Microbial Genomics

Microbial Genomics Pan-genomic perspective on the evolution of the Staphylococcus aureus USA300 epidemic --Manuscript Draft-Manuscript Number:

MGEN-D-16-00016R1

Full Title:

Pan-genomic perspective on the evolution of the Staphylococcus aureus USA300 epidemic

Article Type:

Research Paper

Section/Category:

Microbial evolution and epidemiology: Population Genomics

Corresponding Author:

Dorota Jamrozy, Ph.D Wellcome Trust Sanger Institute UNITED KINGDOM

First Author:

Dorota Jamrozy, Ph.D

Order of Authors:

Dorota Jamrozy, Ph.D Simon R Harris, PhD Naglaa Mohamed, PhD Sharon J Peacock, PhD, FRCP Charles Y Tan, PhD Julian Parkhill, PhD Annaliesa S Anderson, PhD Matthew T G Holden, PhD

Abstract:

Staphylococcus aureus USA300 represents the dominant community-associated methicillin-resistant S. aureus lineage in the United States (US), where it is a major cause of skin and soft tissue infections. Previous comparative genomic studies have described the population structure and evolution of USA300 based on geographically restricted isolate collections. Here, we investigated the USA300 population by sequencing genomes of a geographically distributed panel of 191 clinical S. aureus isolates belonging to clonal complex 8 (CC8), derived from the Tigecycline Evaluation and Surveillance Trial program. Isolates were collected at 12 healthcare centres across 9 US states in 2004, 2009 or 2010. Reconstruction of evolutionary relationships revealed that CC8 was dominated by USA300 isolates (154/191, 81%), which were heterogeneous and demonstrated limited phylogeographic clustering. Analysis of the USA300 core genomes revealed an increase in median pairwise SNP distance from 62 to 98 between 2004 and 2010, with a stable pattern of above average dN/dS ratios. The phylogeny of the USA300 population indicated that early diversification events led to the formation of nested clades, which arose through cumulative acquisition of predominantly non-synonymous SNPs in various coding sequences. The accessory genome of USA300 was largely homogenous and consisted of elements previously associated with this lineage. We observed an emergence of SCCmec negative and ACME negative USA300 isolates amongst more recent samples, and an increase in the prevalence of φSa5 phage. Together, the analysed S. aureus USA300 collection revealed an evolving pan-genome through increased core genome heterogeneity and temporal variation in the frequency of certain accessory elements

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Pan-genomic perspective on the evolution of the Staphylococcus aureus USA300 epidemic

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ABSTRACT Staphylococcus aureus USA300 represents the dominant community-associated methicillinresistant S. aureus lineage in the United States (US), where it is a major cause of skin and soft tissue infections. Previous comparative genomic studies have described the population structure and evolution of USA300 based on geographically restricted isolate collections. Here, we investigated the USA300 population by sequencing genomes of a geographically distributed panel of 191 clinical S. aureus isolates belonging to clonal complex 8 (CC8), derived from the Tigecycline Evaluation and Surveillance Trial program. Isolates were collected at 12 healthcare centres across 9 US states in 2004, 2009 or 2010. Reconstruction of evolutionary relationships revealed that CC8 was dominated by USA300 isolates (154/191, 81%), which were heterogeneous and demonstrated limited phylogeographic clustering. Analysis of the USA300 core genomes revealed an increase in median pairwise SNP distance from 62 to 98 between 2004 and 2010, with a stable pattern of above average dN/dS ratios. The phylogeny of the USA300 population indicated that early diversification events led to the formation of nested clades, which arose through cumulative acquisition of predominantly non-synonymous SNPs in various coding sequences. The accessory genome of USA300 was largely homogenous and consisted of elements previously associated with this lineage. We observed an emergence of SCCmec negative and ACME negative USA300 isolates amongst more recent samples, and an increase in the prevalence of Sa5 prophage. Together, the analysed S. aureus USA300 collection revealed an evolving pan-genome through increased core genome heterogeneity and temporal variation in the frequency of certain accessory elements.

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DATA SUMMARY

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Whole-genome sequence reads and assemblies of Staphylococcus aureus isolates described in this study have been deposited in the European Nucleotide Archive. The accession numbers together with related metadata are provided in Table S1 of Supplementary Material.

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I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ☒

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IMPACT STATEMENT

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The expansion of Staphylococcus aureus USA300 and its dominance as a communityassociated MRSA clone within the United States has prompted interest in the genomics of this pathogen. The resolving power of whole genome sequencing has already shed light on the nature of USA300 transmission within defined communities. However, a question of the broader population structure remains. Here, we investigated whole genome sequences of epidemiologically diverse CC8 isolates, inclusive of 154 USA300 samples. Based on this collection we investigated the wider population structure of USA300 and other CC8 isolates and assessed temporal variation in core and accessory genomes. We report that the USA300 population has become increasingly heterogeneous between 2004 and 2010 at the core genome level, accompanied by subtle changes in the accessory genome involving loss of ACME and SCCmec elements and an increase in the prevalence of Sa5 prophage. These findings demonstrate that successful lineages of MRSA are not genetically static and continue to evolve, which may involve loss of molecular markers previously considered to define the lineage. While further surveillance of USA300 genomes is required to confirm wider SCCmec and ACME loss, such genomic changes might impact on the future epidemiology of this important S. aureus clone.

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INTRODUCTION

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Staphylococcus aureus is a major cause of human disease worldwide. One of the most successful lineages of this bacterial species is clonal complex 8 (CC8), from which a number of major methicillin-resistant S. aureus (MRSA) clones have emerged (Deurenberg & Stobberingh, 2008). This includes several pandemic MRSA clones associated with causing healthcare-associated (HA) infections as well as community-associated (CA) infections (Monecke et al., 2011). HA-MRSA CC8 clones include historic Archaic and Iberian clones as well as the more contemporaneous USA500 clone, whereas the current USA300 epidemic clone has emerged as the dominant cause of CA infections in the United States (US).

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The first reports of USA300 date back to the early 2000s, when it was identified in a number of localised outbreaks of CA skin and soft tissue infections (SSTI) (Control & Prevention, 2001, Control & Prevention, 2003, McDougal et al., 2003, Tenover et al., 2006). Additional reports followed which demonstrated the widespread dissemination of USA300 across the US (Begier et al., 2004, Kazakova et al., 2005, Miller et al., 2005). Other than SSTI, USA300 has also been reported as a causative agent of CA pneumonia, sepsis, infective endocarditis and osteomyelitis (Francis et al., 2005, Gonzalez et al., 2005, Haque et al., 2007, Seybold et al., 2007). In addition to being responsible for a rising incidence of CA infections, USA300 has also been increasingly reported in association with nasal colonisation (Freitas et al., 2010, Tenover et al., 2008) and HA infections (Gonzalez et al., 2006, Patel et al., 2007, Seybold et al., 2006). In the healthcare setting, USA300 was reported to emerge alongside established


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HA-MRSA clones (Chua et al., 2008, Limbago et al., 2009, Pasquale et al., 2013, Tattevin et al., 2009) or to replace them (Carey et al., 2010, Liu et al., 2008).

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The striking epidemiology of USA300 within the US has prompted a number of genomic studies aimed at the identification of genetic features that might have promoted its transmission, host colonisation and disease pathogenesis. Early whole genome sequence analysis of a single USA300 strain revealed that the accessory genome included a Staphylococcal Cassette Chromosome mec (SCCmec) type IVa element, an arginine catabolic mobile element (ACME) type I, a S. aureus pathogenicity island 5 (SaPI5) and a PantonValentine leukocidin-carrying Sa2 prophage (Diep et al., 2006). These four mobile genetic elements (MGEs) represent common molecular markers of USA300, although analysis of a wider population demonstrated that their carriage is not ubiquitous (Uhlemann et al., 2014). Isolates belonging to USA300 also frequently carry two plasmids: a 27 kb plasmid carrying genes that confer resistance to penicillin (blaZ), erythromycin (msrA and mphBM) bacitracin (bcrA), kanamycin (aphA-3) and streptothricin (sat), and a 3.1 kb cryptic plasmid (Kennedy et al., 2010). The contribution of MGEs to the success of USA300 has been a subject of debate and generation of the hypothesis that the accessory genome has promoted the enhanced transmission of USA300 (Li et al., 2009). In particular, the ACME element has been suggested to promote the survival of USA300 on human skin and persistence within cutaneous abscesses (Thurlow et al., 2013). In contrast, the pathogenesis of USA300 may have been mediated by subtle genetic changes within the core genome (Highlander et al., 2007) together with modulated expression of core virulence determinants such as -toxin, phenolsoluble modulins (PSMs) and an accessory gene regulator (agr) (Li et al., 2009). Core genome SNPs might also result in a significantly different virulence phenotypes between USA300 isolates, associated with variable exoprotein expression (Kennedy et al., 2008).

