Characteristics of coagulase-negative staphylococci influencing virulence, antimicrobial resistance and inhibition of other mastitis pathogens

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Virulence characteristics of coagulase-negative staphylococci (CNS) isolates, the most frequently isolated bacteria from bovine udders, have not been well documented. We have currently identified 9 CNS isolates that inhibit in vitro growth of Gram-positive bacteria, particularly Staphylococcus aureus, and expect to identify more. This inhibition is likely conferred by bacteriocins. Multiple variants of these bacteriocins exist within a given strain, each with corresponding immunity genes that protect the producing strains from their own bacteriocidal/bacteriostatic products. Objectives of this research program are threefold. ***1) Candidate genes with known bacteriocin or immunity activity of major pathogens will be identified in silico using whole-genome sequences generated from the 9 in vitro inhibitory CNS isolates in comparison with non-inhibitory isolates. Prevalence of discovered bacteriocins will be confirmed in all 500 sequenced CNS genomes. Furthermore, the presence of bacterocin genes in these isolates will be confirmed phenotypically. Next, targeted mutagenesis will be used to confirm the role of identified genes in the observed inhibition of major pathogens. Finally, novel inhibitory genes and mechanisms will be identified by random transposon mutagenesis screens on some select isolates.***2) Expression of bacteriocins and corresponding immunity genes will be studied by qPCR in pure and co-cultures of CNS isolates and S. aureus to evaluate the potential of the strain as a probiotic. As bacterial gene expression may be differentially regulated in vivo, we will extend our in vitro bacteriocin expression assays into a murine model of mastitis. Additionally, intravital microscopy (IVM) will be used to image gene expression in infected mammary tissue, with the aid of bacteriocin promotor and fluorescent reporter gene constructs.***3) The in vivo inhibitory effect of CNS strains or proteins will be assessed using both murine and bovine models of mastitis to characterize how inhibitory CNS strains and major pathogens interact within the context of a soft-tissue infection. Mice infected intramammarily with CNS strains will be assayed for persistence of infection using whole-body imaging and IVM to visualize and characterize, in real-time, host immune response, tissue inflammation, and bacterial dynamics in live animals. Next, coinfection with CNS and S. aureus isolates will allow us to understand how 2 bacterial strains interact, localize and segregate in vivo. Finally, the role of identified bacteriocins in inhibition of major pathogens by CNS will be confirmed by directly comparing the inhibitory effect of wildtype CNS strains to specific mutant strains deficient for candidate bacteriocins. Persistence of CNS infections in cow udders will be studied and expression and production of protective bacteriocins will be quantified in vivo in a bovine mastitis model.**