Biofilm Formation by Enterococcus species of Bovine Mammary Gland and Environmental Origins

In vitro biofilm formation was compared among Enterococcus species of bovine origin. One hundred-seventeen enterococcal isolates were tested. Isolates originated from aseptically collected bovine quarter milk samples and bedding samples from a single commercial dairy. Isolates from bovine quarter milk samples originated from mammary glands with clinical mastitis, cows with high somatic cell counts, and postpartum milk samples. Bacterial species tested were Enterococcus faecium (n=80), Enterococcus casseliflavus (n=28), and Enterococcus faecalis (n=9). The bacterial species significantly contributed to the ability of organisms to produce biofilm. Enterococcus faecalis biofilm assay values were greater than for either E. faecium, E. casseliflavus, or negative controls. Biofilm results did not differ among E. faecium, E. casseliflavus, or negative controls. Origin of isolates did not affect biofilm formation. Assay values were comparable among isolates of bovine mammary gland origin and those of isolates from the environment. The ability to form biofilm in vitro does not appear to a pathogenicity factor for Enterococcus species associated with bovine intramammary infections. INTRODUCTION Enterococcus species often produce biofilms or large communities of bacteria living on a solid surface within a self-produced matrix (5). The production of biofilm in vitro by Enterococcus faecalis isolates from human clinical settings was correlated with pathogenicity and virulence of strains (4). The percentages of Enterococcus faecalis isolated from either infections or indwelling medical devices producing biofilm ranged from 94 to 100% (1). Enterococci are capable of infecting humans, companion animals, and livestock. Enterococci are normal gastrointestinal flora of dairy cows, but are also opportunistic pathogens that can cause bovine mastitis. Enterococci can infect humans and domestic animals because of their many virulence factors associated with biofilm formation including gelatinase, aggregation substance, capsule formation and enterococcal surface protein. Many strains are resistant to one or more antibiotics, including vancomycin (2). Biofilms are thought to contribute to this resistance. The three most commonly isolated species of enterococci from human and bovine clinical samples are E. faecalis, E. faecium, and E. casseliflavus. Unlike strains from clinical human settings, enterococcal strains isolated from bovine mammary glands, bedding, manure and feed have not been tested for biofilm formation. In vitro growth responses of enterococci in mammary secretions were different between isolates from the bovine mammary glands and those from the environment (3). The relationships between biofilm formation by enterococci and indirect and direct measures of pathogenicity within bovine mammary glands are undefined. OBJECTIVES 1) compare in vitro biofilm formation between isolates of bovine and environmental origins. 2) compare in vitro biofilm formation among Enterococcus species MATERIALS AND METHODS Isolates A total of 117 enterococcal isolates of bovine origin from a single commercial herd of approximately 1200 lactating cows managed in shaded dry lot corrals were tested for in vitro biofilm formation. The number of isolates by bacterial species and origin are in Table 1. Isolates of bovine origin from the commercial herd were from quarter milk samples collected using aseptic technique from high somatic cell count milk (SCC), mammary quarters with clinical mastitis prior to antibiotic therapy (CM), and cows within 3 days after parturition (Fresh). Isolates tested were also from dried manure bedding samples (Bedding) collected monthly from the commercial herd. TABLE 1. Enterococcus species isolates tested for in vitro biofilm formation. Origin of isolates* Enterococcus faecium Enterococcus casseliflavus Enterococcus faecalis TOTAL