Draft Genome Sequences of Staphylococcus aureus Strains Isolated from Subclinical Bovine Mastitis in Brazil

Here, we present the draft genome sequences of four Staphylococcus aureus strains isolated from mastitic milk collected from animals with subclinical manifestations. Three of them were typed as sequence type 126 (ST126), a genotype with no genome sequence available. ST126 is found in several herds of southern Brazil and is described as a bovine pathogen strongly associated with milk around the world.

S taphylococcus aureus is one of the main pathogens isolated from bovine mastitis infections (1). Intense efforts have been made to understand the molecular mechanisms of bacterial pathogenesis (2,3) and to link bacterial characteristics with the specific clinical manifestations of bovine mastitis (4). These strain-specific markers could be used to track relevant strains in herds that would positively affect animal health and welfare.
We monitored two herds in the State of Minas Gerais, Brazil, for the presence of subclinical mastitis for 6 months. Bacteria identified as S. aureus were genotyped by multilocus variablenumber tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). Four isolates were selected for genome sequencing. SAU-302 and SAU-1364 were isolated from two cows with a persistent subclinical infection, while SAU-170 and SAU-1269 were isolated from two cows with subclinical infections for a month. The infection was considered persistent if it was detected after three or more consecutive months from the same animal.
The protein sets were functionally annotated using BLAST searches (http://blast.ncbi.nlm.nih.gov/), and approximately 77% of the proteins of each strain were assigned Clusters of Orthologous Groups (COG) families (6).
In genotyping analysis, SAU-170, SAU-302, and SAU-1269 were classified as sequence type 126 (ST126), and SAU-1364 was classified as ST1 by multilocus sequence type (MLST) analysis (http://saureus.mlst.net/misc/info.asp). ST1 is a type isolated from bovine and human infections (7). ST126 is a prevalent genotype found in several herds in southern Brazil (8,9) and was described elsewhere as a bovine pathogen strongly associated with milk (7). We also genotyped the strains against the reference genome of S. aureus RF122 (accession no. NC_007622), a strain representative of the major clone involved in severe bovine mastitis worldwide (10). We identified single-nucleotide polymorphisms (SNPs) that resulted in amino acid changes in the coded protein. A total of 6,273 SNPs were detected in SAU-170, 5,481 in SAU-302, 7,420 in SAU-1269, and 2,491 in SAU-1364 (CLC bio; minimum, 20X coverage [11]). These new genomes add information to the repertoire of genes described for strains associated with subclinical mastitis that will be useful in studies to elucidate molecular mechanisms of pathogenesis.

ACKNOWLEDGMENTS
We thank the Núcleo de Análise de Biomoléculas (NuBioMol) for support during sequencing and the Programa de Desenvolvimento da Pecuária Leiteira (PDPL) for providing milk samples for bacterial isolation.