BACKGROUND
for the past 75 years, successive waves of antibiotic-resistant staphylococcus aureus strains have posed significant challenges to clinicians and public health officials  <cit> .
beta-lactamase-mediated s. aureus resistance developed within a decade of widespread penicillin use, and two years after the introduction of methicillin, methicillin-resistant s. aureus strains  appeared.
more recently, mrsa has emerged as one of the preeminent public health threats in the united states and worldwide.
in fact, all-cause s. aureus infections have rapidly increased during the past decade  with mrsa currently accounting for >50% of staphylococcal disease  <cit> .
more deaths were attributed in  <dig> in the u.s. to s. aureus than hiv/aids  <cit> .
mrsa also represents a significant economic burden with an estimated annual u.s. cost of  <dig> billion dollars  <cit> .
historically the mrsa threat consisted of hospital-acquired strains  usually affecting individuals with associated risk factors .
community-associated mrsa strains  have recently emerged as the predominant cause of mrsa disease  <cit> .
distinct genetic profiles suggest that ca-mrsa and ha-mrsa evolved independently.
while methicillin-resistance occurs at a fitness cost to the organism in ha-mrsa strains, ca-mrsa strains have a selective advantage and generally affect previously healthy individuals  <cit> .
ca-mrsa strains carry unique drug resistance genes, and widespread clinical experience suggests they may possess increased virulence compared to most ha-mrsa.
while recombination is rare among staphylococci, broad diversity exists amongst subspecies.
among ca-mrsa strains, one clonal isolate, usa <dig>  has become predominant in the united states, representing the majority of all mrsa infections and almost all community-associated staphylococcal infection in much of the u.s. <cit> .
its prevalence is also increasing in europe  <cit> .
usa300’s broad range of clinical manifestations include asymptomatic colonization, skin and soft tissue infections , and life-threatening, severe disease   <cit> .
given this clinical diversity, there may exist genetic differences that could serve as useful predictors of ca-mrsa-related disease phenotypes or virulence.
classical genotyping methodologies such as pfge and mlst rely on the evaluation of highly conserved housekeeping genes representative of the vertical gene pool and resultantly provide insufficient resolution to predict disease phenotypes.
recent reports suggest that there are additional genetic components that contribute to invasiveness not captured by current typing technologies  <cit> .
in an attempt to understand the population genetic architecture of ca-mrsa isolates and possibly resolve genomic differences relating to mrsa invasiveness, we sequenced the whole genomes of  <dig> ca-mrsa clinical isolates.
by leveraging novel de novo assembly methods along with the historical time-stamps associated with the infections, we sought to determine whether there was evidence for ca-mrsa selection both across all coding genes as well as biological pathways implicated to mediate virulence.
RESULTS
clinical collection
during 2001– <dig>   <dig> children with mrsa infections were identified at rady children’s hospital in san diego, california.
to prevent the inclusion of hospital acquired strains we excluded  <dig> isolates  .
among  <dig> remaining children,  <dig>  had severe infections  and  <dig>  had ssti.
<dig> ca-mrsa ssti-associated isolates of the same chronological distribution as that of the severe isolates were selected for genotypic comparison.
the majority of the clinical diagnoses of the  <dig> severe ca-mrsa infections consisted of either osteoarticular infections  or complicated pneumonias .
there was no significant difference in patient age, gender or self-identified ethnicity between the severe infection and ssti study groups.
compared with children with ssti, children with severe infections had significantly longer hospital stays, longer duration of intravenous antibiotic therapy, and total duration of antibiotics .
of all children with ca-mrsa severe infections,  <dig>  children died, another  <dig>  required intensive care prior to recovery, and  <dig> others  were hospitalized for ≥ 10 days prior to recovery.
further, two children , both with osteomyelitis, had long-term sequelae  .
all children in the ca-mrsa ssti group were hospitalized <10 days and had an uncomplicated recovery during hospitalization.