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Recently, the genomics of USA300 have been studied on a wider scale, through whole genome sequencing of larger collections derived from specific geographic regions (Alam et al., 2015, Uhlemann et al., 2014). This led to significant epidemiological findings such as the identification of households as long-term reservoirs of USA300. Comparative genomic analysis has also allowed the diversification of the USA300 lineage to be studied, including evolutionary events surrounding the emergence of the USA300 clade as well as microevolution within the population (Uhlemann et al., 2014). This exemplifies the power of whole genome sequencing for comprehensive analysis of bacterial pathogens, as it allows an in-depth interrogation of phylogenetic relationships, identification of genetic features associated with virulence and antimicrobial resistance and the reconstruction of significant evolutionary events (Harris et al., 2010, Holden et al., 2013). While previous studies describing the comparative genomics of USA300 focused on geographically restricted isolate collections, here we describe whole genome sequence analysis of S. aureus CC8 derived from 12 US healthcare centres located across 9 states. The isolates were collected in 2004, 2009 or 2010 and derived from the multi-centre Tigecycline Evaluation and Surveillance Trial (T.E.S.T) study and represent consecutive clinical isolates (methicillin-susceptible and methicillin-resistant) from both the community and hospital settings. As USA300 has become widespread across the US, an analysis of a collection from geographically distinct locations has allowed us to study a broader population of epidemiologically unrelated USA300 isolates as well as their microevolution over time. For this we interrogated the


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phylogeny of the CC8 lineage, with particular focus on the USA300 clade, followed by analysis of temporal variation within core and accessory genomes as well as investigation of more subtle polymorphisms that define the phylogenetic structure of USA300 population.

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METHODS

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Bacterial isolates

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Isolates analysed here derived from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.), a global multi-centre surveillance study. The trial was initiated in 2004, and since then sites have been added and removed. For the purpose of conducting a longitudinal assessment of S. aureus isolates associated with disease in the US, we prospectively selected US sites that were participating between 2004 and 2010 (5 sites). Further 7 sites active at the time of study were included. A total of 516 S. aureus isolates derived from 2004, 2009 and 2010 were collected and analysed. All isolates belonging to CC8 (n=191), as determined by multi-locus sequence typing, were selected for this work.

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In line with T.E.S.T requirements, the participating sites collected consecutive isolates from patients with a documented infection. Only one isolate per patient was permitted and all body sites were considered an acceptable clinical source. Epidemiological details of the 191 S. aureus CC8 isolates analysed here are presented in Table S1. Briefly, isolates derived from the 12 T.E.S.T. sites that were located across 9 US states (Fig. 1), with 26% (50/191) collected in 2004, 31% (58/191) in 2009 and 43% (83/191) in 2010. For the purpose of this work the collecting sites were called healthcare centre 1 – 12 (HC1 - HC12). The collection consisted of 46 MSSA and 145 MRSA isolates. Isolates were collected from diverse clinical sources, categorised in this work to represent four main infection types defined as wound, invasive, respiratory or other. Isolates were collected from patients aged 0 to 95 in various clinical settings, with samples broadly defined in this study as derived from either inpatient or outpatient.

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Whole genome sequencing

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Genomic DNA was isolated using the Qiagen QIAcube system according to manufacturer’s protocol. Tagged DNA libraries were created using a method adapted from a standard Illumina Indexing protocol, as described previously (Hsu et al., 2015). Whole genome sequencing was performed on the Illumina HiSeq 2000 platform with 100 bp paired-end reads. The Illumina sequence data has been submitted to the European Nucleotide Archive (ENA) and the accession numbers are provided in Table S1.

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Whole genome sequence data analysis

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Single nucleotide polymorphisms (SNPs) were detected by mapping the paired-end reads against the S. aureus USA300_FPR3757 reference genome (GenBank accession NC_007793), using SMALT version 0.5.8. (https://www.sanger.ac.uk/resources/software/smalt/). The


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generated whole genome sequence alignment was curated to exclude accessory regions (mobile genetic elements identified for USA300_FPR3757 reference genome) and variable sites associated with recombination, with the latter detected using Gubbins (Croucher et al., 2014). Based on the curated core genome SNP alignment a maximum likelihood phylogenetic tree was constructed using RAxML version 7.8.6 (Stamatakis, 2014) using generalised time reversible (GTR) model with GAMMA method of correction for among site rate variation and 100 bootstrap replications (Harris et al., 2010). De novo assembly of whole genome sequences was performed using Velvet (Zerbino & Birney, 2008) with Velvet Optimiser (https://github.com/Victorian-Bioinformatics-Consortium/VelvetOptimiser.git) and kmers ranging between 66% and 90% of the read length. Contigs of less than 300 bp were removed. The draft assemblies have been submitted to the ENA with accession numbers provided in Table S1. The assembled contigs were annotated using Prokka (Seemann, 2014). The accessory genome was defined as described previously (Harris et al., 2010).

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Isolate genotyping

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Multi-locus sequence typing was performed on assembled genomes using an in-house developed pipeline based on previously described typing scheme (Enright et al., 2000). Isolates were also spa genotyped by extracting the spa gene variable X region from assembled genomes using previously described primers (Kahl et al., 2005), and determining the spa type with the spaTyper tool (http://spatyper.fortinbras.us). MLST and spa genotypes of all isolates are presented in Table S1. In addition, in silico PCR was used to confirm carriage of prophages (Goerke et al., 2009) and to determine the SCCmec type amongst mecA positive samples (Milheiriço et al., 2007, Milheiriço et al., 2007).

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Statistical analyses

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To test the statistical significance of the temporal variation in the prevalence of USA300 isolates as well as selected mobile genetic elements, the likelihood ratio test was applied. The strength of phylogenetic signal of epidemiological trait was measured using phylo.d() function in R package caper based on 1000 permutations. The function computes a D value, which provides a measure of trait dispersion for categorical characters (Fritz & Purvis, 2010). A negative D statistic indicates that a trait is phylogenetically clustered whereas a positive D statistic indicates that a trait is over dispersed across the phylogenetic tree.

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RESULTS AND DISCUSSION

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CC8 population structure overview and temporal variation

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Whole genome sequences of 191 clinical S. aureus isolates belonging to CC8 were analysed, the majority of which belonged to ST8 (179/191, 94%; Table S1). A total of ten single-locus variants (SLV) of ST8 were identified, which included six distinct novel STs. The majority of isolates belonged to spa type t008 (146/191, 76%; Table S1). Alignment of the core genomes (after excluding accessory and regions of predicted recombination, resulting in a core


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genome length of 2,592,236 bp) of all CC8 isolates revealed a total of 10651 SNP sites. To identify different sub-lineages, a phylogenetic tree of all CC8 samples was reconstructed with the addition of USA300, USA500 and COL reference genomes (Fig. 2). The tree revealed that the majority of isolates belonged to a single dominant clade (154/191, 81%), which contained the two USA300 reference strains (USA300_FPR3757 and USA300-ISMMS1) and thus was identified to represent the USA300 genotype. The population also contained four isolates that clustered with the USA500 reference strain (2395 USA500) and as such represented the USA500 clone. Six further isolates were closely related to the USA500 clade but descended from a distinct node and were labelled here USA500-like. The majority of these USA500 and USA500-like isolates (7/10) originated from a single healthcare centre (HC10, state of Georgia). The reconstructed phylogeny and detection of USA300 and USA500 genotypes in the context of a diverse CC8 population revealed that the two are only distantly related, in contrast to a previous suggestion that USA500 represents a progenitor of the USA300 clone (Li et al., 2009). A number of non-clonal MSSA CC8 isolates were more closely related to the USA300 clade than the USA500 sub-lineage suggesting that the two MRSA clones arose independently. The structure of CC8 phylogeny presented here is in line with a recently published overview of evolutionary relationships between various CC8 genomes (Boyle-Vavra et al., 2015).

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Isolates belonging to the USA300 clade were detected amongst samples from all healthcare centres. Some genotypic heterogeneity was observed within the USA300 clade, which contained six SLVs of ST8. We also observed a level of spa type diversity amongst USA300 isolates. While the majority belonged to spa type t008 (132/154, 86%), 10 other spa types were identified, including t024 (8/154, 5%) and t681 (3/154, 2%). The detected genotype diversity of USA300 isolates based on multi- and single-locus genotyping underscores the benefit of applying core genome sequence phylogeny for comprehensive identification of clinically significant sub-lineages such as S. aureus USA300.

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Focusing on the USA300 clade only, we investigated the strength of phylogenetic signal in the distribution of isolates based on the healthcare centre of origin, year of isolation, patient type and infection type. For this we calculated the D statistic, which provides a measure of trait dispersion. A random distribution of isolates based on geographic origin was observed for the majority of healthcare centres (D range 0.17-1.22), with exception of HC1 (D = -0.68). Phylogenetic clustering was also observed for isolates collected in 2004 (D = -0.08) with random distribution of isolates from 2009 and 2010 (D = 0.86 and D = 0.85, respectively). No phylogenetic signal could be observed based on patient type (outpatient, D = 0.73; inpatient, D = 0.91) or infection type (wound, D = 0.48; respiratory, D = 0.67; invasive, D = 1.1).

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We next investigated whether the CC8 population structure and the distribution of USA300 genotype varied at different sampling time points. For this, isolates collected in 2004 together with 2009 and 2010 collections derived from matched healthcare centres were used (Fig. 1). We found that USA300 was the dominant clone in all years. No significant variation in the distribution of USA300 was observed between 2004 (46/50, 92%) and 2009 (23/24, 96%) collections (p = 0.52). However, the 2010 collection (29/40, 73%) revealed a significantly lower frequency of USA300 genotype in comparison with both 2004 (p < 0.05) and 2009 (p < 0.05). This suggests that the significant drop in the prevalence of USA300


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observed for 2010 was likely specific to that year rather than a time trend. Outside of the USA300 clade, the USA500-like clone was observed only amongst more recent samples with a single isolate identified in 2009 (1/24, 4%) and a comparatively higher prevalence in 2010 (4/40, 10%).

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Accessory genome of CC8

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The genomes of 191 S. aureus CC8 isolates were analysed for the distribution of variable regions, followed by annotation to define the accessory component. Overall, a diverse mobile genetic repertoire was identified across the lineage, although isolates belonging to USA300, USA500 and USA500-like clones revealed presence of clade-associated MGEs (Fig. 2).

