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dc.creatorMelo, Dayanne Araújo de-
dc.creator.Latteshttp://lattes.cnpq.br/1205965922491865por
dc.contributor.advisor1Souza, Miliane Moreira Soares de-
dc.contributor.advisor1Latteshttp://lattes.cnpq.br/0865211214618618por
dc.contributor.advisor-co1Coelho, Irene da Silva-
dc.contributor.referee1Coelho, Shana Mattos de Oliveira-
dc.contributor.referee2Marval, Márcia Giambiagi de-
dc.date.accessioned2020-04-08T01:35:53Z-
dc.date.issued2013-02-27-
dc.identifier.citationMELO, Dayanne Araújo de. Implicações da utilização de parâmetros humanos na detecção do gene mecA em Staphylococcus spp. isolados de mastite bovina e seus impactos na predição da resistência aos beta-lactâmicos em ambientes de produção leiteira. 2013. 2013. 63 f. Dissertação (Mestrado em Ciências Veterinárias) - Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica, 2013.por
dc.identifier.urihttps://tede.ufrrj.br/jspui/handle/jspui/3417-
dc.description.resumoStaphylococcus aureus apresenta um alto nível de resistência antimicrobiana, especialmente aos beta-lactâmicos, o que favorece a sua persistência no rebanho. A detecção do gene mecA é considerado uma predição desta resistência. No entanto, evidências nas pesquisas, revelam que a detecção do gene mecA não desempenha um papel significativo em relação a isolados de estafilococos de origem bovina. O presente trabalho foi realizado para investigar a presença de um homólogo, mecALGA251 e para estudar as regiões conservadas do gene mecA, a fim de desenvolver primers mais específicos. A amostragem compreende 38 S. aureus isolados a partir do leite (bovino), dois Staphylococcus coagulase-negativo (ECN) de mãos e narinas de ordenhadores, e também dois isolados de S. sciuri e um S. lentus, isolados de cavalos. Uma cepa padrão ATCC 43300 isolada de humano foi utilizada como controle. A detecção fenotípica da resistência à meticilina foi realizada através das técnicas de Ágar “screen” com oxacilina e difusão em disco simples com cefoxitina. Foram realizadas PCRs para detecção dos genes mecA e mecALGA251. Foi encontrada resistência à oxacilina em 20 isolados de S. aureus e em cinco ECN nos ensaios fenotípicos, porém somente nos isolados do ECN, de origem humana e de cavalo, o gene mecA foi detectado. Isolados de bovinos mecA-negativos também deram negativo para gene mecALGA251. A elaboração de primers, baseada em sequências de diferentes regiões conservadas do gene mecA foi realizada em duas etapas distintas. Na primeira etapa, os primers foram sintetizados com base nas sequências de nucleotídeos do gene mecA de Staphylococcus spp. (HE681097) de origem humana. Só foi observada a amplificação com estes primers em isolados oriundos de ordenhadores e de cavalos, porém nos isolados de origem bovina, foi encontrado resultado positivo apenas para os primers mecAint1. Com os primers definidos na segunda etapa, baseados na seqüência do gene mecA de S. sciuri (AY820253) de origem bovina, foi possível a amplificação somente nos isolados provenientes de vacas leiteiras. Através da comparação da sequência do gene mecA, considerando as diferentes origens, bovina, humana e de cavalos, observou-se diferenças pontuais, porém, significativas nas sequências de nucleotídeos da região de anelamento do primer elaborado por Murakami et al. (1991), o que pode levar à falha de detecção do gene mecA em isolados de bovinos. Também foi analisada a presença de genes reguladores do sistema mec, não sendo possível esta detecção em isolados de origem bovina. Portanto, mais estudos são necessários para entender melhor como esse mecanismo funciona para estes isolados.por
dc.description.abstractStaphylococcus aureus high antimicrobial resistance level, especially to beta-lactams, favors its persistence in herd environment. The detection of the mecA gene is considered a prediction of this resistance. Otherwise, research evidence does not support that mecA gene plays such a significant role concerning to bovine staphylococci isolates. The present work was carried out to investigate the presence of a homologue, mecALGA251 and to study the conserved regions in mecA gene in order to develop a more specific primer. Sampling comprises 38 S. aureus from milk (bovine), two Coagulase-Negative Staphylococcus (CNS) from milker’s hands and noses, and also two S. sciuri and one S. lentus from horses. A human standard strain (ATCC43300) was used as control. Phenotypic methicillin-resistance detection was carried out through oxacillin agar-screening and cefoxitin disk-diffusion tests. PCR were carried out to detect mecA and mecALGA251. Although, twenty isolates of S. aureus and five CNS were found oxacillin-resistant at the phenotypical assays, only the CNS isolates, from human and horse origin, tested positive for mecA gene. Bovine mecA-negative isolates also tested negative for mecALGA251 gene. A two-step procedure for primers design took in account sequences of distinct conserved regions in the mecA gene. In the first step, the newly synthesized primers were based on the nucleotide sequences of a human Staphylococcus spp. mecA gene (HE681097). Those primers were successful in amplifying mecA gene segment of human and horse Staphylococcus but mostly failed in bovine strains that tested positive only for the first internal segment set primers (mecAint1). Otherwise, the second-step set primers based on sequence of bovine S. sciuri mecA gene (AY820253) only yielded mecA gene segments for bovine strains. Comparison of the assembled contigs of mecA gene, from bovine, human and horse origins, displayed punctual but as far as we can see, significant differences in the nucleotide sequences, emphasizing the primer annealing site established by Murakami et al. (1991), what can lead to the mecA gene detection impairment in bovine isolates. It was also analyzed the occurrence of the regulatory genes of mec system, not detected in bovine isolates. Therefore, further studies are necessary to better understand how this mechanism works in these isolates.eng
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dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brasil.por
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dc.languageporpor
dc.publisherUniversidade Federal Rural do Rio de Janeiropor
dc.publisher.departmentInstituto de Veterináriapor
dc.publisher.countryBrasilpor
dc.publisher.initialsUFRRJpor
dc.publisher.programPrograma de Pós-Graduação em Ciências Veterináriaspor
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dc.rightsAcesso Abertopor
dc.subjectresistência aos beta-lactâmicospor
dc.subjectmastite bovinapor
dc.subjectmecA.por
dc.subjectantimicrobial resistanceeng
dc.subjectbovine mastitiseng
dc.subjectmecA geneeng
dc.subject.cnpqMedicina Veterináriapor
dc.titleImplicações da utilização de parâmetros humanos na detecção do gene mecA em Staphylococcus spp. isolados de mastite bovina e seus impactos na predição da resistência aos beta-lactâmicos em ambientes de produção leiteira. 2013por
dc.title.alternativeImplications of the use of human parameters for the mecA gene detection to predict beta-lactam resistance in bovine Staphylococcus spp. isolated from mastitis. 2013eng
dc.typeDissertaçãopor
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