Caracterização molecular de genes de resistência a cefalosporinas de espectro estendido em Escherichia coli isoladas de frango e carnes de frango
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2016-11-03
Autores
Casella, Tiago [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A resistência a cefalosporinas de espectro estendido (ESC) mediada pela produção de β-lactamases de espectro estendido (ESBL) por Enterobacteriaceae é um problema de saúde pública global. Neste contexto, o aumento da resistência a ESC entre Escherichia coli isoladas de animais de produção é uma questão importante, uma vez que bactérias comensais de animais de produção podem contaminar os produtos alimentícios e chegar ao intestino humano via cadeia alimentar. O Brasil é um importante produtor e o maior exportador de carne de frango no mundo. Assim, é de extrema importância monitorar a presença dessas bactérias neste setor alimentar. No presente estudo, foi realizada uma comparação do genótipo de resistência a ESC em E. coli isoladas a partir de carnes de frango no Brasil e na França, considerando que não há relação comercial de carne de frango entre esses dois países. Esta abordagem pode ser útil para compreender as dinâmicas dos fatores de resistência na cadeia de produção de frangos. Além disso, linhagens isoladas do trato gastrointestinal de frangos no Brasil foram também estudadas. Para isso, amostras de carnes de frango de diferentes pontos do varejo e swabs de cloaca de frangos de três diferentes fazendas foram coletadas no Estado de São Paulo, Brasil. Também, amostras de carnes de frango de diferentes pontos do varejo de Lyon, França, foram amostradas. As amostras de carne e os swabs de cloaca de frangos foram inoculados em ágar MacConkey acrescido com ESC. As colônias foram identificadas por sistema automatizado, e a suscetibilidade aos antimicrobianos foi testada por disco-difusão. PCR para detecção de genes blaESBL e blapAmpC e para a determinação dos grupos filogenéticos de E. coli foram realizadas. Os genes bla foram sequenciados e os plasmídeos foram caracterizados pelo esquema PBRT e por southern blot/hibridação de géis de PFGE-S1. Quase todas as carnes de frango apresentaram, ao menos, uma linhagem de E. coli resistente a ESC, e 53,8% dos frangos analisados também estavam colonizados por E. coli resistente a ESC. Resistência a aminoglicosídeos, fenicóis, quinolonas, sulfonamidas, tetraciclina e trimetoprim também foi detectada. Um total de 60 linhagens isoladas de amostras do Brasil foram estudadas, e o gene blaCTX-M-2 foi o principal responsável pela resistência a ESC, mas blaCTX-M-55, blaCMY-2, blaCTX-M-15 e blaCTX-M-8 também foram detectados. De 77 linhagens isoladas de amostras de carne de frango da França, o gene blaCTX-M-1 foi o principal responsável pela resistência a ESC, mas blaTEM-52, blaCMY-2 e blaSHV-12 também foram detectados. A maioria dos blaCTX-M-2 estão associados à ISCR1 e a integrons complexos de classe 1, e blaCTX-M-1, blaCTX-M-15, blaCTX-M-55, e os oito blaCMY-2 do Brasil estão precedidos pela ISEcp1. O gene blaCTX-M-8 está associado à IS10. Nas amostras do Brasil, um gene blaCTX-M-2 é carreado por um plasmídeo IncHI2/P, blaCTX-M-8 por um plasmídeo IncI1, um gene blaCTX-M-15 por um plasmídeo IncX1, e os genes blaCTX-M-55 por plasmídeos IncFII ou IncN/FII. Nas amostras de carnes de frango da França, os genes blaCTX-M-1 são carreados por plasmídeos IncI1 ou IncFII, blaTEM-52 por plasmídeos IncI1 ou IncX1, blaCMY-2 por plasmídeos IncA/C ou IncK, e o gene blaSHV-12 por um plasmídeo IncI1. Todos os quatro principais grupos filogenéticos de E. coli (A, B1, B2, D) foram detectados, porém o filogrupo D foi predominante entre as linhagens do Brasil, e os filogrupos D, A e B1 foram igualmente distribuídos nas linhagens recuperadas na França. Em resumo, E. coli presentes na cadeia de produção de frangos carreiam genes de ESBL e de AmpC plasmideal, e genes, plasmídeos e linhagens resistentes a antimicrobianos parecem ser específicos em cada região geográfica.