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In total, five different prophage types were identified. The most common was Sa3, found in 96% of isolates although variation in carriage of prophage-encoded virulence factors was observed (Fig. S1). Nearly all Sa3-positive USA300 isolates carried the immune evasion cluster (IEC) composed of scn, chp and sak genes, which was also detected amongst some non-USA300 isolates. The second most common prophage, due to the dominance of USA300 within this population, was Sa2. All but one USA300 isolate carried the Panton-Valentine leukocidin-positive Sa2USA300, which was also detected in three non-USA300 isolates. Three further prophages were identified within the analysed CC8 population: Sa1, Sa5 and Sa7. All three were detected at considerably lower frequency than Sa3 or Sa2. Prophage Sa1 was detected sporadically but across the entire CC8 population whereas Sa7 was prevalent mostly amongst non-USA300 MRSA isolates and found in all USA500 and majority of USA500-like (5/6) samples. In contrast, prophage Sa5 was identified predominantly within the USA300 clade (47/154, 30%), and was randomly distributed (D=1.66) across the USA300 phylogeny suggesting multiple acquisition events rather than prevalence due to clonal propagation of Sa5-positive isolates. No putative virulence genes were identified within sequence of any of the three prophages. We investigated whether acquisition of these prophages might affect the bacterial host thorough gene inactivation at the insertion site. Analysis of the chromosomal environment surrounding the site of prophage insertion revealed that Sa1 and Sa7 inserted within intergenic locations downstream of the ironsulphur cluster assembly protein gene sufB (between coding sequences corresponding to USA300_FPR3757 SAUSA300_0822 and SAUSA300_0823), and the heme transport protein gene isdB, (between coding sequences corresponding to USA300_FPR3757 SAUSA300_1027 and SAUSA300_1028) respectively. In contrast, prophage Sa5 were found inserted within a coding sequence responsible for a putative metal-binding protein with iron-sulphur oxidoreductase domain (SAUSA300_1858), which would likely interfere with its expression.

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Other accessory virulence genes amongst analysed CC8 isolates were associated with various pathogenicity islands, with SaPI5 identified in majority of USA300 isolates (139/154, 90%). Nearly all USA500 (3/4) and USA500-like (5/6) isolates carried the SaPI3, which similarly to SaPI5, encodes the sek and seq enterotoxin genes.

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The majority of USA300 isolates carried ACME (137/154, 89%) and SCCmec type IVa elements (130/154, 84%), which were not found in any other isolates. Where the elements were absent this was predicted to have resulted from a deletion event, with corresponding


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isolates dispersed across the phylogenetic tree. In line with a previous report (Uhlemann et al., 2014), an association between the absence of SCCmec and ACME was observed, with majority of ACME negative USA300 isolates also lacking the SCCmec (13/17, 76%). This is not surprising considering the proximal location of these elements in the USA300 chromosome. Nearly all SCCmec positive USA300 isolates carried a complete type IVa element although two isolates carried unusual recombinant variants of SCCmec IVa, which arose through putative recombination of SCCmec with ACME or another SCCmec element (Fig. S2). We also identified one isolate carrying a remnant SCC element lacking the mecA gene that exhibited similarity to SCCpbp4 from Staphylococcus epidermidis ATCC 12228. Finally, non-USA300 mecA-positive isolates carried either SCCmec IVd or SCCmec IVg. The carriage of SCCmec correlated with the methicillin susceptibility phenotype (Table S1) except for a single SCCmec positive isolate, which was identified as MSSA (PFESA2067). Further investigation revealed presence of a single base insertion in mecA gene resulting in a frame shift mutation and premature stop codon, which would likely lead to a loss of mecA expression.

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The analysed CC8 population revealed a high prevalence of an accessory region recognised as integrative conjugative element 6013 (ICE6013) (Smyth & Robinson, 2009). The element was detected in all USA300 and USA500 isolates but was absent in all USA500-like samples. The ICE6013 does not appear to carry any specific virulence or resistance genes and lacks insertion specificity (Smyth & Robinson, 2009), integrating at a number of sites amongst the analysed CC8 isolates, including both inter- and intragenic positions (Fig. S3). In the USA300 isolates however, ICE6013 was only found inserted within a putative membrane protein gene (SAUSA300_0606), and thus an insertional inactivation of this particular coding sequence might provide selective advantage to USA300. Interestingly, this particular ICE6013 insertion site differs from the USA300_FPR3757 reference genome (Diep et al., 2006), which also contains this element, but is identical to ICE6013 location within the USA300-ISMMS1 genome (Altman et al., 2014).

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There was only a sporadic carriage of resistance-associated transposon elements such as tetM-positive Tn6086, ermA- and spc-positive Tn554, and bla operon-carrying Tn552. All USA500-like isolates carried a 3.3 kb insertion element containing the trimethoprim resistance gene dfrG, not found elsewhere in this collection. Amongst USA300 isolates the antimicrobial resistance genes were carried predominantly on plasmids with the majority of samples (146/154, 95%) carrying a large plasmid resembling a previously reported 27 kb pUSA300HOUMR (Highlander et al., 2007). The plasmid contains genes that confer resistance to various antimicrobials including penicillin (blaZ), kanamycin (aphA-3), streptothricin (sat), bacitracin (bcrA), and erythromycin (msrA, mphBM), and homologous plasmids have been previously reported in other USA300 isolates (Kennedy et al., 2010, Uhlemann et al., 2014). However, amongst the 146 USA300 isolates carrying the pUSA300HOUMR-like plasmid 12 carried a variant that lacked some of the resistance genes described above, with at least five different variants detected (Fig. S1). The pUSA300HOUMR-like plasmid was not confined to the USA300 clade within this collection, and was detected in several other isolates. A few USA300 isolates (8/154, 5%) carried a circa 34 kb plasmid, which partly resembled the 37 kb pUSA03 that contains genes conferring resistance to erythromycin (ermC) and mupirocin (ileS-2) (Diep et al., 2006). At least three variants of this pUSA03-like plasmid were observed (Fig. S1). All lacked the ermC gene, and


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only some carried the ileS-2 resistance determinant. In addition some of the ileS-2 positive variants also carried the kanamycin resistance gene addD. Also detected amongst USA300 isolates (3/154, 2%) was a 2.5 kb ermC-positive pWBG751-like plasmid and a small tetracycline resistance (tetK) plasmid (8/154, 5%), resembling the 4.4 kb pUSA02 (Diep et al., 2006).

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Temporal variation in the accessory genome

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We further analysed whether the variable distribution of selected MGEs was associated with the year of sampling. For this, we compared the distribution of SCCmec, ACME and Sa5 amongst USA300 isolates collected in 2004 with the 2009 and 2010 collections from matched healthcare centres (Table 1). All USA300 isolates carried SCCmec in 2004, with a single negative isolate in 2009. The frequency of SCCmec carriage was lowest amongst 2010 isolates, which represented a significant drop in comparison with both 2004 (p < 0.05) and 2009 (p < 0.05) groups. Emergence of SCCmec negative USA300 isolates resulting from a sporadic loss of the element has been previously reported, however at lower frequency than observed here (Uhlemann et al., 2014). As such the reduced frequency of SCCmec amongst USA300 isolates from 2010 might be specific to that year. Nevertheless a reported shift in -lactams in favour of MRSA-active drugs when treating SSTI might have influenced the epidemiology of CA-MRSA infections (Talan et al., 2011). Lower frequency of beta-lactam usage could result in a lower selective pressure to maintain SCCmec leading to gradual loss of this element. While USA300 has been recognised predominantly as a MRSA lineage, infections caused by MSSA USA300 have also been reported suggesting that transmission of USA300 may not be entirely dependent on SCCmec carriage (McCaskill et al., 2007, Talan et al., 2011). In support of hypothesis that a shift in epidemiology of MRSA might have occurred around 2010, we also observed an increase in prevalence of non-USA300 SCCmec negative isolates at this sampling time point.

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As described above, carriage of ACME was associated with the distribution of SCCmec amongst USA300 isolates. Therefore in line with SCCmec prevalence, loss of ACME was only observed in 2009 and 2010 samples, both groups showing a significantly lower number of ACME positive USA300 isolates when contrasted with the 2004 group (p < 0.05). Although the prevalence of ACME negative USA300 isolates was more notable in 2010, the difference between 2009 and 2010 was not significant (p = 0.39).

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The sample size of our collection was relatively low and thus the observed here temporal variation in the distribution of SCCmec and ACME among USA300 isolates and the apparent loss of these elements in more recent samples would require further surveillance. However, the occurrence of SCCmec and ACME negative USA300 isolates was not limited to a particular healthcare centre (Table 1). Furthermore, the lack of phylogenetic association between USA300 isolates lacking either SCCmec or ACME suggests that forces driving the loss of these important MGEs are acting upon the wider USA300 population.

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In addition to changes in prevalence of SCCmec and ACME, we observed a trend of increasing prophage Sa5 frequency, although only the difference between 2004 and 2010 isolates was statistically significant (p < 0.05). The overall frequency of Sa5 carriage by


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USA300 isolates collected in 2009 and 2010 was higher than the prevalence reported for New York City isolates (16%) (Uhlemann et al., 2014). However, the presence of Sa5 prophage within the latter collection was associated with a particular USA300 sub-clade, suggesting that carriage of this prophage might contribute to expansion of more successful sub-populations (Uhlemann et al., 2014).

