CONICET | Buscador de Institutos y Recursos Humanos

Staphylococcus aureus is a major agent of contagious bovine mastitis and its ability to produce biofilms is considered a risk factor for persistent infections. Chitosan (Ch) is an acetylglucosamine polymer obtained from chitin, the second most abundant polysaccharide in nature. This biomaterial has antibacterial activity and inhibits biofilm formation on medical devices. This study aimed to examine in vitro biofilm formation in S. aureus isolated from bovine, and to evaluate the antibacterial activity of Ch as well as their efficacy in preventing biofilm formation. Samples from fifteen farms representing around 20% of dairy cows were randomly selected and analyzed by classical microbiological methods. The isolates were identified as S. aureus by PCR-amplified of 16S rRNA sequences. In the S. aureus isolated from ten samples we determined: a) the biofilm production in Congo red agar (CRA) cultures (qualitative assay); b) the amount of biofilm by the tissue culture plate (TCP) method and crystal violet (CV) staining (quantitative assays) and d) the expression of the biofilm-associated protein (Bap) gene by PCR, involved in biofilm formation only in bovine strains. We found that 8 out of 10 isolated S. aureus produced slime on CRA, which according to the TCP method and CV staining were classified as high (3:10) or low (6:10) biofilm producers. The Bap gene was found in only one of the 10 isolated strains. The effect of Ch was evaluated using the wild-type bovine mastitis hyperbiofilm S. aureus V329 strain. The inhibition of bacterial growth by Ch 100-800 ug/ml was tested in broth cultures during 16 hs. Compared with cultures without Ch, the growth rate was 31.7% at 100ug/ml and 8.5% at 800ug/ml (p<0,05). The UFC/ml showed a 2,4 log decrease with Ch 800ug/ml (p<0,05). The biofilm formation of V329 was reduced in a dose depended manner in CRA supplemented with 100-800 ug/ml Ch. An inhibition of of 3.2% (100ug/ml) to 42.8% (800ug/ml) (p<0,05) for biofilm formation were observed in V329 cultures grown in TCP with Ch (100-800 ug/ml) after 24h of incubation and CV stained. We determined the biofilm viability by the tetrazole MTT assay, and it was also significatively impaired by Ch with 7.2% reduction at 100ug/ml to 71.9% at 800ug/ml (p<0,05). The findings show the ability of S. aureus strains isolated from bovine mastitis to produce biofilm, which could be associated with persistent infections. Moreover, Ch exhibited effective bacteriostatic activity, inhibited biofilm formation and impaired biofilm viability. The effects of thispolysaccharide could contribute to eliminate the pathogen in the bovine mammary gland, acting a new agent for an alternative therapeutic required for bacterial control.