multilocus pcr coupled to electrospray ionization-mass spectrometry  genotyping revealed that all  <dig> severe infection and ssti-associated ca-mrsa isolates were of a single genotype: pcr/esi-ms genotype  <dig>  which corresponds to pfge usa300/usa <dig> and sequence type  <dig> designations  <cit> .
we selected  <dig> isolates representing the entire collection period for whole genome sequencing .
in addition, two replicate dna samples from the usa <dig> strain fpr <dig>  were sequenced to serve dual functions of quality control and assistance during sequence assembly  <cit> .
sequencing and data summary
we sequenced ca-mrsa isolates using the illumina genome analyzer ii.
in order to maximize sequencing capacity, we sequenced  <dig> or  <dig> isolates per lane using nucleotide barcodes, with an average of 181 mb of sequence per isolate.
we used a custom pipeline that leveraged remapping protocols using the aligner bwa  <cit>  against the closely related reference sequence usa300-fpr <dig> as well as de novo assembly with abyss  <cit>  to successfully call polymorphisms ranging from single nucleotide variants  to large structural rearrangements.
of the core genome defined as the curated fpr <dig> sequence, we sequenced to an average depth of 57-fold with  <dig> % of bases across all genomes covered >5x and  <dig> % >20x .
a -  <dig> snv &  <dig> insertion confined to a single  <dig> kb stretch were removed from all subsequent analysis.
in order to prevent false positive calls, we conservatively masked 3% of the genome where placing or assembling short reads could not be confidently performed.
all single nucleotide events as well as large insertions and deletions that originated in the masked region were discarded.
we also detected the presence of all four previously identified usa <dig> plasmids in our isolates.
the most common combination was the presence of pusa <dig> and pusahou <dig> in  <dig> of the isolates .
the remaining  <dig> strains represented an additional  <dig> combinations that is not delineated by the phylogenetic structure of the population.
as noted, in addition to the  <dig> clinical ca-mrsa isolates, we sequenced the fpr <dig> reference strain in duplicate.
perfect concor-dance was obtained between the duplicates with each sample having  <dig> detected variants compared to the reference sequence.
of the  <dig> variants,  <dig> were previously reported as probable errors in the reference sequence  <cit> .
we identified  <dig> new variants in fpr <dig>   <dig> of which were seen in all clinical isolates, supporting their status of true variants.
the remaining two variants were visually inspected and determined as true variants.
we also detected one large rearrangement in the reference sequence consisting of a  <dig>  kb relocation.
this rearrangement had been previously reported in a sepa-rate usa <dig> strain  <cit>  and was also detected in all clinical isolates.
we verified the relocation using pcr and presume it to be an error in the original assembly of the fpr <dig> reference sequence.
with respect to the clinical ca-mrsa isolates, the severe and ssti groups each carried a mean of  <dig> and  <dig> single nucleotide variants , respectively  .
of the  <dig> snv sites, 88% were private to a single isolate with 94% occurring at an allele frequency of less than 10% .
we performed multiple sequence alignment on all  <dig> isolates as well as the resequenced fpr <dig> strain and constructed a maximum-likelihood phylogenetic tree .
there is no obvious differentiation between severe and ssti isolates with respect to their placements on the tree.
however, there are two distinct haplogroups defined by  <dig> variants distributed across the genome.
this suggests a decisive split in the lineage of the isolates from an ancestral clone.
we screened our entire collection of severe  and ssti  isolates for one of the eight haplogroup-defining variants and saw a modest association between the more recently arisen haplogroup b with the less severe  isolates .
in both groups we observed a distinct temporal correlation supporting that the isolates are evolving away from a single common clone over time.
we estimated an average mutation rate of  <dig> ×10- <dig>  per site per year for severe isolates and  <dig> ×10- <dig>  per site per year for ssti isolates .
these values are in close concordance with a previously reported estimate of  <dig> ×10- <dig> per site per year in hospital-acquired s. aureus  <cit> .
among snvs, there was a transition: transversion ratio of  <dig> : <dig> and a higher frequency of - >  mutations, with a  <dig>  fold greater occurrence, suggesting that ca-mrsa is actively introducing additional at bias in an already gc depleted genome .