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The analysed here USA300 population demonstrated a stable temporal prevalence of antimicrobial resistance-associated MGEs. All isolates collected in 2004 carried the multiple drug resistance pUSA300HOUMR-like plasmid, which was also found in majority of 2009 (20/23, 87%) and 2010 isolates (28/29, 97%) from matched healthcare centres. A dip in pUSA300HOUMR-like plasmid prevalence amongst 2009 isolates was specific to a single centre and thus unlikely to represent a wider trend. It has been previously observed that carriage of aminoglycoside and bacitracin resistance genes associated with the pUSA300HOUMR-like plasmid might mediate resistance to certain topical antimicrobial agents available over-the-counter in the US (Suzuki et al., 2011). It is likely, therefore, that a continuous selective pressure has been maintaining this element within the USA300 population.

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We also investigated whether the chromosomally-inserted MGEs carried by USA300 (Sa2, Sa3, SaPI5, ACME, SCCmec IVa and ICE6013) diversified over time leading to temporal variation in the distribution of pairwise SNP distances. To discount recombination-mediated variation, alignments of each MGE sequence were first queried for the presence of recombination regions and if detected, the corresponding isolate was removed from the analysis (up to two isolates for ICE6013, Sa3 and SCCmec IVa were removed). We found that the analysed MGEs were well conserved between the isolates, with a median pairwise SNP distance for all elements equal to 1. When comparing the median of pairwise SNP distances for individual MGEs between the 2004, 2009 and 2010 isolates, temporal variation was observed for all except ACME and ICE6013 elements (Fig. 3). For all an increase of median pairwise SNP distance by 1 was observed at either or both later sampling time points.

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Outside the USA300 clade, it was noteworthy that the temporally distributed USA500-like sub-population differed markedly in its accessory genome from the closely related USA500 clone and could be distinguished based on SCCmec type, carriage of dfrG insertion fragment and absence of the ICE6013.

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Core genome microevolution of USA300 clade

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We investigated further the core genomes of USA300 isolates and found that the population formed a heterogeneous clade with a median pairwise SNP distance of 88 and a range of 0 165 SNPs. We then investigated if USA300 population has become more heterogeneous over time by comparing pairwise SNP distances of isolates collected in 2004 with the panels from 2009 and 2010 (isolates from matched healthcare centres only). We observed an increase in median pairwise SNP distance values between 2004 (62, MAD = 13) and 2009 (103, MAD = 13) as well as 2010 (98, MAD = 15) groups. As previously reported (Kennedy et al., 2008), the USA300 isolates demonstrate an elevated ratio of non-synonymous-to-synonymous SNPs (dN/dS), associated with the recent clonal expansion of this lineage and accumulation of


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SNPs that have not yet been eliminated by purifying selection (Rocha et al., 2006). We observed a dN/dS median of 0.71 (MAD = 0.19) across USA300 core genomes, in comparison to the median 0.22 (MAD = 0.01) dN/dS ratio for non-USA300 isolates. In order to determine the dN/dS ratio of mutations that were acquired independently by the USA300 isolates after each diverged from the last common ancestor, we determined the dN/dS ratios on terminal branches of the USA300 clade. For clonal or very closely related isolates (up to 2 pairwise SNPs distance) a single representative was selected, and isolates that accumulated less than a total of 10 SNPs since divergence from the last common ancestor, or showed either no synonymous or no non-synonymous mutations were excluded. A total of 139 USA300 isolates were analysed and revealed a median dN/dS ratio of 2.6 (MAD = 1.1), which corresponded to a previously reported dN/dS ratio for another USA300 collection (Kennedy et al., 2008). Analysis of temporal changes in dN/dS ratio revealed equally high median values for 2004 (2.33, MAD = 1.23), 2009 (2.5, MAD = 0.4) and 2010 (2.8, MAD = 1.5) altogether indicating that purifying selection has not yet acted upon the USA300 population (Rocha et al., 2006).

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To interrogate more closely the phylogenetic structure of the USA300 clade, we repeated the phylogeny reconstruction for USA300 isolates only (Fig. 4). Genomic divergence within the USA300 clade, based on branch lengths, was most pronounced on terminal branches and thus was strain-specific. In line with previous studies describing the population structure of USA300 isolates, the generated phylogeny revealed low resolution of inter-strain evolutionary relationships due to short internal branches. We therefore investigated further the clustering patterns of the innermost branches descending immediately from the root of USA300 clade, by focusing on branches that after each evolutionary split gave rise to a majority of the descending population. This revealed a subtle and sequential diversification process involving the formation of nested clades (Fig. 4). Based on the collection analysed in this study, at least six fine-scale divergence events descending from the root of the clade could be identified, each involving acquisition of up to five SNPs (Table 2). Where branches emerged due to multiple SNPs, in each case the mutations were independent in relation to their genomic position and thus did not represent a recombination. Nearly all mutations occurring in protein coding sequence were non-synonymous (11/12) and involved genes of various functions. Four of the non-synonymous SNPs (NS-SNP) occurring across three distinct branches were identified in genes associated with antimicrobial resistance. This included a gene encoding a putative drug resistance transporter ErmB/QacA (SAUSA300_2126), a gene encoding an efflux pump (SAUSA300_2489), recently associated with resistance to linoleic and arachidonic acids that are present on the skin and contribute to the low pH of this environment (Alnaseri et al., 2015) and gyrA (SAUSA300_006) and parC/grlA (SAUSA300_1251) genes associated with fluoroquinolone resistance. The emergence of fluoroquinolone resistance in the USA300 population was previously described and estimated to have occurred around 1995 (Uhlemann et al., 2014). Amongst other genes affected by non-synonymous SNPs was a putative virulence determinant (SAUSA300_1322), which belongs to the cvfC operon and contributes to control of haemolysin production and detergent resistance through positive regulation of thyA gene (Ikuo et al., 2010).


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To further resolve the observed USA300 tree topology and formation of nested clades, we have reconstructed the phylogeny of the study USA300 isolates together with 329 genomes of previously sequenced USA300 isolates derived from New York city (Uhlemann et al., 2014). The tree topology of this expanded USA300 collection also revealed a nested clade population structure (Fig. S4). The clade-defining mutations corresponded to the polymorphisms described above (Table 2) demonstrating that the early diversification changes identified in this work can be detected across wider USA300 population.

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Conclusions

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This study provides an overview of the S. aureus CC8 population structure based on whole genome sequence analysis of clinical isolates derived from 9 US states, collected in 2004, 2009 and 2010. Unsurprisingly, it was dominated by the USA300 clone. The non-USA300 isolates represented a fifth of the CC8 population and included the USA500 clone as well as a closely related USA500-like genotype with a distinct accessory genome composition, which emerged at the later sampling time point. The USA500 clone emerged prior to the appearance and dissemination of USA300 genotype but has been diminishing in prevalence since (David et al., 2015, Nelson et al., 2015). This trend was also observed in our collection, with the majority of USA500 isolates identified amongst the 2004 samples. Furthermore, the distribution of both USA500 and USA500-like isolates was geographically restricted as most originated from a single site (HC10). This is in contrast to geographically dispersed USA300 isolates. There may also be additional regional differences associated with USA500 that we did not detect owing to the majority of USA500 isolates being detected from just one site. For example Benson et al. (2014) identified that most of USA500 isolates obtained from patients in New York area harboured the IS256 element located in close proximity to the rot gene. In our analysis we identified the presence of this IS element in the genomes of all USA500 isolates and a single USA500-like strain. However, insertion of IS256 next to rot was observed for only one of the USA500 strains.

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The analysis of temporal variation in population structure of CC8 isolates indicated a significant drop in the prevalence of USA300 around 2010, in comparison with both 2004 and 2009. However, due to the limitations of our study such as narrow sampling time points, small sample size and lack of isolates representing the Western US we could not verify if this dip in frequency of USA300 clone represented a broader event or a time trend. A recent review reported that there has been an overall increase in spatio-temporal prevalence of USA300 across the US between 2000 and 2013 in the context of MRSA epidemiology (Carrel et al., 2015). However, regional differences as well as year-on-year fluctuations were also observed demonstrating a need for a national surveillance program that would allow more accurate monitoring of the epidemiology of clinically significant S. aureus clones such as USA300.

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Based on this geographically diverse collection of CC8 isolates, we investigated the USA300 clade for evidence of microevolution. The observed limited phylogeographic clustering suggests a general lack of geographical expansion events resulting from emergence of locally adapted clones. This is in agreement with findings that at a local community level, multiple introductions of USA300 occur (Uhlemann et al., 2014). Analysis of core genomes revealed that USA300 isolates are becoming more heterogeneous as defined by a temporal increase


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in pairwise SNP distances, which is underlined by a stable pattern of elevated dN/dS ratios. However, an analysis of the wider phylogenetic relationships defined by branches descending immediately from the root of the clade revealed the presence of subtle polymorphisms shared across the population. The pattern of accumulative SNP acquisition as defined by the formation of nested clades suggests that the USA300 ancestral isolates continued to diversify shortly after the emergence of the clade but prior to its clonal expansion. Although determining the phenotypic significance of the acquired SNPs is beyond the scope of this work, we speculate that at least some might represent adaptive evolution such as development of antimicrobial resistance.

