Caracterização molecular de Escherichia coli, isolada de leite de vacas com mastite clínica
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Data
2019-08-09
Autores
Casale, Fernanda Cristina de Campos
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Universidade Estadual Paulista (Unesp)
Resumo
A mastite bovina é uma das doenças que mais causam prejuízos às propriedades leiteiras e, apesar dos programas de controle, Escherichia coli destaca-se como importante patógeno ambiental causador desta enfermidade na sua forma clínica. Isolados obtidos de infecções nas mamas são classificadas como E. coli patogênica mamária (MPEC). O objetivo deste estudo foi caracterizar geneticamente isolados de E. coli a partir de leite de vacas com mastite clínica utilizando técnicas moleculares, bem como investigar os padrões de aderência e resistência aos antimicrobianos dos isolados, para isso a investigação dos grupos filogenéticos, genes de virulência, presença de clones (por Pulsed field gel electrophoresis, PFGE), tipos de adesão em células HeLa, resistência aos antimicrobianos e genes relacionados com essa resistência foram realizados em 110 E. coli isoladas de leite de vacas com mastite clínica de diferentes fazendas dos estados de São Paulo, Minas Gerais e Paraná. De acordo com a presença dos genes específicos arpA, chuA, yjaA e TspE4.C2, os isolados foram classificados principalmente nos grupos comensais A (50,9 %) e B1 (38,2 %), seguidos dos grupos D (2,7 %), C (1,8 %) e B2/E/F/Escherichia clade I (0,9 %). Três isolados (2,7 %) não foram categorizados em nenhum desses grupos, classificados como de grupo filogenético desconhecido. Nenhum dos 110 isolados apresentou genes que caracterizam E. coli diarreiogênicas (DEC), entretanto os genes astA e shf, geralmente encontrados em E. coli enteroagregativa, foram identificados em 7,3 % (8/110) e 10,3 % (3/29) dos isolados, respectivamente. Entre os genes que caracterizaram E. coli extra-intestinal (ExPEC), fimH (93,6 %) foi o mais frequente, seguidos pelos genes traT (77,3 %), ompT (68,2 %), ecpA (65,5 %), irp2 (34,5 %), fimA (28,2 %), sitA (26,4 %), cdt (10 %), KpsMTII/iroN/iucD (3,6 %), hlyA/ireA/ibe10 (1,8 %) e papA/papC/sat/vat (0,9 %). Pela análise de PFGE, observou-se 103 pulsotipos e seis clones, quatro dos quais obtidos na mesma fazenda em animais diferentes e um no mesmo animal, em diferentes momentos da lactação, indicando casos de persistência de E. coli nas infecções intramárias. Segundo os parâmetros de adesão em células HeLa, 26 (23,6 %) isolados aderiram de forma agregativa, enquanto quatro (3,6 %) aderiram de forma difusa; o restante dos isolados foram caracterizados com aderência não característica, não aderentes ou promoveram o destacamento do tapete celular. Em relação à ação dos antimicrobianos, 19,1 % dos isolados foram resistentes à tetraciclina e a maioria (99,1 %), foram sensíveis à cefoxitina. Ainda, cinco (4,5 %) isolados foram produtores de ESBL em teste fenotípico, ocorrendo a presença dos genes blaTEM (31,8 %), blaCTX-M-2/ blaCTX-M-8 (1,8 %) e blaCTX-M-15 (0,9 %). O gene blaCMY-2 foi relatado em um (0,9 %) isolado de E. coli. Conclui-se que os isolados de E. coli de mastite clínica bovina foram bastante heterogêneos em suas identidades gênicas, não sendo possível apresentar um padrão de virulência característico para o patotipo MPEC, pois os genes ecpA, papA, shf, ibe10, ompT, ireA, cdt, sat e blaCTX-M-8, já descritos genericamente em ExPEC, foram observados nos isolados de mastite pela primeira vez nesse trabalho. Não foram identificadas DEC e por isso, os isolados de mastite apresentam pequeno potencial de risco zoonótico, transmissível pelo leite bovino, bem como os genes associados a processos diarreiogênicos não parecem não apresentar influência no estabelecimento de mastite clínica Além disso, embora grande parte dos isolados não tenham produzido β-lactamases no teste fenotípico, a presença de cepas carreadoras desses genes no ambiente representa preocupação em saúde pública, em virtude do aumento de resistência de E. coli obtidas de animais.