small deletions  were 2-fold more frequent than small insertions  among the  <dig> clinical isolates.
as expected, we found that indels were most likely to occur in homopolymer stretches, consistent with polymerase slippage during replication  <cit> .
large deletions were detected in ten of the isolates.
the largest deletion , observed in a severe isolate, included a large portion of both the sccmec and acme regions but retained pbp <dig> .
the acme region has previously been postulated to be one of the loci responsible for usa 300’s increased pathogenicity.
while this locus may play a role in pathogenicity, our data suggest that the entire acme region may not be required for severe infection.
there was also a common  <dig> kb deletion completely removing staphylococcal pathogenicity island sapi <dig> observed in  <dig> severe and  <dig> ssti isolates.
the deletion appeared on three separate bootstrapped supported branches of the phylogenetic tree, suggesting either recombination or three independent deletion events.
large insertions  and additional contigs accounted for  <dig> kb of novel sequence across  <dig> isolates .
for  <dig> isolates, we placed  <dig> insertions onto the core genome consisting of  <dig> kb of the additional sequence.
of the  <dig> placed insertions,  <dig> are large mobile genetic elements  of prophage origin.
there were four unique insertion sites for the  <dig> mges observed:  <dig> private appearing in only one isolate sequenced,  <dig> site shared by two isolates, and  <dig> site found in seven isolates at position  <dig>  mb on the reference sequence .
of the  <dig> isolates sharing the large insertion located at  <dig>  mb the phylogenetic reconstruction suggests only two isolates share the insertion due to a single common ancestor with the remainder representing independent events.
this insertion and the previously discussed  <dig> kb deletion represented two of the  <dig> events we detected as exhibiting homoplasy within our data .
unlike previous observations, we did not observe any convergent events with obvious implications for drug resistance  <cit> .
this is not surprising in light of the fact that prior studies characterized ha-mrsa isolates that are presumably under different selective pressures than our pediatric ca-mrsa isolates from previously well children.
of the  <dig> snvs exhibiting homoplasy, only three affected protein-coding sequence directly.
however,  <dig> of the  <dig> snvs fell within or directly adjacent to transcriptional regulators, or within putative regulatory binding sequence.
this suggests that regulatory mechanisms may play an equally critical role in ca-mrsa pathogenesis as point mutations conferring functional protein-coding changes.
functional distribution of mutations
to assess the distribution of functional differences in our collection we annotated all non-ubiquitous va-riants of  <dig> bp or smaller into three distinct classes: non-synonymous coding, synonymous coding, non-coding .
in line with our discovery of a greater overall abundance of variants in the ssti isolates we saw a significantly larger number of both synonymous and non-coding variants in the ssti isolates when compared to the severe isolates.
for synonymous changes there were  <dig> variants in the ssti isolates compared to  <dig> in the severe isolates .
for non-coding changes we detected  <dig> changes in ssti and  <dig> in the severe isolates .
for non-synonymous variants the trend was similar with an over abundance of variants in the ssti isolates  although this difference did not pass our threshold for significance .
the higher proportion of synonymous variants in the ssti isolates relative to the difference in non-synonymous variants did create an overall lower dn/ds ratio in our ssti isolates .
however, we are cautious of any classical interpretation of the dn/ds given the highly clonal nature of the samples and the small population size; both of which have been shown to cause fluctuation in the dn/ds calculations  <cit> .
pathway analysis
under the hypothesis that the ssti isolates are evolving away from a virulent phenotype we looked for an enrichment of genes harboring unique nonsense, ns-csnvs or deleterious frame shift mutations among the ssti isolates in genes related to virulence.
since no single gene harbored enough variants across sufficient number of isolates for direct testing, we sought to determine if there was enrichment across all virulence-associated genes classified as being involved in “pathogenesis” or “toxin production and resistance” by the jcvi cmr database  <cit> .
this list consisted of  <dig> genes encompassing  <dig> % of the total coding genome.
