Extensive Genomic Diversity among Bovine-Adapted Staphyloco…

Genomic Diversity of Bovine-Adapted Staphylococcus aureus

additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the “ authors contributions ” section. Competing Interests: The authors of this manuscript declare the following competing interests: S. Monecke is an employee of Alere Technologies GmbH, the company that manufactures the microarray used in this study. This had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

referred to as contagious or environmental species depending on their behaviour within dairy herds. Contagious pathogens generally spread from cow to cow with the infected udder being the primary source of infection whereas environmental pathogens, which are found in the envi- ronment where the cow resides, spread directly to the udder from the environment [2]. Staphy- lococcus aureus , a major mastitis pathogen is commonly considered a contagious pathogen, although recently it has been recognized that its epidemiological behaviour is not clear cut, with strains demonstrating contagious and/or environmental transmission patterns [3]. S . aureus presents an important economic problem for the global dairy industry [4, 5] and a poor cure rate has been identified as a significant hurdle for dairy producers [6]. Antimicrobial resistance of S . aureus is also an increasingly important issue. This bacterium has developed resistance to multiple classes of antibiotics including methicillin and other β -lactams [7, 8] and the horizontal transfer of antimicrobial resistance determinants between livestock and human- associated isolates is an increasing public health concern [9]. Previous studies, which compared diverse strains of S . aureus , identified genes common to all strains and these comprise the core genome [10, 11]. The remainder of the genome, termed the variable genome, is composed of strain-specific accessory genes often involved in virulence and the ability to colonise specific hosts or environments [12]. Specific lineages are adapted to colonise particular mammalian hosts [12, 13], however, host range barriers are not absolute with some lineages demonstrating a broad host range, while host shifts have also been reported [14]. The ability of S . aureus to adapt to a specific host is influenced by the acquisition of mobile genetic elements, gene diversification and decay [14]. Understanding the combinations of genes which are responsible for the success of dominant clonal lineages of S . aureus , and knowledge of the factors required for host specificity and virulence are important for under- standing the pathogenesis, management and treatment of S . aureus mastitis. In this study, the genetic heterogeneity of 126 S . aureus isolates from cases of clinical masti- tis in Ireland was evaluated both at the core and variable genome levels. The clonality of the isolates, the presence of important genes associated with virulence and the antimicrobial sus- ceptibility of the isolates to antibiotics commonly used to treat mastitis was also determined. Finally a genomic rearrangement in a subgroup of isolates belonging to clonal complex (CC) 97 was characterised. All 126 S . aureus isolates used in this study were recovered from milk samples taken from cows presenting clinical mastitis between February 2010 and February 2011 from 26 farms in Ire- land. Sample collection and bacterial isolation methods have been described previously [15]. Staphylococci were identified based on colony morphology, Gram stain, haemolysis, catalase test and growth on Baird Parker and Mannitol Salt agar plates. Putative S . aureus were distin- guished from coagulase negative Staphylococci by the above tests as well as the coagulase test and the API Staph strip (BioMerieux). Isolates were routinely grown in Trypticase Soy broth (TSB) or on Trypticase Soy agar for further study. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was carried out on all isolates using the disk diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [16]. Penicillin (6 μ g/10 IU), ampicillin (10 μ g), amoxycillin and clavulanic acid (20 μ g + 10 μ g), oxacillin (1 μ g), tetracycline (30 μ g), kanamycin (30 μ g), neomycin (30 IU), ceftiofur (30 μ g), enrofloxacin (5 μ g), erythromycin (15 μ g), clindamycin (2 μ g) cefalexin (30 μ g) and Materials and Methods Bacterial isolates

PLOS ONE | DOI:10.1371/journal.pone.0134592 August 28, 2015

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