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Our data also suggests temporal changes in the composition of USA300 accessory genome involving small-scale loss of SCCmec and ACME elements and increased frequency of Sa5 carriage. These findings are subject to the previously described limitations of this study and thus might not be indicative of a continuous temporal trend of USA300 accessory genome re-shuffle. Still, the analysed here collection represents an unbiased sample of S. aureus CC8 population derived from diverse clinical sources, providing unique insight into the broader epidemiology and evolution of USA300 in the context of its parental lineage. This allowed us to capture the apparent instability of SCCmec and ACME carriage amongst more recent USA300 isolates. This is significant considering some strategies for identification of USA300 isolates based on defined genetic markers such as carriage of SCCmec or ACME (Alam et al., 2015, David et al., 2013), which might hinder identification of emerging USA300 variants that have lost these MGEs leading to their under-reporting. Also, taking into consideration the putative role of SCCmec and ACME in the successful dissemination of S. aureus USA300 it will be important to understand what leads to the loss of these elements. We speculate that the loss of SCCmec may have been driven by changes in antimicrobial prescription practices in the US. It is more challenging to hypothesize what factors could have contributed to the drop in ACME prevalence. However, the observation that absence of SCCmec and ACME coincided in majority of isolates, suggests that the elements are frequently lost in a single deletion event. This scenario is supported by previous findings that the CcrAB recombinases of SCCmec can mobilise ACME as well as catalyse an excision of the entire ACME-SCCmec composite island (Diep et al., 2008). While further work is required to investigate how this might impact on the epidemiology of USA300, the analysis presented here of spatially diverse USA300 isolates gives a new insight into the wider evolutionary changes occurring within this highly successful sub-lineage, and provides ground for further surveillance of this clone.

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ACKNOWLEDGEMENTS

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We thank the core sequencing and informatics teams at the Sanger Institute for their assistance as well as the following employees of Pfizer for their helpful suggestions: Li Hao, Robert Donald and Elizabeth Bieger. SRH and JP were supported by Wellcome Trust grant


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098051. This work was funded by Pfizer Vaccine Research and Development. NM, CYT and ASA are employees of Pfizer and may own stock in the company.

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ABBREVIATIONS

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ACME: arginine catabolic mobile element; bp: base pair; CA-MRSA: community-associated methicillin-resistance Staphylococcus aureus; CC: clonal complex; HA-MRSA: hospitalassociated methicillin-resistant Staphylococcus aureus; HC: healthcare centre; ICE6013: integrative conjugative element 6013; MAD – median absolute deviation; MGE: mobile genetic element; MRSA: methicillin-resistance Staphylococcus aureus; MSSA: methicillinsusceptible Staphylococcus aureus; SaPI: staphylococcal pathogenicity island; SCCmec: Staphylococcal Cassette Chromosome mec; SNP: single nucleotide polymorphism; SLV: single locus variant; ST: sequence type; T.E.S.T.: Tigecycline Evaluation and Surveillance Trial

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REFERENCES

556 557 558

Alam, M. T., Read, T. D., Petit, R. A., Boyle-Vavra, S., Miller, L. G., Eells, S. J., Daum, R. S. & David, M. Z. (2015). Transmission and microevolution of USA300 MRSA in US households: evidence from whole-genome sequencing. Mbio 6, e00054-00015.

559 560 561 562

Alnaseri, H., Arsic, B., Schneider, J. E., Kaiser, J. C., Scinocca, Z. C., Heinrichs, D. E. & McGavin, M. J. (2015). Inducible expression of a resistance-nodulation-division-type efflux pump in Staphylococcus aureus provides resistance to linoleic and arachidonic acids. J Bacteriol 197, 1893-1905.

563

Altman, D., Sebra, R., Hand, J., Attie, O., Deikus, G., Carpini, K., Patel, G., Rana, M.,

564

Arvelakis, A. & Grewal, P. (2014). Transmission of Methicillin‐Resistant Staphylococcus

565 566

aureus via Deceased Donor Liver Transplantation Confirmed by Whole Genome Sequencing. Am J Transplant 14, 2640-2644.

567 568 569 570

Begier, E. M., Frenette, K., Barrett, N. L., Mshar, P., Petit, S., Boxrud, D. J., Watkins-Colwell, K., Wheeler, S., Cebelinski, E. A. & Glennen, A. (2004). A high-morbidity outbreak of methicillin-resistant Staphylococcus aureus among players on a college football team, facilitated by cosmetic body shaving and turf burns. Clin Infect Dis 39, 1446-1453.

571 572 573

Benson, M. A., Ohneck, E. A., Ryan, C., Alonzo, F., Smith, H., Narechania, A., Kolokotronis, S. O., Satola, S. W., Uhlemann, A. C. & Sebra, R. (2014). Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element. Mol Microbiol 93, 664-681.

574 575 576 577

Boyle-Vavra, S., Li, X., Alam, M. T., Read, T. D., Sieth, J., Cywes-Bentley, C., Dobbins, G., David, M. Z., Kumar, N. & Eells, S. J. (2015). USA300 and USA500 Clonal Lineages of Staphylococcus aureus Do Not Produce a Capsular Polysaccharide Due to Conserved Mutations in the cap5 Locus. mBio 6, e02585-02514.


578 579 580

Carey, A. J., Della-Latta, P., Huard, R., Wu, F., Graham, P. L., Carp, D. & Saiman, L. (2010). Changes in the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit. Infect Control 31, 613-619.

581 582

Carrel, M., Perencevich, E. N. & David, M. Z. (2015). USA300 Methicillin-Resistant Staphylococcus aureus, United States, 2000–2013. Emerg Infect Dis 21, 1973.

583 584 585

Chua, T., Moore, C. L., Perri, M. B., Donabedian, S. M., Masch, W., Vager, D., Davis, S. L., Lulek, K., Zimnicki, B. & Zervos, M. J. (2008). Molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates in urban Detroit. J Clin Microbiol 46, 2345-2352.

586 587 588

Control, C. f. D. & Prevention (2001). Methicillin-resistant Staphylococcus aureus skin or soft tissue infections in a state prison--Mississippi, 2000. MMWR Morbidity and mortality weekly report 50, 919.

589 590 591

Control, C. f. D. & Prevention (2003). Methicillin-resistant Staphylococcus aureus infections among competitive sports participants--Colorado, Indiana, Pennsylvania, and Los Angeles County, 2000-2003. MMWR Morbidity and mortality weekly report 52, 793.

592 593 594

Croucher, N. J., Page, A. J., Connor, T. R., Delaney, A. J., Keane, J. A., Bentley, S. D., Parkhill, J. & Harris, S. R. (2014). Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res, gku1196.

595 596 597 598 599

David, M. Z., Taylor, A., Lynfield, R., Boxrud, D. J., Short, G., Zychowski, D., Boyle-Vavra, S. & Daum, R. S. (2013). Comparing pulsed-field gel electrophoresis with multilocus sequence typing, spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for Panton-Valentine leukocidin, arcA, and opp3 in methicillin-resistant Staphylococcus aureus isolates at a US medical center. J Clin Microbiol 51, 814-819.

600 601 602 603

David, M. Z., Acree, M. E., Sieth, J. J., Boxrud, D. J., Dobbins, G., Lynfield, R., Boyle-Vavra, S. & Daum, R. S. (2015). Pediatric Staphylococcus aureus Isolate Genotypes and Infections from the Dawn of the Community-Associated Methicillin-Resistant S. aureus Epidemic Era in Chicago, 1994 to 1997. J Clin Microbiol 53, 2486-2491.

604 605

Deurenberg, R. H. & Stobberingh, E. E. (2008). The evolution of Staphylococcus aureus. Infect Genet Evol 8, 747-763.

606 607 608 609

Diep, B. A., Gill, S. R., Chang, R. F., Phan, T. H., Chen, J. H., Davidson, M. G., Lin, F., Lin, J., Carleton, H. A. & Mongodin, E. F. (2006). Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. The Lancet 367, 731-739.

610 611 612 613 614

Diep, B. A., Stone, G. G., Basuino, L., Graber, C. J., Miller, A., des Etages, S.-A., Jones, A., Palazzolo-Ballance, A. M., Perdreau-Remington, F. & Sensabaugh, G. F. (2008). The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus. J Infect Dis 197, 1523-1530.

615 616 617

Enright, M. C., Day, N. P., Davies, C. E., Peacock, S. J. & Spratt, B. G. (2000). Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones ofStaphylococcus aureus. J Clin Microbiol 38, 1008-1015.


618 619 620 621

Francis, J. S., Doherty, M. C., Lopatin, U., Johnston, C. P., Sinha, G., Ross, T., Cai, M., Hansel, N. N., Perl, T. & Ticehurst, J. R. (2005). Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin Infect Dis 40, 100-107.

622 623 624

Freitas, E. A., Harris, R. M., Blake, R. K. & Salgado, C. D. (2010). Prevalence of USA300 strain type of methicillin-resistant Staphylococcus aureus among patients with nasal colonization identified with active surveillance. Infect Control 31, 469-475.

625 626

Fritz, S. A. & Purvis, A. (2010). Selectivity in mammalian extinction risk and threat types: a new measure of phylogenetic signal strength in binary traits. Conserv Biol 24, 1042-1051.

627 628 629

Goerke, C., Pantucek, R., Holtfreter, S., Schulte, B., Zink, M., Grumann, D., Bröker, B. M., Doskar, J. & Wolz, C. (2009). Diversity of prophages in dominant Staphylococcus aureus clonal lineages. J Bacteriol 191, 3462-3468.

630 631 632 633

Gonzalez, B. E., Martinez-Aguilar, G., Hulten, K. G., Hammerman, W. A., Coss-Bu, J., AvalosMishaan, A., Mason, E. O. & Kaplan, S. L. (2005). Severe staphylococcal sepsis in adolescents in the era of community-acquired methicillin-resistant Staphylococcus aureus. Pediatrics 115, 642-648.

634 635 636

Gonzalez, B. E., Rueda, A. M., Shelburne, S. A., Musher, D. M., Hamill, R. J. & Hultén, K. G. (2006). Community-associated strains of methicillin-resistant Staphylococccus aureus as the cause of healthcare-associated infection. Infect Control 27, 1051-1056.