we tested against only private variation among the isolates to prevent overt bias from the few mutations that are shared.
there were  <dig> putative functional variants found in the ssti isolates with  <dig> in genes related to virulence .
this represents a  <dig>  fold increase over the expected value if the placement of variation was random across the genome .
in contrast the severe isolates carried close to the expected number at  <dig> va-riants out of  <dig>  although we cannot claim a difference between the two groups given the number of isolates sampled .
discussion
to address a major public health crisis that involves an infectious disease, it is essential that we track and better understand the biology of the causative pathogen.
high-throughput dna sequencing technologies provide a platform for tracking pathogens as they propagate and permit insights into virulence determinants which could be targeted pharmacologically.
we sequenced the complete genomes of  <dig> emerging ca-mrsa isolates, half of which caused lethal or severe infections.
as expected, we found evidence that all isolates of ca-mrsa are accumulating genetic variation over time, indicative of an evolution away from an ancestral clone.
this rate corresponded to what has been seen in a similar study for ha-mrsa suggesting that, at least at a population wide level, these two distinct classes are evolving at a similar rate  <cit> .
however at higher resolution these two groups likely have distinct pressures being applied to them given their differing environments.
one such example is the lack of homoplasy within genes related to antimicrobial resistance.
in addition we did not observe differences between the dn/ds ratios of the core and non-core portions of the genome as previously reported  <cit> .
however, given the different population of samples and the smaller sample set that our data represents, neither discordance is troubling.
we did detect that the accumulated genetic variation appears to be higher among ca-mrsa isolates causing the milder ssti phenotypes.
this distance also manifested itself by association of a distinct haplogroup, which evolved from the parental strain, with the less severe sample.
further, pathways involved in virulence are enriched among genes harboring coding variations among the clinical isolates of lesser clinical severity.
taken together, these data suggest that the invasiveness of usa  <dig> may be dependent, to some degree, on the genetic distance a particular clone is away from the clonal parental usa  <dig> strain.
usa  <dig> was first described a little over a decade ago suggesting its emergence is recent  <cit> .
the strain is a result of a two step process; first through the acquisition of the sccmex iv complex which resulted in the clonal type usa <dig>  followed later by the acquisition of several genes including the pvl and acme loci  <cit> .
given that staphylococcus aureus is a natural member of our nasal flora, as demonstrated by pervasive asymptomatic carriage  <cit> , there is likely genetic pressure on the organism to maintain some degree of a commensal relationship and as a result dampen the hyper-virulent characteristic which arose from a single recombination event.
this “wave” of clonality wherein an epidemic clone decreases it pathogenicity and as a result its association with infections independent of control measures has been seen in other mrsa strains  <cit> .
thus, the increase in genetic diversity of usa <dig> ssti isolates may be an evolutionary progression away from being the hyper-virulent clonal type that usa <dig> represents.
the fact that we did not find any single genetic variant or set of genetic variants readily capable of distingui-shing severe- and ssti associated ca-mrsa isolates speaks to the complexity of mrsa pathogenicity and underscores the need for more sophisticated ways to identify disease-causing ca-mrsa strains via genomic screens.
while we focused our analysis to only a small number of ca-mrsa isolates displaying distinct clinical phenotypes, we are optimistic that surveying a large number of isolates  in the future will provide the power to detect evolutionary pressures at the single gene level.
of course there are two genomes at play during an infection and, as a result, a greater understanding of human genetic variation is also pivotal for the complete picture of how genetic architecture influences our interaction with microbial pathogens.
it is also important to recognize that numerous non-genetic influences for virulence exist; as such to effectively investigate the genetics of pathogenicity it is critical to properly select the cases as to mitigate such effects of predisposition.
CONCLUSIONS
technological advances and continued decreases in the cost of sequencing are allowing the whole genome reconstruction of large collections of pathogens.
these data sets offer valuable insight into the clonal population structure as well clues into the evolutionary pressures applied to the pathogen and will provide a foundation for the use of whole genome sequencing in a clinical setting.
in the future, the genomic identity of individual isolates may be increasingly compared to global pathogen databases allowing the rapid identification of strains likely to cause severe disease, possess drug-resistance, and additional traits of critical utility to inform clinical decisions.

