Análise da frequência de genes de resistência aos antimicrobianos em isolados bacterianos provenientes da microbiota intestinal de animais de corte e de matrizes aquáticas
Data
2022-01-28
Autores
Franchikoski, Milena Fajani 
Orientadores
Silva, Rodrigo Cayô
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Em maio de 2015, a Organização Mundial da Saúde (OMS) lançou um plano de ação global contra a resistência aos antimicrobianos. Atualmente, é reconhecido que a inclusão de antimicrobianos não essenciais na alimentação animal com a finalidade de promover o crescimento e a engorda dos animais voltados à pecuária intensiva constrói a problemática de que o uso de antimicrobianos em animais de corte pode ser vinculado diretamente com o aumento e a persistência de cepas multirresistentes em humanos. O conceito de Saúde Única se baseia na correlação de saúde humana, saúde animal e meio-ambiente, o que os estudos baseados neste conceito são essenciais no combate ao fenômeno da resistência bacteriana. Sendo assim, o presente estudo teve por objetivo avaliar a presença de cepas bacterianas resistentes aos antimicrobianos na microbiota intestinal de diferentes animais de corte e em matrizes aquáticas. Para isso foram coletadas previamente em oito propriedades localizadas no município de Toledo, Paraná, swabs retais de bovinos, suínos, criados para consumo próprio ou para grandes frigoríficos, além de amostras de água de tanques de peixes para consumo, nascente de irrigação, e do rio paraná. As amostras foram processadas no Laboratório ALERTA/UNIFESP em meio cromogênico acrescidos de antimicrobianos e bancadas em criotubos contendo TSB acrescido 15% de glicerol a -20ºC. Posteriormente, os isolados foram identificados por MALDI-TOF MS, tiveram o seu perfil de sensibilidade determinado por difusão em disco. A detecção de genes de resistência adquiridos aos β-lactâmicos, às fluoroquinolonas e aos aminoglicosídeos foi realizada por PCR. Ao foram recuperados 296 isolados, sendo a maioria obtidas de amostras de fezes de suínos (Criação Comercial) de gado leiteiro (Criação Comercial). A grande maioria (63,5%) dos isolados foram recuperados na presença de antimicrobianos -lactâmicos, enquanto um menor número de isolados foram recuperados na presença dos aminoglicosídeos amicacina+gentamicina (9,1%). Um total de 79,4% (n=235/296) foram identificados pela técnica de MALDI-TOF MS, dos quais oito isolados (3,4%) foram identificados como Candida krusei (n = 6) e Trichosporon asahii (n = 2). Entre os isolados não identificados pelo MALDI-TOF MS, 65,6% eram provenientes de fezes. Ao todo 19 gêneros bacterianos foram identificados entre os 227 isolados bacterianos pelo MALDI-TOF MS, sendo os mais frequentes: Escherichia spp. (49,3%), Stenotrophomonas spp. (11,4%), Acinetobacter spp. (9,7%) e Pseudomonas spp. (7,9%). Dos 227 isolados bacterianos, foram selecionados 98 isolados (43%) que apresentavam fenótipo de resistência antimicrobiana de interesse. As maiores taxas de resistência observadas foram: sulfametoxazol/trimetoprima (33,7%), tobramicina (29,6%), tetraciclina (28,6%) e ciprofloxacino (27,5%). Ao todo, 79,6% dos isolados apresentaram resistência à pelo menos um dos 17 antimicrobianos testados pela técnica de difusão em disco. Dentre os 11 genes de resistência aos antimicrobianos encontrados, a a maioria conferia resistência às fluoroquinolonas (80,5%) e aos B-lactâmicos (33,3%). Os principais genes de resistência encontrados foram qnrS, qepA e blaTEM-like. Uma correlação entre os genes de resistência aos antimicrobianos encontrados e os antimicrobianos utilizados nas terapias profiláticas e/ou terapêuticas dos animais avaliados foi observada.