Resistance to extended spectrum cephalosporins (ESC) mediated by the production of extended-spectrum β-lactamases (ESBL) by Enterobacteriaceae is a global public health concern. In this context, the raise in ESC-resistance among Escherichia coli isolated from food-producing animals is an important issue, since commensal bacteria from producing animals may contaminate food products and reach the human gut through the food chain. Brazil is an important producer and the greatest exporter of chicken meat in the world, so, it is of utmost importance to monitor the presence of these bacteria in this food sector. In this study, a comparison of the resistance genotype to ESC in E. coli isolated from chicken meat produced in Brazil and France was performed, considering that there is no commercial relationship of chicken meats between these countries. This approach may be useful to understand the dynamics of the resistance factors in chicken production chain. Also, isolates from cloacal swabs collected in Brazil were studied. For this, samples of chicken meat from different markets and chicken cloacal swabs from three separate farms were sampled in the State of São Paulo, Brazil. In addition, chicken meat samples were acquired from markets in Lyon, France. Meat samples and cloacal swabs were inoculated on MacConkey agar supplemented with ESC. Colonies were identified by an automated system, and antimicrobial susceptibility was tested by disc diffusion. PCRs for detection of blaESBL and blapAmpC genes were performed and phylogenetic groups were determined. The bla genes were sequenced, and plasmids were characterized by rep-typing and southern blot/hybridization on S1-PFGE gels. Almost all pieces of chicken meat presented at least one ESC-resistant E. coli, and 53.8% of chickens were also colonized by ESC-resistant E. coli. Resistance to phenicols, quinolones, aminoglycoside and sulphonamides was also detected. A total of 60 strains isolated from Brazilian samples was studied and the blaCTX-M-2 gene was the main responsible for the ESC-resistance, but blaCTX-M-55, blaCMY-2, blaCTX-M-15 and blaCTX-M-8 were also detected. From 77 strains isolated from chicken meat samples from France, the blaCTX-M-1 gene was the main responsible for ESC-resistance, but blaTEM-52, blaCMY-2 and blaSHV-12 were also detected. Most of blaCTX-M-2 were associated to ISCR1 and complex class 1 integrons, and blaCTX-M-1, blaCTX-M-15, blaCTX-M-55, and the eight blaCMY-2 from Brazil were preceded by ISEcp1. The blaCTX-M-8 gene was associated to IS10. In samples from Brazil, one blaCTX-M-2 gene was harbored by an IncHI2/P plasmid, blaCTX-M-8 was carried by an IncI1 plasmid, one blaCTX-M-15 was carried by an IncX1 plasmid, and the blaCTX-M-55 genes were harbored by IncFII or IncN/FII plasmids. In chicken meats from France, the blaCTX-M-1 genes were harbored by IncI1 or IncFII plasmids, blaTEM-52 were carried by IncI1 or IncX1 plasmids, blaCMY-2 were carried by IncA/C or IncK plasmids, and the blaSHV-12 gene was harbored by an IncI1 plasmid. All four major phylogenetic groups (A, B1, B2, D) were present, but phylogroup D was predominant among strains from Brazil, and phylogroups D, A and B1 were equally distributed in strains recovered in France. In summary, E. coli in the chicken production chain carry genes for ESBL and plasmidic AmpC, and genes, plasmids and antimicrobial resistant strains seem to be specific for each geographic region.
Resistance to extended spectrum cephalosporins (ESC) mediated by the production of extended-spectrum β-lactamases (ESBL) by Enterobacteriaceae is a global public health concern. In this context, the raise in ESC-resistance among Escherichia coli isolated from food-producing animals is an important issue, since commensal bacteria from producing animals may contaminate food products and reach the human gut through the food chain. Brazil is an important producer and the greatest exporter of chicken meat in the world, so, it is of utmost importance to monitor the presence of these bacteria in this food sector. In this study, a comparison of the resistance genotype to ESC in E. coli isolated from chicken meat produced in Brazil and France was performed, considering that there is no commercial relationship of chicken meats between these countries. This approach may be useful to understand the dynamics of the resistance factors in chicken production chain. Also, isolates from cloacal swabs collected in Brazil were studied. For this, samples of chicken meat from different markets and chicken cloacal swabs from three separate farms were sampled in the State of São Paulo, Brazil. In addition, chicken meat samples were acquired from markets in Lyon, France. Meat samples and cloacal swabs were inoculated on MacConkey agar supplemented with ESC. Colonies were identified by an automated system, and antimicrobial susceptibility was tested by disc diffusion. PCRs for detection of blaESBL and blapAmpC genes were performed and phylogenetic groups were determined. The bla genes were sequenced, and plasmids were characterized by rep-typing and southern blot/hybridization on S1-PFGE gels. Almost all pieces of chicken meat presented at least one ESC-resistant E. coli, and 53.8% of chickens were also colonized by ESC-resistant E. coli. Resistance to phenicols, quinolones, aminoglycoside and sulphonamides was also detected. A total of 60 strains isolated from Brazilian samples was studied and the blaCTX-M-2 gene was the main responsible for the ESC-resistance, but blaCTX-M-55, blaCMY-2, blaCTX-M-15 and blaCTX-M-8 were also detected. From 77 strains isolated from chicken meat samples from France, the blaCTX-M-1 gene was the main responsible for ESC-resistance, but blaTEM-52, blaCMY-2 and blaSHV-12 were also detected. Most of blaCTX-M-2 were associated to ISCR1 and complex class 1 integrons, and blaCTX-M-1, blaCTX-M-15, blaCTX-M-55, and the eight blaCMY-2 from Brazil were preceded by ISEcp1. The blaCTX-M-8 gene was associated to IS10. In samples from Brazil, one blaCTX-M-2 gene was harbored by an IncHI2/P plasmid, blaCTX-M-8 was carried by an IncI1 plasmid, one blaCTX-M-15 was carried by an IncX1 plasmid, and the blaCTX-M-55 genes were harbored by IncFII or IncN/FII plasmids. In chicken meats from France, the blaCTX-M-1 genes were harbored by IncI1 or IncFII plasmids, blaTEM-52 were carried by IncI1 or IncX1 plasmids, blaCMY-2 were carried by IncA/C or IncK plasmids, and the blaSHV-12 gene was harbored by an IncI1 plasmid. All four major phylogenetic groups (A, B1, B2, D) were present, but phylogroup D was predominant among strains from Brazil, and phylogroups D, A and B1 were equally distributed in strains recovered in France. In summary, E. coli in the chicken production chain carry genes for ESBL and plasmidic AmpC, and genes, plasmids and antimicrobial resistant strains seem to be specific for each geographic region.
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Palavras-chave
Escherichia coli, ESBL, pAmpC, Frango