 for the past 75 years, successive waves of antibiotic-resistant staphylococcus aureus strains have posed significant challenges to clinicians and public health officials. beta-lactamase-mediated s. aureus resistance developed within a decade of widespread penicillin use, and two years after the introduction of methicillin, methicillin-resistant s. aureus strains appeared. more recently, mrsa has emerged as one of the preeminent public health threats in the united states and worldwide. in fact, all-cause s. aureus infections have rapidly increased during the past decade with mrsa currently accounting for >50% of staphylococcal disease. more deaths were attributed in in the u.s. to s. aureus than hiv/aids. mrsa also represents a significant economic burden with an estimated annual u.s. cost of billion dollars. historically the mrsa threat consisted of hospital-acquired strains usually affecting individuals with associated risk factors. community-associated mrsa strains have recently emerged as the predominant cause of mrsa disease. distinct genetic profiles suggest that ca-mrsa and ha-mrsa evolved independently. while methicillin-resistance occurs at a fitness cost to the organism in ha-mrsa strains, ca-mrsa strains have a selective advantage and generally affect previously healthy individuals. ca-mrsa strains carry unique drug resistance genes, and widespread clinical experience suggests they may possess increased virulence compared to most ha-mrsa. while recombination is rare among staphylococci, broad diversity exists amongst subspecies. among ca-mrsa strains, one clonal isolate, usa has become predominant in the united states, representing the majority of all mrsa infections and almost all community-associated staphylococcal infection in much of the u.s.. its prevalence is also increasing in europe. usa300’s broad range of clinical manifestations include asymptomatic colonization, skin and soft tissue infections, and life-threatening, severe disease. given this clinical diversity, there may exist genetic differences that could serve as useful predictors of ca-mrsa-related disease phenotypes or virulence. classical genotyping methodologies such as pfge and mlst rely on the evaluation of highly conserved housekeeping genes representative of the vertical gene pool and resultantly provide insufficient resolution to predict disease phenotypes. recent reports suggest that there are additional genetic components that contribute to invasiveness not captured by current typing technologies. in an attempt to understand the population genetic architecture of ca-mrsa isolates and possibly resolve genomic differences relating to mrsa invasiveness, we sequenced the whole genomes of ca-mrsa clinical isolates. by leveraging novel de novo assembly methods along with the historical time-stamps associated with the infections, we sought to determine whether there was evidence for ca-mrsa selection both across all coding genes as well as biological pathways implicated to mediate virulence.  clinical collection during 2001– children with mrsa infections were identified at rady children’s hospital in san diego, california. to prevent the inclusion of hospital acquired strains we excluded isolates. among remaining children, had severe infections and had ssti. ca-mrsa ssti-associated isolates of the same chronological distribution as that of the severe isolates were selected for genotypic comparison. the majority of the clinical diagnoses of the severe ca-mrsa infections consisted of either osteoarticular infections or complicated pneumonias. there was no significant difference in patient age, gender or self-identified ethnicity between the severe infection and ssti study groups. compared with children with ssti, children with severe infections had significantly longer hospital stays, longer duration of intravenous antibiotic therapy, and total duration of antibiotics. of all children with ca-mrsa severe infections, children died, another required intensive care prior to recovery, and others were hospitalized for ≥ 10 days prior to recovery. further, two children, both with osteomyelitis, had long-term sequelae. all children in the ca-mrsa ssti group were hospitalized <10 days and had an uncomplicated recovery during hospitalization. multilocus pcr coupled to electrospray ionization-mass spectrometry genotyping revealed that all severe infection and ssti-associated ca-mrsa isolates were of a single genotype: pcr/esi-ms genotype which corresponds to pfge usa300/usa and sequence type designations. we selected isolates representing the entire collection period for whole genome sequencing. in addition, two replicate dna samples from the usa strain fpr were sequenced to serve dual functions of quality control and assistance during sequence assembly. sequencing and data summary we sequenced ca-mrsa isolates using the illumina genome analyzer ii. in order to maximize sequencing capacity, we sequenced or isolates per lane using nucleotide barcodes, with an average of 181 mb of sequence per isolate. we used a custom pipeline that leveraged remapping protocols using the