637 638 639

Haque, N., Davis, S., Manierski, C., Vager, D., Donabedian, S., Perri, M., Sabbagh, R., Cheema, F. & Zervos, M. (2007). Infective endocarditis caused by USA300 methicillinresistant Staphylococcus aureus (MRSA). Int J Antimicrob Agents 30, 72-77.

640 641 642

Harris, S. R., Feil, E. J., Holden, M. T., Quail, M. A., Nickerson, E. K., Chantratita, N., Gardete, S., Tavares, A., Day, N. & Lindsay, J. A. (2010). Evolution of MRSA during hospital transmission and intercontinental spread. Science 327, 469-474.

643 644 645

Highlander, S. K., Hultén, K. G., Qin, X., Jiang, H., Yerrapragada, S., Mason, E. O., Shang, Y., Williams, T. M., Fortunov, R. M. & Liu, Y. (2007). Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus. BMC Microbiol 7, 99.

646 647 648 649

Holden, M. T., Hsu, L.-Y., Kurt, K., Weinert, L. A., Mather, A. E., Harris, S. R., Strommenger, B., Layer, F., Witte, W. & de Lencastre, H. (2013). A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic. Genome Res 23, 653-664.

650 651 652

Hsu, L.-Y., Harris, S. R., Chlebowicz, M. A., Lindsay, J. A., Koh, T.-H., Krishnan, P., Tan, T.-Y., Hon, P.-Y., Grubb, W. B. & Bentley, S. D. (2015). Evolutionary dynamics of methicillinresistant Staphylococcus aureus within a healthcare system. Genome Biol 16, 81.

653 654

Ikuo, M., Kaito, C. & Sekimizu, K. (2010). The cvfC operon of Staphylococcus aureus contributes to virulence via expression of the thyA gene. Microb Pathog 49, 1-7.

655 656 657 658

Kahl, B. C., Mellmann, A., Deiwick, S., Peters, G. & Harmsen, D. (2005). Variation of the polymorphic region X of the protein A gene during persistent airway infection of cystic fibrosis patients reflects two independent mechanisms of genetic change in Staphylococcus aureus. J Clin Microbiol 43, 502-505.


659 660 661

Kazakova, S. V., Hageman, J. C., Matava, M., Srinivasan, A., Phelan, L., Garfinkel, B., Boo, T., McAllister, S., Anderson, J. & Jensen, B. (2005). A clone of methicillin-resistant Staphylococcus aureus among professional football players. N Engl J Med 352, 468-475.

662 663 664 665

Kennedy, A. D., Otto, M., Braughton, K. R., Whitney, A. R., Chen, L., Mathema, B., Mediavilla, J. R., Byrne, K. A., Parkins, L. D. & Tenover, F. C. (2008). Epidemic communityassociated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proceedings of the National Academy of Sciences 105, 1327-1332.

666 667 668 669 670

Kennedy, A. D., Porcella, S. F., Martens, C., Whitney, A. R., Braughton, K. R., Chen, L., Craig, C. T., Tenover, F. C., Kreiswirth, B. N. & Musser, J. M. (2010). Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences. J Clin Microbiol 48, 4504-4511.

671 672 673 674

Li, M., Diep, B. A., Villaruz, A. E., Braughton, K. R., Jiang, X., DeLeo, F. R., Chambers, H. F., Lu, Y. & Otto, M. (2009). Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus. Proceedings of the National Academy of Sciences 106, 5883-5888.

675 676 677 678

Limbago, B., Fosheim, G. E., Schoonover, V., Crane, C. E., Nadle, J., Petit, S., Heltzel, D., Ray, S. M., Harrison, L. H. & Lynfield, R. (2009). Characterization of methicillin-resistant Staphylococcus aureus isolates collected in 2005 and 2006 from patients with invasive disease: a population-based analysis. J Clin Microbiol 47, 1344-1351.

679 680 681 682

Liu, C., Graber, C. J., Karr, M., Diep, B. A., Basuino, L., Schwartz, B. S., Enright, M. C., O'Hanlon, S. J., Thomas, J. C. & Perdreau-Remington, F. (2008). A population-based study of the incidence and molecular epidemiology of methicillin-resistant Staphylococcus aureus disease in San Francisco, 2004–2005. Clin Infect Dis 46, 1637-1646.

683 684 685 686

McCaskill, M. L., Mason Jr, E. O., Kaplan, S. L., Hammerman, W., Lamberth, L. B. & Hultén, K. G. (2007). Increase of the USA300 clone among community-acquired methicillinsusceptible Staphylococcus aureus causing invasive infections. The Pediatric infectious disease journal 26, 1122-1127.

687 688 689 690

McDougal, L. K., Steward, C. D., Killgore, G. E., Chaitram, J. M., McAllister, S. K. & Tenover, F. C. (2003). Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41, 5113-5120.

691 692 693

Milheiriço, C., Oliveira, D. C. & de Lencastre, H. (2007). Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus:‘SCCmec IV multiplex’. J Antimicrob Chemother 60, 42-48.

694 695 696

Milheiriço, C., Oliveira, D. C. & de Lencastre, H. (2007). Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother 51, 3374-3377.

697 698 699 700

Miller, L. G., Perdreau-Remington, F., Rieg, G., Mehdi, S., Perlroth, J., Bayer, A. S., Tang, A. W., Phung, T. O. & Spellberg, B. (2005). Necrotizing fasciitis caused by communityassociated methicillin-resistant Staphylococcus aureus in Los Angeles. N Engl J Med 352, 1445-1453.


701 702 703

Monecke, S., Coombs, G., Shore, A. C., Coleman, D. C., Akpaka, P., Borg, M., Chow, H., Ip, M., Jatzwauk, L. & Jonas, D. (2011). A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One 6, e17936.

704 705 706 707

Nelson, M. U., Bizzarro, M. J., Baltimore, R. S., Dembry, L. M. & Gallagher, P. G. (2015). Clinical and Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Neonatal Intensive Care Unit in the Decade Following Implementation of an Active Detection and Isolation Program. J Clin Microbiol, JCM. 00470-00415.

708 709 710 711

Pasquale, T. R., Jabrocki, B., Salstrom, S.-J., Wiemken, T. L., Peyrani, P., Haque, N. Z., Scerpella, E. G., Ford, K. D., Zervos, M. J. & Ramirez, J. A. (2013). Emergence of methicillinresistant Staphylococcus aureus USA300 genotype as a major cause of late-onset nosocomial pneumonia in intensive care patients in the USA. Int J Infect Dis 17, e398-e403.

712 713 714

Patel, M., Kumar, R. A., Stamm, A. M., Hoesley, C. J., Moser, S. A. & Waites, K. B. (2007). USA300 genotype community-associated methicillin-resistant Staphylococcus aureus as a cause of surgical site infections. J Clin Microbiol 45, 3431-3433.

715 716 717

Rocha, E. P., Smith, J. M., Hurst, L. D., Holden, M. T., Cooper, J. E., Smith, N. H. & Feil, E. J. (2006). Comparisons of dN/dS are time dependent for closely related bacterial genomes. J Theor Biol 239, 226-235.

718

Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics, btu153.

719 720 721 722

Seybold, U., Kourbatova, E. V., Johnson, J. G., Halvosa, S. J., Wang, Y. F., King, M. D., Ray, S. M. & Blumberg, H. M. (2006). Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care—associated blood stream infections. Clin Infect Dis 42, 647-656.

723 724 725

Seybold, U., Talati, N., Kizilbash, Q., Shah, M., Blumberg, H. & Franco-Paredes, C. (2007). Hematogenous osteomyelitis mimicking osteosarcoma due to Community Associated Methicillin-Resistant Staphylococcus aureus. Infection 35, 190-193.

726 727

Smyth, D. S. & Robinson, D. A. (2009). Integrative and sequence characteristics of a novel genetic element, ICE6013, in Staphylococcus aureus. J Bacteriol 191, 5964-5975.

728 729

Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312-1313.

730 731 732

Suzuki, M., Yamada, K., Nagao, M., Aoki, E., Matsumoto, M., Hirayama, T., Yamamoto, H., Hiramatsu, R., Ichiyama, S. & Iinuma, Y. (2011). Antimicrobial ointments and methicillinresistant Staphylococcus aureus USA300. Emerg Infect Dis 17, 1917-1920.

733 734 735

Talan, D. A., Krishnadasan, A., Gorwitz, R. J., Fosheim, G. E., Limbago, B., Albrecht, V. & Moran, G. J. (2011). Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008. Clin Infect Dis 53, 144-149.

736 737

Tattevin, P., Diep, B. A., Jula, M. & Perdreau-Remington, F. (2009). Methicillin-resistant Staphylococcus aureus USA300 clone in long-term care facility. Emerg Infect Dis 15, 953.

738 739 740 741

Tenover, F. C., McDougal, L. K., Goering, R. V., Killgore, G., Projan, S. J., Patel, J. B. & Dunman, P. M. (2006). Characterization of a strain of community-associated methicillinresistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol 44, 108-118.


742 743 744 745

Tenover, F. C., McAllister, S., Fosheim, G., McDougal, L. K., Carey, R. B., Limbago, B., Lonsway, D., Patel, J. B., Kuehnert, M. J. & Gorwitz, R. (2008). Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004. J Clin Microbiol 46, 2837-2841.

