Caracterização fenotípica, genotípica e estudo de mecanismos de virulência de Enterococcus spp.

Abstract

Bovine mastitis is the most frequent and cost-generating illness of dairy cows allover the world. Many different microorganisms can infect the mammary gland. Enterococci are one of the environmental cause of mastitis. These opportunistic bacteria are a part of normal physiological gut flora in humans and animals. There are no sufficient data from Brazil about the occurrence of Enterococcus spp. in mastitic cow’s milk or about their susceptibility to antimicrobials, biofilm formation and interaction with bovine mammary epithelial cells. This investigation was undertaken to evaluate the occurrence of enterococci in cows with subclinical mastitis and to assess their antimicrobial resistance, genetic diversity, biofilm formation and the ability to interact with bovine mammary epithelial cells (MAC-T). Atotal of 11 samples were identified through genotypic and phenotypic tests. Data showed that Enterococcus faecalis (ddl - E. faecalis) was the predominant species (73%), followed by E. mundtii (sodA - E. mundtii; 27%). Antimicrobial susceptibility tests showed that the highest level of resistance was observed with tetracycline (82%), erythromycin (72%) and quinupristin-dalfopristin (63%). The presence of the vanA, vanB and vanC1/2 genes were not detected. Biofilm formation was performed using crystal violet staining at different times (6, 24 and 48h). Results demonstrated that all strains were biofilm producers in microplates. However, E. mundtii presented lower biofilm production when compared with E. faecalis. The biofilm formation was higher in bovine milk than in TSB medium after 24h incubation. PFGE analysis discerned 3 clones. For interaction assays with MAC-T, 3 strains were selected according to the different PFGE-types. All strains were able to adhere and invade MAC-T. These results indicated that biofilm formation by Enterococcus, especially in bovine milk might contribute in the pathogenesis during intramammary infection favoring the epithelial adhesion and invasion.