aligner bwa against the closely related reference sequence usa300-fpr as well as de novo assembly with abyss to successfully call polymorphisms ranging from single nucleotide variants to large structural rearrangements. of the core genome defined as the curated fpr sequence, we sequenced to an average depth of 57-fold with % of bases across all genomes covered >5x and % >20x. a - snv & insertion confined to a single kb stretch were removed from all subsequent analysis. in order to prevent false positive calls, we conservatively masked 3% of the genome where placing or assembling short reads could not be confidently performed. all single nucleotide events as well as large insertions and deletions that originated in the masked region were discarded. we also detected the presence of all four previously identified usa plasmids in our isolates. the most common combination was the presence of pusa and pusahou in of the isolates. the remaining strains represented an additional combinations that is not delineated by the phylogenetic structure of the population. as noted, in addition to the clinical ca-mrsa isolates, we sequenced the fpr reference strain in duplicate. perfect concor-dance was obtained between the duplicates with each sample having detected variants compared to the reference sequence. of the variants, were previously reported as probable errors in the reference sequence. we identified new variants in fpr of which were seen in all clinical isolates, supporting their status of true variants. the remaining two variants were visually inspected and determined as true variants. we also detected one large rearrangement in the reference sequence consisting of a kb relocation. this rearrangement had been previously reported in a sepa-rate usa strain and was also detected in all clinical isolates. we verified the relocation using pcr and presume it to be an error in the original assembly of the fpr reference sequence. with respect to the clinical ca-mrsa isolates, the severe and ssti groups each carried a mean of and single nucleotide variants, respectively. of the snv sites, 88% were private to a single isolate with 94% occurring at an allele frequency of less than 10%. we performed multiple sequence alignment on all isolates as well as the resequenced fpr strain and constructed a maximum-likelihood phylogenetic tree. there is no obvious differentiation between severe and ssti isolates with respect to their placements on the tree. however, there are two distinct haplogroups defined by variants distributed across the genome. this suggests a decisive split in the lineage of the isolates from an ancestral clone. we screened our entire collection of severe and ssti isolates for one of the eight haplogroup-defining variants and saw a modest association between the more recently arisen haplogroup b with the less severe isolates. in both groups we observed a distinct temporal correlation supporting that the isolates are evolving away from a single common clone over time. we estimated an average mutation rate of ×10- per site per year for severe isolates and ×10- per site per year for ssti isolates. these values are in close concordance with a previously reported estimate of ×10- per site per year in hospital-acquired s. aureus. among snvs, there was a transition: transversion ratio of: and a higher frequency of - >  mutations, with a fold greater occurrence, suggesting that ca-mrsa is actively introducing additional at bias in an already gc depleted genome. small deletions were 2-fold more frequent than small insertions among the clinical isolates. as expected, we found that indels were most likely to occur in homopolymer stretches, consistent with polymerase slippage during replication. large deletions were detected in ten of the isolates. the largest deletion, observed in a severe isolate, included a large portion of both the sccmec and acme regions but retained pbp. the acme region has previously been postulated to be one of the loci responsible for usa 300’s increased pathogenicity. while this locus may play a role in pathogenicity, our data suggest that the entire acme region may not be required for severe infection. there was also a common kb deletion completely removing staphylococcal pathogenicity island sapi observed in severe and ssti isolates. the deletion appeared on three separate bootstrapped supported branches of the phylogenetic tree, suggesting either recombination or three independent deletion events. large insertions and additional contigs accounted for kb of novel sequence across isolates. for isolates, we placed insertions onto the core genome consisting of kb of the additional sequence. of the placed insertions, are large mobile genetic elements of prophage origin. there were four unique insertion sites for the mges observed: private appearing in only one isolate sequenced, site shared by two isolates, and site found in seven isolates at position mb on the reference sequence. of the isolates sharing the large insertion located at mb the phylogenetic reconstruction suggests only two isolates share the insertion due to a single