746 747 748 749

Thurlow, L. R., Joshi, G. S., Clark, J. R., Spontak, J. S., Neely, C. J., Maile, R. & Richardson, A. R. (2013). Functional modularity of the arginine catabolic mobile element contributes to the success of USA300 methicillin-resistant Staphylococcus aureus. Cell host & microbe 13, 100107.

750 751 752 753

Uhlemann, A.-C., Dordel, J., Knox, J. R., Raven, K. E., Parkhill, J., Holden, M. T., Peacock, S. J. & Lowy, F. D. (2014). Molecular tracing of the emergence, diversification, and transmission of S. aureus sequence type 8 in a New York community. Proceedings of the National Academy of Sciences 111, 6738-6743.

754 755

Zerbino, D. R. & Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18, 821-829.

756 757

Zhang, H., Gao, S., Lercher, M. J., Hu, S. & Chen, W.-H. (2012). EvolView, an online tool for visualizing, annotating and managing phylogenetic trees. Nucleic Acids Res 40, W569-W572.

758 759 760

DATA BIBLIOGRAPHY

761 762 763 764 765 766 767 768 769 770 771 772 773

1. Altman, D.R., Sebra, R., Hand, J., Attie, O., Deikus, G., Carpini, K.W.D., Patel, G., Rana, M., Arvelakis, A., Grewal, P. and Dutta, J. GenBank accession NZ_CP007176 (2014). 2. Benson, M.A., Ohneck, E.A., Ryan, C., Alonzo, F., Smith, H., Narechania, A., Kolokotronis, S.O., Satola, S.W., Uhlemann, A.C., Sebra, R. and Deikus, G. GenBank accession NZ_CP007499 (2014). 3. Diep, B.A., Gill, S.R., Chang, R.F., Phan, T.H., Chen, J.H., Davidson, M.G., Lin, F., Lin, J., Carleton, H.A., Mongodin, E.F. and Sensabaugh, G.F. GenBank accession NC_007793 (2006). 4. Gill, S.R., Fouts, D.E., Archer, G.L., Mongodin, E.F., DeBoy, R.T., Ravel, J., Paulsen, I.T., Kolonay, J.F., Brinkac, L., Beanan, M. and Dodson, R.J. GenBank accession NC_002951 (2005). 5. Uhlemann, A.C., Dordel, J., Knox, J.R., Raven, K.E., Parkhill, J., Holden, M.T., Peacock, S.J. and Lowy, F.D. ENA accession PRJEB2870 (2014).


Research paper template

774 775

FIGURES AND TABLES

776 777 778

Table 1. Temporal variation in the distribution of selected mobile genetic elements among USA300 isolates. Based on isolates derived from healthcare centres selected for temporal analysis (HC1, HC6, HC7, HC10).

779 780

Table 2. Descriptive summary of SNPs identified on branches of early divergence nested clades across the phylogenetic tree of analysed USA300 isolates. The phylogeny is presented in Fig. 4

781 782 783 784

Figure 1. Geographic distribution of healthcare centres (HC) that collected isolates analysed in this study. The geographic location on the map represents state coordinates, where the corresponding HC was based. Plots are scaled to represent total number of isolates from corresponding site. HCs that provided isolates used in temporal analyses are marked with a star.

785 786 787 788 789 790 791 792 793 794 795 796 797

Figure 2. Midpoint-rooted phylogenetic tree of the 191 analysed S. aureus CC8 isolates. Four reference genomes were included (corresponding nodes marked with a star). Branches of USA300, USA500 and USA500-like clades have been highlighted. The tree has been annotated to show the association between phylogenetic distribution and healthcare centre (HC) of origin (excluding reference genomes), year of collection (excluding reference genomes), patient type (excluding reference genomes), infection type (excluding reference genomes) and carriage of mobile genetic elements (MGE). The latter shows the distribution of identified prophages, pathogenicity islands (where novel sequence was identified name has been assigned based on virulence gene content), transposons and other elements such as ICE6013 and dfrG insertion element, and plasmids (name assigned based on close similarity to previously identified plasmid). Further details on variable carriage of virulence genes within Sa2 and Sa3 prophages, as well as variable distribution of antimicrobial resistance genes within pUSA300HOUMR-like and pUSA03-like plasmids have been provided in Fig. S1. The tree was annotated using EvolView (Zhang et al., 2012)

798 799 800

Figure 3. Temporal variation in pairwise SNP distance distributions between chromosomallyinserted MGEs carried by USA300 isolates. Based on isolates derived from healthcare centres selected for temporal analysis (HC1, HC6, HC7, HC10).

801 802 803 804 805

Figure 4. Mid-point rooted phylogenetic tree of S. aureus USA300 isolates. Two most closely related non-USA300 isolates were included as an out-group (isolates PFESA2332 and PFESA1492, branches coloured in red). Also, two reference genomes were included and corresponding nodes are marked with a star. Branches were highlighted to show consecutive clades of the nested clade structure, and labelled from 1 to 6. SNPs that define each labelled clade are described in Table 2.

806


Table_1

Click here to download Table Table_1.docx

*

HC1 HC6 HC7 HC10 Total p† p‡ p§

N 14 6 8 18 46

2004 SCCmec ACME 14 14 6 6 8 8 18 18 46 46

φSa5 2 1 3 2 8

*

N na 12 10 1 23

2009 SCCmec ACME na na 12 12 9 8 1 1 22 21 0.19 A

R228H

1915352

Intergenic

-

-

A -> G

-

3

386867

Intergenic

-

-

A -> T

-

4

906507

Intergenic

-

-

C -> A

-

1693739

Non-synonymous

SAUSA300_1543

oxygen-independent coproporphyrinogen III oxidase

C -> T

A145T

1908169

Non-synonymous

SAUSA300_1725

transaldolase

C -> T

V6I

2230768

Synonymous

SAUSA300_2068

hypothetical protein

T -> C

-

2690087

Non-synonymous

SAUSA300_2489

antibiotic transport-associated protein

G -> T

Q795K

7282

Non-synonymous

SAUSA300_0006

DNA gyrase GyrA

C -> T

S84L

1374405

Non-synonymous

SAUSA300_1251

DNA topoisomerase ParC/GrlA

C -> A

S80Y

837108

Non-synonymous

SAUSA300_0750

hypothetical protein

T -> G

D17E

1074815

Non-synonymous

SAUSA300_0980

putative membrane protein

A -> G

N71D

5 6


Figure 1 50

Click here to download Figure Fig1.pdf

HC8 HC9

45

HC2

HC4 HC12 HC5

40 HC3 HC6

Temporal analysis HC

35 HC10 HC7

Isolate_count

30

10 HC1

HC11

20 30

25 -120


-100

-80

0.01 Figure 2

Clades

Prophages

COL

USA300

USA500_2395

USA500

USA300_ISMMS1

USA500-like

USA300_FPR3757

Pathogenicity islands

Transposons Click here to download Figure Fig2_revised.pdf

ACME & SCCmec

& other elements

2004

HC1

2009

HC2

2010

0.01 m es HC3

HC4

Reference genom es

1 7

HC5 COL HC6 USA500_2395

HC7 USA300_ISMMS1 HC8 USA300_FPR3757

ST 8 3007 3008

MS/MR

HC9

MSSA

HC HC10

MRSA

HC1 HC11 HC2 HC12 HC3

YearHC4 2004 HC5 2009 HC6

HC

Year of isolat ion HC

Healt hcare cent re HC1 HC6 HC7 HC10

2010 HC7 HC8

Pat ient t ype HC9ient Inpat HC10 Out pat ient HC11

Infect ion t ype HC12 Wound

Respirat ory Year 2004 Invasive Ot2009 her 2010

MGE present Pat ient t ype Inpat ient Out pat ient

Infect ion t ype Wound Respirat ory Invasive Ot her

MGE present

0.01 substitutions/site

Plasmids

Ye ar Pa I In ent fe ty φS cIo pe a n φS 1 ty pe a φS 2 a3 φS a φS 5 a Sa 7 PI Sa 3 P Sa I5 PI Sa _se P K-‐ Sa I_ea seQ P r _t AC I_e st M ar SC E _se C_ Cm se SC ec L Cm IV SC ec a Cm IV SC e d C c Tn me IVg 5 c Tn 54 NT 5 Tn 52 6 df 086 rG IC _IE E p 601 US 3 pU A30 SA 0H pU 01 OU -‐l pUSA0 ike MR -‐li SA 2-‐l ke pW 03 ike -‐ l B i k pW G7 e B 5 SA G7 1-‐lik P0 57 e 48 -‐lik A-‐ e lik e

Reference genom es

0.1


Figure 3

Click here to download Figure Fig3.pdf ɸSa3 2004 2009 2010

2004 2009 2010

ɸSa2USA300

1

2

3

4

5

0

1

2

3

4

Pairwise SNP distance

SaPI5

ACME

5

2004 2009 2010


2004 2009 2010

0

1

2

3

4

0

2

4

6

8

10


SCCmec IVa

ICE6013

12

2004 2009 2010


2004 2009 2010

0

0

2

4

6


8 10 0.0 0.5 1.0 Downloaded from www.microbiologyresearch.org by

IP: 107.172.249.178 On: Sun, 15 May 2016 15:08:00

1.5

2.0


2.5

3.0

Figure 4

Click here to download Figure Fig4.pdf

0.01

0.01 Reference genom es

COL USA500_2395 Reference genom es

COL USA300_ISMMS1

1

USA500_2395 USA300_FPR3757

ST

USA300_ISMMS1

8USA300_FPR3757 0.003 substitutions/site

2

250

ST976 8

1150

250 2253 976 2319

3

3000 1150 3001 2253 3007

2319

4

3008

3000

3010

3001

3015

3007 Year3008 of isolat ion 2004

5

3010 2009 3015 2010

6

Healt cention re Year hcare of isolat HC1 2004 HC2

2009

HC3

2010

HC4 HC5

Healt hcare cent re


Supplementary Material Files

Click here to download Supplementary Material Files Supplementary_Files_revised.pdf

