Analysis of the MRSA252 Genome Figure 2. EvoPrint and EvoUnique comparative analysis of 13 different S. aureus isolates reveals that only the human MRSA252 and contagious bovine mastitis RF122 chromosomes contain the malolactic enzyme gene. A) An EvoPrint generated from pairwise alignments of MRSA252 DNA (6,497 bp) and aligning regions from 12 other S. aureus genomes (S. aureus COL; S. aureus MSSA476, S. aureus Mu50; S. aureus MW2; S. aureus N315; S. aureus NCTC 8325; S. aureus RF122; S. aureus USA300; S. aureus JH1; S. aureus JH9; S. aureus Mu3 and S. aureus Newman) reveals high homology between all aligning regions except for a central 1,981 bp region. Uppercase black-colored letters identify MRSA252 sequences that align with all genomes and lowercase gray-colored bases indicate nucleotides that do not align with at least one of the genomes. The boxed ORF sequence encodes the 545 amino acid malolactic enzyme. B) Generated from the same analysis, the MRSA252 EvoUnique print indicates that only one of the 12 genomes included in the analysis contains homologous sequences that span the MRSA252 malolactic enzyme gene locus (SAR0824). Uppercase red-colored SNPs are unique to the MRSA252 sequence; green-colored letters represent bases that align with only one of the 12 genomes (S. aureus RF122) and lowercase gray-colored letters represent sequences that are common to two or more alignments. (C) Protein database homology searches identify the RF122 malolactic enzyme as sharing the highest identity with the MRSA252 enzyme. Note: no homologies to other malolactic enzyme encoding genes were detected in other S. aureus species. doi:10.1371/journal.pone.0003074.g002 However, our analysis reveals multiple instances of HGT events between RF122 and MRSA252 that are adjacent to MGE and bacteriophage sequences. In most cases, the flanking vector sequences are in MRSA252 and not in RF122, suggesting that MRSA252 or one of its related clones was the recipient in the HGT exchange. One likely scenario is that the DNA exchanges may have occurred during human and/or bovine co-infections. In light of the documented cases of direct transmission of MRSA between cows and humans [45] and the isolation in cows of a MRSA related to human epidemic strain Irish 01 [4], this possibility is highly probable. Clearly, more studies are required to determine how and where the putative HGT events took place. Nevertheless, the finding of unique virulence factor genes in a human pathogen whose potential source(s) may have originated from different causative agents of contagious bovine mastitis, including Listeria monocytogenes and S. saprophyticus, suggests that there may be a common epidemiological association between these bacteria and that co-infections are a likely point of origin for these exchanges. A similar concern regarding co-infection has been raised over the possibility of HGT of vancomycin resistance from enterococcus to S. aureus [46]. Taken together, these findings suggest that HGT events may be more prevalent between human and livestock bacteria than previously recognized and that animal husbandry practices that enhance contact between human and livestock pathogens should be avoided. Further work is required to elucidate the details and ramifications of these exchanges. Materials and Methods EvoPrinter analysis EvoPrinter algorithms consist of a series of web-accessed tools for discovering and comparing conserved or uniquely shared sequences within orthologous DNAs [21,22] (http://evoprinter. PLoS ONE | www.plosone.org 5 August 2008 | Volume 3 | Issue 8 | e3074