common ancestor with the remainder representing independent events. this insertion and the previously discussed kb deletion represented two of the events we detected as exhibiting homoplasy within our data. unlike previous observations, we did not observe any convergent events with obvious implications for drug resistance. this is not surprising in light of the fact that prior studies characterized ha-mrsa isolates that are presumably under different selective pressures than our pediatric ca-mrsa isolates from previously well children. of the snvs exhibiting homoplasy, only three affected protein-coding sequence directly. however, of the snvs fell within or directly adjacent to transcriptional regulators, or within putative regulatory binding sequence. this suggests that regulatory mechanisms may play an equally critical role in ca-mrsa pathogenesis as point mutations conferring functional protein-coding changes. functional distribution of mutations to assess the distribution of functional differences in our collection we annotated all non-ubiquitous va-riants of bp or smaller into three distinct classes: non-synonymous coding, synonymous coding, non-coding. in line with our discovery of a greater overall abundance of variants in the ssti isolates we saw a significantly larger number of both synonymous and non-coding variants in the ssti isolates when compared to the severe isolates. for synonymous changes there were variants in the ssti isolates compared to in the severe isolates. for non-coding changes we detected changes in ssti and in the severe isolates. for non-synonymous variants the trend was similar with an over abundance of variants in the ssti isolates although this difference did not pass our threshold for significance. the higher proportion of synonymous variants in the ssti isolates relative to the difference in non-synonymous variants did create an overall lower dn/ds ratio in our ssti isolates. however, we are cautious of any classical interpretation of the dn/ds given the highly clonal nature of the samples and the small population size; both of which have been shown to cause fluctuation in the dn/ds calculations. pathway analysis under the hypothesis that the ssti isolates are evolving away from a virulent phenotype we looked for an enrichment of genes harboring unique nonsense, ns-csnvs or deleterious frame shift mutations among the ssti isolates in genes related to virulence. since no single gene harbored enough variants across sufficient number of isolates for direct testing, we sought to determine if there was enrichment across all virulence-associated genes classified as being involved in “pathogenesis” or “toxin production and resistance” by the jcvi cmr database. this list consisted of genes encompassing % of the total coding genome. we tested against only private variation among the isolates to prevent overt bias from the few mutations that are shared. there were putative functional variants found in the ssti isolates with in genes related to virulence. this represents a fold increase over the expected value if the placement of variation was random across the genome. in contrast the severe isolates carried close to the expected number at va-riants out of although we cannot claim a difference between the two groups given the number of isolates sampled. discussion to address a major public health crisis that involves an infectious disease, it is essential that we track and better understand the biology of the causative pathogen. high-throughput dna sequencing technologies provide a platform for tracking pathogens as they propagate and permit insights into virulence determinants which could be targeted pharmacologically. we sequenced the complete genomes of emerging ca-mrsa isolates, half of which caused lethal or severe infections. as expected, we found evidence that all isolates of ca-mrsa are accumulating genetic variation over time, indicative of an evolution away from an ancestral clone. this rate corresponded to what has been seen in a similar study for ha-mrsa suggesting that, at least at a population wide level, these two distinct classes are evolving at a similar rate. however at higher resolution these two groups likely have distinct pressures being applied to them given their differing environments. one such example is the lack of homoplasy within genes related to antimicrobial resistance. in addition we did not observe differences between the dn/ds ratios of the core and non-core portions of the genome as previously reported. however, given the different population of samples and the smaller sample set that our data represents, neither discordance is troubling. we did detect that the accumulated genetic variation appears to be higher among ca-mrsa isolates causing the milder ssti phenotypes. this distance also manifested itself by association of a distinct haplogroup, which evolved from the parental strain, with the less severe sample. further, pathways involved in virulence are enriched among genes harboring coding variations among the clinical isolates of lesser clinical severity. taken together, these