between different bacterial genera; transfer of vancomycin resistance has been shown to occur between Enterococcus faecalis and S. aureus [18]. Other examples of transfer under conditions of co- infection include the transfer of antibiotic resistance from E. coli to the plague bacillis, Yersinia pestis [19], and transfer of vancomycin resistance from porcine to human Enterococcus faecium [20]. Multi-genome alignments play an important role in the identification of unique or uniquely shared HGT DNA sequences among related or evolutionary distant bacteria, and hence can be used to detect more recently acquired virulence factor genes in emerging pathogens [10]. To expedite the search for HGT sequences, we have adapted the web-accessed comparative genomics tool EvoPrinter for the rapid screening of chromosomal and MGE DNA [21,22]. Evoprinter works rapidly: the alignment time for a 40 kb sequence to 17 staphylococcal genomes is accomplished in less than 20 sec. EvoPrinter algorithms serve as an initial search tool to help identify DNA sequences that are not uniformly shared by other related bacteria. Whereas BLAST sequence homology searches highlight sequence similarities, bacterial EvoPrinter algorithms highlight sequence differences between DNAs that may otherwise go unnoticed in a BLAST search, especially when large sequence files are searched. The uninterrupted EvoPrinter readouts allow for rapid visual screening of up to 40 kb of DNA, without having to sort through the multiple pairwise BLAST alignments that include both orthologs and less related sequence comparisons. EvoPrinter is currently formatted for the automated comparative analysis of 17 staphy- lococcal, 20 streptococcal and 22 enteric bacterial genomes and can be used to detect HGT sequences among bacterial chromosomes and their MGEs. Staphylococcal genomes included in the EvoPrinter automated analysis currently include 13 S. aureus, two S. epidermidis, one S. haemolyticus and one S. saprophyticus. Our search for HGT DNA sequences within the genomes of different human MRSA isolates has led to the discovery that one of the human strains, the hospital-acquired epidemic MRSA252 [23], uniquely shares multiple DNA sequence blocks with three different causative agents of contagious bovine mastitis but not with other human isolates. These putative HGT sequences encode virulence factors that are also incorporated into the chromosomes or plasmids of the bovine S. aureus ET3 strain RF122, S. saprophyticus and Listeria monocytogenes. Analysis of the MRSA252 complete genome EvoUnique profile (compared to 12 other S. aureus isolates), identified over 20 different regions that were either unique to the MRSA252 genome or uniquely shared with just one other S. aureus isolate included in the analysis, the bovine RF122. Taken together, the multiple uniquely shared DNAs indicate that the human MRSA252 or another related epidemic MRSA strain may have undergone repeated HGT events with different bovine pathogens. Results and Discussion The comparative analysis of the bacterial chromosomes and their MGEs described in this study was performed using the EvoPrinterHD alignment algorithms. Evoprinter functions as a tool for the rapid discovery of unique DNA sequence differences among multiple bacterial genomes and their MGEs. Specifically, an EvoDifference profile identifies sequences present in a group of related genomes that are lacking in a single genome, and an EvoUnique profile highlights sequences that are either unique or uniquely shared by a subset of bacteria included in the analysis. To identify uniquely shared DNA sequence blocks and unique single nucleotide polymorphisms (SNPs) among subsets of 13 different S. aureus genomes, we generated EvoUnique profiles of their chromosomes. Our initial MRSA252 EvoUnique profile alerted us Analysis of the MRSA252 Genome to multiple instances of exclusive sequence sharing with other S. aureus chromosomes, indicated by green uppercase letters (Figure 1). To our surprise, the MRSA252 EvoUnique profile revealed that the highlighted DNAs were almost exclusively shared with a contagious bovine mastitis strain known as RF122. Notably, MRSA252 and RF122 chromosomes share 14 different unique DNA sequence blocks that are not present in any of the other S. aureus or in any of the other Staphylococci included in the EvoPrinter database (Figure 1, Table 1 and Table S1; see materials and methods for complete list of genomes). The different sequence blocks, which are from 144 to 4,950 bp in length, exhibit between 93% and 99% pairwise identity, with most having 97% or greater identity. Complete EvoUnique profiles of the MRSA252 and RF122 chromosomes are available at the EvoPrinter website. Considerable information has been acquired about the biology of these two pathogens [23,24,25,27,28]. Comparative analysis of the human and bovine S. aureus strains has revealed that they are phylogenetically distinct from one another and from other S. aureus strains [23,25] and that their pathogenicity-associated genes have undergone significant genetic divergence [23,24,25]. MRSA252 is a sub-clone of the hospital epidemic EMRSA-16 clone [23]. The EMRSA-16 clone and its representative members are responsible for half of all MRSA infections in the U.K., and it is now considered one of the most clinically important global lineages within the U.S. [23]. The MRSA252 chromosome contains a 58.8 kb SCCmec element that carries multiple antibiotic resis- tance genes [23]. MRSA252 also contains the Tn552 transposon that harbors penicillin-resistance genes, which are components of the inducible S. aureus b-lactamase operon [26]. The RF122 isolate belongs to the ST151 sub-clone of the bovine ET3 clone [27], and members of this subclone display greater virulence than other ET3 sub-clones in a mouse model of mastitis [28]. In addition, comparative analysis of the different bovine ET3 clonal subtypes revealed that multiple episodes of HGT may have occurred within the ET3 lineage [28]. Database searches reveal that many of the MRSA252-RF122 unique sequence blocks span genes that encode virulence factors, metabolic enzymes or novel protein encoding sequences found in other bacteria (Table 1). For example, among the Staphylococcal genomes included in this analysis, a sequence encoding the malolactic enzyme gene (annotated respectively as SAR0824 and SAB0723 in the MRSA252 and RF122 genomes) is unique to MRSA252 and RF122 but present in many fermentation bacteria (Figure 2 and Table 1). The shared sequence, consisting of 1,981 bp, is located at synonymous genomic locations and exhibits 97.8% sequence identity (Figures 1 and 2). Malolactic enzyme is a component of the anaerobic respiration pathway and confers bacterial virulence by enabling survival in the anaerobic environ- ment of deep tissue abscesses [29]. Given that abscess formation is an important aspect of human MRSA pathogenesis [30], the acquisition of this enzyme is most likely of clinical importance. The EvoUnique profile of the malolactic enzyme region also demonstrates the utility of this approach for the detection of single nucleotide polymorphisms (SNPs). For example, the red-colored bases in Figure 2B represent SNPs that are unique in MRSA252 and not present in any of the other genomes analyzed, including RF122. The EvoUnique profile of the first 50 kb of the MRSA252 chromosome identifies 229 SNPs that distinguishes it from the other isolates (data not shown). The speed and base-pair resolution of this approach should prove invaluable when markers are sought to distinguish between different MRSA isolates. All of the MRSA252-RF122 shared DNAs exhibit features of HGT events [6]; the acquired DNA is not found in other closely related strains and the sequences flanking the unique DNA share PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e3074