Caracterizaçao de estirpes de Staphylococcus spp isoladas em ambiente de ordenha e no leite bubalino
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Data
2017-02-03
Autores
Pizauro, Lucas José Luduverio [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Tendo em vista a importância e o crescente interesse na produção de leite de búfala e seus derivados e da ocorrência de Staphylococcus coagulase negativa (SCN) como patógenos da mastite tanto em bovinos como em bubalinos. O presente estudo objetivou avaliar genes de virulência, a resistência a antimicrobianos, bem como metodologias para correta identificação destes SCN em ambiente de ordenha e no leite de bubalinos. Foram colhidas 320 amostras de leite de quartos mamários de 80 búfalas escolhidas aleatoriamente, 20 amostras de narinas e 20 amostras da boca dos bezerros bubalinos, 16 amostras das mãos dos ordenhadores e 64 amostras de insufladores das teteiras, coletadas durante a ordenha. Vinte e sete cepas de Staphylococcus coagulase negativa foram positivas para o gene eno, 10 para o gene ebps, 10 para o gene fnbA. Em relação aos genes relacionados com a produção de enterotoxinas, apenas uma cepa foi positiva para o gene sea, uma para o gene see e para os genes relacionados a resistência antimicrobiana, uma cepa foi positiva para o gene mecA. A identificação das espécies isoladas foi realizada utilizando-se a metodologia de MALDI-TOF MS e confirmada por iniciadores espécie-especifico desenhados neste estudo, exceto para S. agnetis o qual foi erroneamente identificado como S. hyiucs por espectofotometria de massa. Neste trabalho a identificação destas duas espécies foi confirmada por sequenciamento genômico de um isolado representativo. Foram observadas quatro amostras resistentes à clindamicina, nove à vancomicina, uma ao cloranfenicol, sete à rifampicina, quatro a cefepime, sete à oxacilina, 17 à penicilina, 13 à eritromicina, 15 ao cotrimoxazole e três à tetraciclina. Resistência a dois ou mais princípios ativos antimicrobianos simultaneamente foi observada em 21 isolados. Os principais SCN isolados foram os S. chromogenes, S. agnetis e S. epidermidis. A detecção de genes relacionados à adesão e à produção de enterotoxinas e toxinas do choque toxico tornam estas cepas um risco potencial à saúde pública. A técnica de MALDI-TOF MS permite o diagnóstico rápido e preciso para algumas espécies de SCN, entanto o método deve ser utilizado com precaução ao avaliar amostras veterinárias. Os iniciadores desenhados para o gene cydB neste estudo podem ser utilizados para PCR convencional ou em tempo real
Due to the importance and the growing interest in buffalo milk production and its derivate and the concurrency of coagulase negative staphylococci as major mastitis pathogens both in cattle and in buffalo. This study aimed to search for virulence genes, antimicrobial resistance, and to evaluate methodologies for the identification of these microorganisms in samples of buffalo milk and from milking environment. For this, a total of 320 milk samples were collected from mammary quarters of 80 randomly selected buffaloes, 20 samples from nostrils and 20 samples from buffalo calves 'mouths, 16 samples from milking hands and 64 samples from liners were collected at the time of milking. Twenty-seven strains of coagulase negative staphylococci were positive for the eno gene, ten for ebpS gene, ten for the fnbA. Regarding genes related to enterotoxins production. Only one strain was positive for the sea and see gene and one for the mecA gene. The identification of the isolates was correctly done by MALDI-TOF MS and subsequently confirmed by species specific primers, except for S. agnetes that was wrongly identified as S. hyiucs. This identification was confirmed by genomic sequencing of a representative isolate from each species. There were four strains resistant to clindamycin, nine to vancomycin, one to chloramphenicol, seven to rifanmpicina, four to cefepime, seven to oxacillin, 17 to penicillin, 13 to erythromycin, 15 to cotrimoxazole and three to tetracycline. Furthermore, resistance to two or more antibiotics were observed in 21 isolates. The present study results may contribute to incidence prevention and control of mastitis in buffalo, caused by coagulase negative Staphylococcus. The main SCN species isolated were S. chromogenes, S. agnetis and S. epidermidis., the detection of genes related to adhesion and the production of enterotoxins make these strains a potential public health risk. The MALDI-TOF MS technique allows rapid and accurate diagnosis for some SCN, but the method should be used with caution when evaluating veterinary samples. The primers designed for the c and B gene in this study can be used for conventional or real-time PCR.
Due to the importance and the growing interest in buffalo milk production and its derivate and the concurrency of coagulase negative staphylococci as major mastitis pathogens both in cattle and in buffalo. This study aimed to search for virulence genes, antimicrobial resistance, and to evaluate methodologies for the identification of these microorganisms in samples of buffalo milk and from milking environment. For this, a total of 320 milk samples were collected from mammary quarters of 80 randomly selected buffaloes, 20 samples from nostrils and 20 samples from buffalo calves 'mouths, 16 samples from milking hands and 64 samples from liners were collected at the time of milking. Twenty-seven strains of coagulase negative staphylococci were positive for the eno gene, ten for ebpS gene, ten for the fnbA. Regarding genes related to enterotoxins production. Only one strain was positive for the sea and see gene and one for the mecA gene. The identification of the isolates was correctly done by MALDI-TOF MS and subsequently confirmed by species specific primers, except for S. agnetes that was wrongly identified as S. hyiucs. This identification was confirmed by genomic sequencing of a representative isolate from each species. There were four strains resistant to clindamycin, nine to vancomycin, one to chloramphenicol, seven to rifanmpicina, four to cefepime, seven to oxacillin, 17 to penicillin, 13 to erythromycin, 15 to cotrimoxazole and three to tetracycline. Furthermore, resistance to two or more antibiotics were observed in 21 isolates. The present study results may contribute to incidence prevention and control of mastitis in buffalo, caused by coagulase negative Staphylococcus. The main SCN species isolated were S. chromogenes, S. agnetis and S. epidermidis., the detection of genes related to adhesion and the production of enterotoxins make these strains a potential public health risk. The MALDI-TOF MS technique allows rapid and accurate diagnosis for some SCN, but the method should be used with caution when evaluating veterinary samples. The primers designed for the c and B gene in this study can be used for conventional or real-time PCR.
Descrição
Palavras-chave
Alinhamento, Buballus bubalis, Genômica, Genes de virulência, MALDI-TOF MS, Alignment, Genomic, Virulence genes