gene sequence can be suggested as an additional tool for differentiation of the two groups in B. fragilis, providing highly reproducible and reliable data in B. fragilis taxonomy.