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

Abstract

Staphylococcus 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.