In May 2015, the World Health Organization (WHO) launched a global action plan against antimicrobial resistance. Currently, it is recognized that the inclusion of non-essential antimicrobials in animal feed to promote the growth and fattening of animals for extensive livestock raises the problem that the use of antimicrobials in animals can be directly linked to the increase and the persistence of multidrug-resistant strains in humans. The One Health concept is based on the correlation of human and animal health with the environment, which studies based on this concept are essential in combating the phenomenon of bacterial resistance. Therefore, the present study aimed to evaluate the presence of antimicrobial-resistant bacterial strains in the intestinal microbiota of different beef animals and in aquatic matrices. For this, rectal swabs from cattle, swine, raised for their own consumption or for large slaughterhouses, in addition to samples of water from fish tanks for consumption, irrigation source, and from the Parana River. The samples were processed at the ALERTA/UNIFESP Laboratory in chromogenic medium plus antimicrobials and benches in cryotubes containing TSB plus 15% glycerol at -20ºC. Subsequently, the isolates were identified by MALDI-TOF MS and had their sensitivity profile determined by disk diffusion. The detection of acquired resistance genes to β-lactams, fluoroquinolones and aminoglycosides was performed by PCR. A total of 296 isolates were recovered, most of which were obtained from samples of swine feces (production center) and from dairy cattle (production center). The vast majority (63.5%) of the isolates were recovered in the presence of -lactam antimicrobials, while a smaller number of isolates were recovered in the presence of the aminoglycosides amikacin+gentamicin (9.1%). A total of 79.4% (n=235/296) were identified by the MALDI-TOF MS technique, of which eight isolates (3.4%) were identified as Candida krusei (n=6) and Trichosporon asahii (n=2). Among the isolates not identified by MALDI-TOF MS, 65.6% came from feces. A total of 19 bacterial genera were identified by MALDI-TOF MS among the 227 bacterial isolates, the most frequent being: Escherichia spp. (49.3%), Stenotrophomonas spp. (11.4%), Acinetobacter spp. (9.7%), and Pseudomonas spp. (7.9%). Of the 227 bacterial isolates, 98 isolates (43%) were selected for presenting antimicrobial resistance phenotypes of interest. The highest resistance rates observed were sulfamethoxazole/trimethoprim (33.7%), tobramycin (29.6%), tetracycline (28.6%), and ciprofloxacin (27.5%). Altogether, 79.6% of the isolates showed resistance to at least one of the 17 antimicrobials tested by the disk diffusion technique. Among the 11 antimicrobial resistance genes found, the majority conferred resistance to fluoroquinolones (80.5%) and -lactams (33.3%). The main resistance genes found were qnrS, qepA and blaTEM-like. A correlation between the antimicrobial resistance genes found and the antimicrobials used in the prophylactic and/or therapeutic therapies of the evaluated animals was observed.
In May 2015, the World Health Organization (WHO) launched a global action plan against antimicrobial resistance. Currently, it is recognized that the inclusion of non-essential antimicrobials in animal feed to promote the growth and fattening of animals for extensive livestock raises the problem that the use of antimicrobials in animals can be directly linked to the increase and the persistence of multidrug-resistant strains in humans. The One Health concept is based on the correlation of human and animal health with the environment, which studies based on this concept are essential in combating the phenomenon of bacterial resistance. Therefore, the present study aimed to evaluate the presence of antimicrobial-resistant bacterial strains in the intestinal microbiota of different beef animals and in aquatic matrices. For this, rectal swabs from cattle, swine, raised for their own consumption or for large slaughterhouses, in addition to samples of water from fish tanks for consumption, irrigation source, and from the Parana River. The samples were processed at the ALERTA/UNIFESP Laboratory in chromogenic medium plus antimicrobials and benches in cryotubes containing TSB plus 15% glycerol at -20ºC. Subsequently, the isolates were identified by MALDI-TOF MS and had their sensitivity profile determined by disk diffusion. The detection of acquired resistance genes to β-lactams, fluoroquinolones and aminoglycosides was performed by PCR. A total of 296 isolates were recovered, most of which were obtained from samples of swine feces (production center) and from dairy cattle (production center). The vast majority (63.5%) of the isolates were recovered in the presence of -lactam antimicrobials, while a smaller number of isolates were recovered in the presence of the aminoglycosides amikacin+gentamicin (9.1%). A total of 79.4% (n=235/296) were identified by the MALDI-TOF MS technique, of which eight isolates (3.4%) were identified as Candida krusei (n=6) and Trichosporon asahii (n=2). Among the isolates not identified by MALDI-TOF MS, 65.6% came from feces. A total of 19 bacterial genera were identified by MALDI-TOF MS among the 227 bacterial isolates, the most frequent being: Escherichia spp. (49.3%), Stenotrophomonas spp. (11.4%), Acinetobacter spp. (9.7%), and Pseudomonas spp. (7.9%). Of the 227 bacterial isolates, 98 isolates (43%) were selected for presenting antimicrobial resistance phenotypes of interest. The highest resistance rates observed were sulfamethoxazole/trimethoprim (33.7%), tobramycin (29.6%), tetracycline (28.6%), and ciprofloxacin (27.5%). Altogether, 79.6% of the isolates showed resistance to at least one of the 17 antimicrobials tested by the disk diffusion technique. Among the 11 antimicrobial resistance genes found, the majority conferred resistance to fluoroquinolones (80.5%) and -lactams (33.3%). The main resistance genes found were qnrS, qepA and blaTEM-like. A correlation between the antimicrobial resistance genes found and the antimicrobials used in the prophylactic and/or therapeutic therapies of the evaluated animals was observed.