data suggest that the invasiveness of usa may be dependent, to some degree, on the genetic distance a particular clone is away from the clonal parental usa strain. usa was first described a little over a decade ago suggesting its emergence is recent. the strain is a result of a two step process; first through the acquisition of the sccmex iv complex which resulted in the clonal type usa followed later by the acquisition of several genes including the pvl and acme loci. given that staphylococcus aureus is a natural member of our nasal flora, as demonstrated by pervasive asymptomatic carriage, there is likely genetic pressure on the organism to maintain some degree of a commensal relationship and as a result dampen the hyper-virulent characteristic which arose from a single recombination event. this “wave” of clonality wherein an epidemic clone decreases it pathogenicity and as a result its association with infections independent of control measures has been seen in other mrsa strains. thus, the increase in genetic diversity of usa ssti isolates may be an evolutionary progression away from being the hyper-virulent clonal type that usa represents. the fact that we did not find any single genetic variant or set of genetic variants readily capable of distingui-shing severe- and ssti associated ca-mrsa isolates speaks to the complexity of mrsa pathogenicity and underscores the need for more sophisticated ways to identify disease-causing ca-mrsa strains via genomic screens. while we focused our analysis to only a small number of ca-mrsa isolates displaying distinct clinical phenotypes, we are optimistic that surveying a large number of isolates in the future will provide the power to detect evolutionary pressures at the single gene level. of course there are two genomes at play during an infection and, as a result, a greater understanding of human genetic variation is also pivotal for the complete picture of how genetic architecture influences our interaction with microbial pathogens. it is also important to recognize that numerous non-genetic influences for virulence exist; as such to effectively investigate the genetics of pathogenicity it is critical to properly select the cases as to mitigate such effects of predisposition.  technological advances and continued decreases in the cost of sequencing are allowing the whole genome reconstruction of large collections of pathogens. these data sets offer valuable insight into the clonal population structure as well clues into the evolutionary pressures applied to the pathogen and will provide a foundation for the use of whole genome sequencing in a clinical setting. in the future, the genomic identity of individual isolates may be increasingly compared to global pathogen databases allowing the rapid identification of strains likely to cause severe disease, possess drug-resistance, and additional traits of critical utility to inform clinical decisions. 
the broad range of disease severity may be influenced by bacterial genetic variation. while all isolates shared remarkable genetic similarity, we found greater overall time-dependent sequence diversity among ssti isolates. in addition, pathway analysis of non-synonymous variations revealed increased sequence diversity in the putative virulence genes of ssti isolates. the majority of the ca-mrsa disease burden consists of skin and soft tissue infections not associated with significant morbidity; however, ca-mrsa also causes severe, invasive infections resulting in significant morbidity and mortality. the results demonstrate the close relatedness of clinically independent ca-mrsa isolates, which carry implications for understanding ca-mrsa epidemiology and combating its spread. we sequenced the complete genomes of ca-mrsa clinical isolates from the predominant north american community acquired clonal type usa.
 community-associated methicillin-resistant staphylococcus aureus is a significant bacterial pathogen that poses considerable clinical and public health challenges. the majority of the ca-mrsa disease burden consists of skin and soft tissue infections not associated with significant morbidity; however, ca-mrsa also causes severe, invasive infections resulting in significant morbidity and mortality. the broad range of disease severity may be influenced by bacterial genetic variation.  we sequenced the complete genomes of ca-mrsa clinical isolates from the predominant north american community acquired clonal type usa. while all isolates shared remarkable genetic similarity, we found greater overall time-dependent sequence diversity among ssti isolates. in addition, pathway analysis of non-synonymous variations revealed increased sequence diversity in the putative virulence genes of ssti isolates.  here we report the first whole genome survey of diverse clinical isolates of the usa lineage and describe the evolution of the pathogen over time within a defined geographic area. the results demonstrate the close relatedness of clinically independent ca-mrsa isolates, which carry implications for understanding ca-mrsa epidemiology and combating its spread. 