Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.

Staphylococcus aureus is the most important and prevalent contagious mammary pathogen; it causes clinical and subclinical intramammary infection with serious economic loss and herd management problems in dairy cows. In vitro studies have shown that Staphylococcus aureus adheres to mammary epithelial cells and extracellular matrix components and invades into mammary epithelial as well as other mammary cells. Staphylococcus aureus strains from intramammary infection produce several cell surface-associated and extracellular secretory products. The exact pathogenic roles of most of the products and their effects on adhesion and invasion are not well evaluated. It is also known that mammary epithelial cell-associated molecules and extracellular matrix components interact with S. aureus during the pathogenesis of mastitis, but their roles on adhesion and invasion have not been characterized. The adhesion of S. aureus to epithelial cells may involve non-specific physicochemical interactions and/or specific interactions between bacterial cell-associated ligands and host cell surface receptors. In vitro adhesion depends on the S. aureus strain, the growth phase of the bacteria, the growth medium and the origin of the epithelial cells. Adhesion is hypothesized to be a prerequisite and crucial early step for mammary gland infection. Staphylococcus aureus invades mammary epithelial cells. It also invades other cells such as endothelial cells and fibroblasts. Bacteria are found enclosed in membrane bound vacuoles in the cytoplasm of mammary epithelial cells. Recent observations indicate that S. aureus escapes from the phagosome into the cytoplasm and induces apoptosis. The invasion into mammary epithelial cells may occur through an endocytic process that requires involvement of elements of the cytoskeleton or by direct binding of bacteria to epithelial cells through a process mediated by specific receptors that needs de novo protein synthesis by both cells. Thus, the recurrent subclinical infection may result from this intracellular existence of bacteria that are protected from host defenses and effects of antibiotics. This review emphasizes on recent findings on S. aureus adhesion to mammary epithelial cells and extracellular matrix components and invasion into mammary epithelial cells.