Molecular characterization and virulence factors of Clostridium difficile in epidemiological multicentric study in Brazil

A doença associada ao Clostridium difficile (CDI) é mundialmente reconhecida como um problema de saúde pública, sendo a tipagem molecular uma ferramenta importante para o entendimento da disseminação da bactéria. Esse trabalho tem como objetivo identificar as STs (sequence type) de isolados de C. difficile através da tipagem molecular por MLST (multilocus sequence type), bem como identificar os fatores de virulência e resistência associados a essas cepas. Foram coletadas amostras de fezes diarreicas oriundas de oito hospitais de três estados brasileiros em dois dias pré-estabelecidos e 80 amostras de carnes embaladas. Para o isolamento da bactéria, utilizou-se o meio seletivo C. difficile CM0601 (Oxoid) suplementado com D-cicloserina e cefoxitina, em atmosfera de anaerobiose. MALDI-TOF MS (Bruker) foi utilizado para a identificação da bactéria em nível de espécie. O MLST foi realizado baseado no sequenciamento dos genes housekeeping: adk, atpA, dxr, glyA, recA, sodA e tpi. Para a análise dos fatores de virulência, sete amostras foram submetidas ao sequenciamento de nova geração pela plataforma Illumina Miseq. Em 13 amostras foi realizado o teste de Concentração Inibitória Mínima (CIM) através do método de ágar diluição para os antimicrobianos metronidazol e vancomicina. Do total de 153 amostras analisadas, foram recuperados das fezes 17 isolados de C. difficile, identificando-se as ST2, ST15, ST35, ST42, ST54, ST67 e oito novas STs. Genes de resistência como ermB, tetM, VanW e nimb, e outros genes associados aos fatores de virulência, como cwp84, cwp66, cwp2, fbpA e secA foram encontrados em cepas toxigênicas. A CIM apresentou isolados resistentes à vancomicina e ao metronidazol. A bactéria não foi encontrada em amostras de carnes. As diferentes STs encontradas no estudo revelam a grande variabilidade de cepas de C. difficile que circulam em hospitais brasileiros, contribuindo para um melhor entendimento da dinâmica de distribuição da bactéria no país.The disease associated with Clostridium difficile (CDI) is recognized worldwide as a public health problem, and molecular typing is an important tool for understanding the dissemination of the bacteria. This work aims to identify STs (sequence type) of C. difficile isolates through molecular typing by MLST (multilocus sequence type), as well as to identify the virulence and resistance factors associated with these strains. Samples of diarrheal stools from eight hospitals in three Brazilian states were collected on two pre-established days and 80 samples of packed meats. For the isolation of the bacteria, the C. difficile CM0601 (Oxoid) selective medium supplemented with D-cycloserine and cefoxitin was used under anaerobic conditions. MALDI-TOF MS (Bruker) was used to identify the bacteria at the species level. MLST was performed based on the sequencing of housekeeping genes: adk, atpA, dxr, glyA, recA, sodA and tpi. For the analysis of the virulence factors, seven samples were submitted to the new generation sequencing by the Illumina Miseq platform. In 13 samples the Minimum Inhibitory Concentration (MIC) test was performed using the agar dilution method for the antimicrobials metronidazole and vancomycin. From the total of 153 analyzed samples, 17 isolates of C. difficile were recovered from the stools and ST2, ST15, ST35, ST42, ST54, ST67 and eight new STs were identified. Resistance genes such as ermB, tetM, VanW and nimb, and other genes associated with virulence factors such as cwp84, cwp66, cwp2, fbpA and secA were found in toxigenic strains. The MIC presented isolates resistant to vancomycin and metronidazole. The bacteria were not found in meat samples. The different STs found in the study reveal the great variability of C. difficile strains circulating in Brazilian hospitals, contributing to a better understanding of the dynamics of bacterial distribution in the country

Genetic relatedness, virulence factors and antimicrobial resistance of C. difficile strains from hospitalized patients in a multicentric study in Brazil

Background: Clostridium (Clostridioides) difficile infection (CDI) is recognized worldwide as a public health concern, related mainly with hypervirulent strains. In Brazil there are few studies about molecular epidemiology of C. difficile, for this reason, we aimed to characterize C. difficile isolates from a large cohort study of three different Brazilian states to identify virulence and resistance genes, specifically genes related to metronidazole and vancomycin resistance. Methods: All 153 fecal samples were submitted to C. difficile culture in CM0601 broth. Identification of suspected colonies was confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF/MS, Brucker Daltonics, Germany). The tcdA and tcdB toxin were searched by PCR. The sequence type (ST) was determinate by multilocus sequencing typing (MLST) and susceptibility profile was performed by agar dilution method. Results: Among the 16 isolates, we identified fourteen different STs, five belonging to Clade 1, one to Clade 2 and eight news STs with high similarity levels. Resistance (ermB, tetM, VanW and nimB) and virulence genes (cwp84, cwp66, cwp2, fbpA and secA) were found in toxigenic strains. Conclusion: Differently from other studies, we found high levels of resistance to vancomycin. These results suggest that the main circulating strains in Brazil belong to Clade 1 and have high pathogenicity and resistance profile

Genetic relatedness, virulence factors and antimicrobial resistance of C. difficile strains from hospitalized patients in a multicentric study in Brazil

Background: Clostridium (Clostridioides) difficile infection (CDI) is recognized worldwide as a public health concern, related mainly with hypervirulent strains. In Brazil there are few studies about molecular epidemiology of C. difficile, for this reason, we aimed to characterize C. difficile isolates from a large cohort study of three different Brazilian states to identify virulence and resistance genes, specifically genes related to metronidazole and vancomycin resistance. Methods: All 153 fecal samples were submitted to C. difficile culture in CM0601 broth. Identification of suspected colonies was confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF/MS, Brucker Daltonics, Germany). The tcdA and tcdB toxin were searched by PCR. The sequence type (ST) was determinate by multilocus sequencing typing (MLST) and susceptibility profile was performed by agar dilution method. Results: Among the 16 isolates, we identified fourteen different STs, five belonging to Clade 1, one to Clade 2 and eight news STs with high similarity levels. Resistance (ermB, tetM, VanW and nimB) and virulence genes (cwp84, cwp66, cwp2, fbpA and secA) were found in toxigenic strains. Conclusion: Differently from other studies, we found high levels of resistance to vancomycin. These results suggest that the main circulating strains in Brazil belong to Clade 1 and have high pathogenicity and resistance profile