Bovine mastitis is one of the diseases that most causes damage to dairy properties and, despite control programs, Escherichia coli stands out as an important environmental pathogen that causes this disease in its clinical form. Isolates obtained from breast infections are classified as mammary pathogenic E. coli (MPEC). The objective of this study was to genetically characterize E. coli isolates from milk of cows with clinical mastitis using molecular techniques, as well as to investigate the adhesion patterns and antimicrobial resistance of isolates, for this purpose the investigation of phylogenetic groups, virulence genes, presence of clones (by Pulsed field gel electrophoresis, PFGE), HeLa cell adhesion types, antimicrobial resistance, and genes related to this resistance were performed on 110 E. coli isolated from milk of cows with clinical mastitis from different state farms from São Paulo, Minas Gerais and Paraná. According to the presence of the specific genes arpA, chuA, yjaA and TspE4.C2, isolates were classified mainly in commensal groups A (50.9 %) and B1 (38.2 %), followed by groups D (2.7 %), C (1.8 %) and B2/E/F/Escherichia clade I (0.9 %). Three isolates (2.7 %) were not categorized in any of these groups, classified as unknown phylogenetic group. None of the 110 isolates presented genes that characterize diarrheagenic E. coli (DEC), however, the astA and shf genes, commonly found in enteroaggregative E. coli, were identified in 7.3 % (8/110) and 10.3 % (3/29) of the isolates, respectively. Among the genes that characterized extraintestinal pathogenic E. coli (ExPEC), fimH (93.6 %) was the most frequent, followed by traT (77.3 %), ompT (68.2 %), ecpA (65, 5 %), irp2 (34.5 %), fimA (28.2 %), sitA (26.4 %), cdt (10 %), KpsMTII/iroN/iucD (3.6 %), hlyA/ireA/ibe10 (1.8 %) and papA/papC/sat/vat (0.9 %). By PFGE analysis, 103 pulsotypes and six clones were observed, four of which were obtained from the same farm in different animals and one in the same animal at different times of lactation, indicating cases of E. coli persistence in intrammary infections. According to adhesion parameters in HeLa cells, 26 (23.6 %) isolates adhered aggregatively, while four (3.6 %) adhered diffusely; the remaining isolates were characterized with non-characteristic adherence, non-adherence or promoted detachment of the cell mat. Regarding the action of antimicrobials, 19.1 % of the isolates were resistant to tetracycline and most (99.1 %) were sensitive to cefoxitin. In addition, five (4.5 %) isolates produced ESBL in phenotypic test, with the presence of blaTEM (31.8 %), blaCTX-M-2/blaCTX-M-8 (1.8 %) and blaCTX-M-15 (0.9 %). The blaCMY-2 gene was reported in one (0.9 %) E. coli isolate. It was concluded that the isolates of E. coli from clinical bovine mastitis were very heterogeneous in their gene identities. It is not possible to present a characteristic virulence pattern for the MPEC pathotype, since the genes ecpA, papA, shf, ibe10, ompT, ireA, cdt, sat and blaCTX-M-8, already described generically in ExPEC, were observed in mastitis isolates for the first time in this work. No DEC were identified, therefore, mastitis isolates have little potential for zoonotic risk, transmissible through bovine milk, and genes associated with diarrhiogenic processes do not seem to have any influence on the establishment of clinical mastitis. In addition, although most isolates have not produced β-lactamases in the phenotypic test, the presence of carrier strains of these genes in the environment is a concern in public health, due to the increased resistance of E. coli obtained from animals.
Bovine mastitis is one of the diseases that most causes damage to dairy properties and, despite control programs, Escherichia coli stands out as an important environmental pathogen that causes this disease in its clinical form. Isolates obtained from breast infections are classified as mammary pathogenic E. coli (MPEC). The objective of this study was to genetically characterize E. coli isolates from milk of cows with clinical mastitis using molecular techniques, as well as to investigate the adhesion patterns and antimicrobial resistance of isolates, for this purpose the investigation of phylogenetic groups, virulence genes, presence of clones (by Pulsed field gel electrophoresis, PFGE), HeLa cell adhesion types, antimicrobial resistance, and genes related to this resistance were performed on 110 E. coli isolated from milk of cows with clinical mastitis from different state farms from São Paulo, Minas Gerais and Paraná. According to the presence of the specific genes arpA, chuA, yjaA and TspE4.C2, isolates were classified mainly in commensal groups A (50.9 %) and B1 (38.2 %), followed by groups D (2.7 %), C (1.8 %) and B2/E/F/Escherichia clade I (0.9 %). Three isolates (2.7 %) were not categorized in any of these groups, classified as unknown phylogenetic group. None of the 110 isolates presented genes that characterize diarrheagenic E. coli (DEC), however, the astA and shf genes, commonly found in enteroaggregative E. coli, were identified in 7.3 % (8/110) and 10.3 % (3/29) of the isolates, respectively. Among the genes that characterized extraintestinal pathogenic E. coli (ExPEC), fimH (93.6 %) was the most frequent, followed by traT (77.3 %), ompT (68.2 %), ecpA (65, 5 %), irp2 (34.5 %), fimA (28.2 %), sitA (26.4 %), cdt (10 %), KpsMTII/iroN/iucD (3.6 %), hlyA/ireA/ibe10 (1.8 %) and papA/papC/sat/vat (0.9 %). By PFGE analysis, 103 pulsotypes and six clones were observed, four of which were obtained from the same farm in different animals and one in the same animal at different times of lactation, indicating cases of E. coli persistence in intrammary infections. According to adhesion parameters in HeLa cells, 26 (23.6 %) isolates adhered aggregatively, while four (3.6 %) adhered diffusely; the remaining isolates were characterized with non-characteristic adherence, non-adherence or promoted detachment of the cell mat. Regarding the action of antimicrobials, 19.1 % of the isolates were resistant to tetracycline and most (99.1 %) were sensitive to cefoxitin. In addition, five (4.5 %) isolates produced ESBL in phenotypic test, with the presence of blaTEM (31.8 %), blaCTX-M-2/blaCTX-M-8 (1.8 %) and blaCTX-M-15 (0.9 %). The blaCMY-2 gene was reported in one (0.9 %) E. coli isolate. It was concluded that the isolates of E. coli from clinical bovine mastitis were very heterogeneous in their gene identities. It is not possible to present a characteristic virulence pattern for the MPEC pathotype, since the genes ecpA, papA, shf, ibe10, ompT, ireA, cdt, sat and blaCTX-M-8, already described generically in ExPEC, were observed in mastitis isolates for the first time in this work. No DEC were identified, therefore, mastitis isolates have little potential for zoonotic risk, transmissible through bovine milk, and genes associated with diarrhiogenic processes do not seem to have any influence on the establishment of clinical mastitis. In addition, although most isolates have not produced β-lactamases in the phenotypic test, the presence of carrier strains of these genes in the environment is a concern in public health, due to the increased resistance of E. coli obtained from animals.
Descrição
Palavras-chave
AmpC, antibiograma, ESBL, infecção intramamária, MPEC, antibiogram, intramammary infection