Table S1. Description of 191 CC8 S. aureus isolates analysed in this study. Public name

ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA1490

ERS410907

MSSA

8

t008

-

USA300

2009

HC8

Wisconsin

Outpatient

Other

PFESA1492

ERS410923

MSSA

8

t008

-

-

2009

HC8

Wisconsin

Inpatient

Other

PFESA1606

ERS410796

MSSA

8

t008

-

-

2009

HC4

New York

Inpatient

Invasive

PFESA1614

ERS410765

MSSA

8

t008

-

USA300

2009

HC4

New York

Outpatient

Invasive

PFESA1788

ERS410716

MSSA

8

t008

-

-

2009

HC5

Pennsylvania

Inpatient

Invasive

PFESA1885

ERS410629

MSSA

8

t008

-

USA300

2009

HC3

Missouri

Inpatient

Other

PFESA1975

ERS411176

MSSA

8

t008

-

USA300

2010

HC10

Georgia

Inpatient

Invasive

PFESA1976

ERS411184

MSSA

8

t008

-

USA300

2010

HC10

Georgia

Inpatient

Respiratory

PFESA2034

ERS432065

MSSA

8

t008

-

USA300

2010

HC9

Wisconsin

Outpatient

Other

PFESA2064

ERS365748

MSSA

8

t008

-

USA300

2010

HC7

Texas

Inpatient

Wound

PFESA2070

ERS365796

MSSA

8

t008

-

USA300

2009

HC7

Texas

Inpatient

Wound

PFESA2083

ERS365797

MSSA

8

t008

-

-

2010

HC1

Florida

Inpatient

Invasive

PFESA2084

ERS365805

MSSA

8

t008

-

USA300

2010

HC1

Florida

Inpatient

Other

PFESA2091

ERS365766

MSSA

8

t008

-

USA300

2010

HC1

Florida

Inpatient

Other

PFESA2161

ERS365815

MSSA

8

t008

-

USA300

2010

HC2

Michigan

Inpatient

Respiratory

PFESA2175

ERS410378

MSSA

8

t008

-

-

2010

HC2

Michigan

Outpatient

Respiratory

PFESA2178

ERS410402

MSSA

8

t008

-

USA300

2009

HC11

Florida

Inpatient

Other

PFESA2180

ERS410418

MSSA

8

t008

-

USA300

2009

HC11

Florida

Inpatient

Wound

PFESA2184

ERS410450

MSSA

8

t008

-

USA300

2009

HC11

Florida

Inpatient

Other

PFESA2196

ERS410451

MSSA

8

t008

-

-

2010

HC12

Pennsylvania

Inpatient

Wound

PFESA2274

ERS365784

MSSA

8

t008

-

USA300

2010

HC6

Tennessee

Inpatient

Invasive

PFESA2298

ERS365754

MSSA

8

t008

-

USA300

2010

HC2

Michigan

Inpatient

Wound

PFESA2303

ERS365794

MSSA

8

t008

-

-

2010

HC2

Michigan

Inpatient

Other

PFESA2332

ERS410342

MSSA

8

t008

-

-

2010

HC8

Wisconsin

Outpatient

Wound

PFESA2413

ERS365826

MSSA

8

t008

-

USA300

2010

HC7

Texas

Inpatient

Respiratory



ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA2417

ERS411049

MSSA

8

t008

-

USA300

2010

HC7

Texas

Inpatient

Respiratory

PFESA2432

ERS410215

MSSA

8

t008

-

-

2010

HC5

Pennsylvania

Inpatient

Invasive

PFESA2449

ERS432202

MSSA

8

t008

-

USA300

2010

HC3

Missouri

Inpatient

Wound

PFESA2453

ERS432139

MSSA

8

t008

-

USA300

2010

HC9

Wisconsin

Outpatient

Wound

PFESA2460

ERS432195

MSSA

8

t008

-

USA300

2010

HC9

Wisconsin

Inpatient

Other

PFESA1306

ERS411147

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Wound

PFESA1308

ERS411163

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Invasive

PFESA1309

ERS411171

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Wound

PFESA1311

ERS411179

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Wound

PFESA1312

ERS411187

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Wound

PFESA1317

ERS411132

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Invasive

PFESA1322

ERS411172

MRSA

8

t008

IVa

USA300

2004

HC7

Texas

Inpatient

Wound

PFESA1326

ERS411112

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1328

ERS411204

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Inpatient

Wound

PFESA1329

ERS410929

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1333

ERS411133

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Inpatient

Invasive

PFESA1335

ERS411213

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Inpatient

Respiratory

PFESA1339

ERS411205

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Inpatient

Wound

PFESA1341

ERS410977

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Other

PFESA1342

ERS410985

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1345

ERS411009

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Other

PFESA1347

ERS411017

MRSA

8

t008

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1350

ERS411173

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Inpatient

Wound

PFESA1352

ERS410946

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Wound

PFESA1353

ERS410954

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Wound



ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA1354

ERS410962

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Wound

PFESA1355

ERS410970

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1357

ERS410986

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1358

ERS410994

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1359

ERS411002

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1360

ERS411010

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1362

ERS411018

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1364

ERS410939

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1365

ERS411157

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Inpatient

Other

PFESA1366

ERS411149

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Inpatient

Other

PFESA1368

ERS411141

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Inpatient

Wound

PFESA1369

ERS410955

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1371

ERS411126

MRSA

8

t008

IVa

USA300

2004

HC10

Georgia

Inpatient

Other

PFESA1391

ERS411150

MRSA

8

t008

IVa

USA300

2004

HC6

Tennessee

Inpatient

Other

PFESA1398

ERS411020

MRSA

8

t008

IVa

USA300

2004

HC6

Tennessee

Outpatient

Wound

PFESA1399

ERS410933

MRSA

8

t008

IVa

USA300

2004

HC6

Tennessee

Outpatient

Wound

PFESA1404

ERS410949

MRSA

8

t008

IVa

USA300

2004

HC6

Tennessee

Outpatient

Other

PFESA1410

ERS411167

MRSA

8

t008

IVa

USA300

2004

HC6

Tennessee

Inpatient

Other

PFESA1476

ERS410890

MRSA

8

t008

IVa

USA300

2010

HC8

Wisconsin

Inpatient

Wound

PFESA1477

ERS410898

MRSA

8

t008

IVa

USA300

2010

HC8

Wisconsin

Outpatient

Other

PFESA1485

ERS410867

MRSA

8

t008

IVa

USA300

2009

HC8

Wisconsin

Inpatient

Other

PFESA1489

ERS410899

MRSA

8

t008

IVa

USA300

2009

HC8

Wisconsin

Outpatient

Wound

PFESA1491

ERS410915

MRSA

8

t008

IVa

USA300

2009

HC8

Wisconsin

Outpatient

Wound

PFESA1497

ERS410868

MRSA

8

t008

IVa

USA300

2009

HC8

Wisconsin

Inpatient

Other

PFESA1498

ERS410876

MRSA

8

t008

IVa

USA300

2009

HC8

Wisconsin

Inpatient

Other



ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA1532

ERS410863

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Other

PFESA1537

ERS410903

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

unknown

Wound

PFESA1538

ERS410911

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Respiratory

PFESA1541

ERS410840

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Other

PFESA1545

ERS410872

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Wound

PFESA1547

ERS410888

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Respiratory

PFESA1579

ERS410747

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Wound

PFESA1580

ERS410755

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Wound

PFESA1583

ERS410771

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA1584

ERS410779

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Wound

PFESA1585

ERS411199

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Inpatient

Wound

PFESA1586

ERS410787

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA1589

ERS410811

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Wound

PFESA1590

ERS411207

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Inpatient

Other

PFESA1591

ERS410819

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA1593

ERS410740

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA1594

ERS410748

MRSA

8

t008

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA1618

ERS410797

MRSA

8

t008

IVa

USA300

2009

HC4

New York

Outpatient

Wound

PFESA1785

ERS410692

MRSA

8

t008

IVa

USA300

2009

HC5

Pennsylvania

Inpatient

Respiratory

PFESA1787

ERS410708

MRSA

8

t008

IVa

USA300

2009

HC5

Pennsylvania

Inpatient

Respiratory

PFESA1791

ERS410645

MRSA

8

t008

IVa

USA300

2009

HC5

Pennsylvania

Inpatient

Respiratory

PFESA1880

ERS410589

MRSA

8

t008

IVa

USA300

2010

HC3

Missouri

Inpatient

Other

PFESA1882

ERS410605

MRSA

8

t008

IVa

USA300

2009

HC3

Missouri

Inpatient

Other

PFESA1883

ERS410613

MRSA

8

t008

IVa

USA300

2010

HC3

Missouri

Inpatient

Wound

PFESA1888

ERS410558

MRSA

8

t008

IVa

USA300

2010

HC3

Missouri

Inpatient

Other



ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA1889

ERS410566

MRSA

8

t008

IVa

USA300

2010

HC3

Missouri

Inpatient

Other

PFESA1977

ERS432053

MRSA

8

t008

IVa

USA300

2010

HC10

Georgia

Outpatient

Other

PFESA1978

ERS432061

MRSA

8

t008

IVa

USA300

2010

HC10

Georgia

Outpatient

Other

PFESA2055

ERS365779

MRSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Invasive

PFESA2057

ERS365795

MRSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Invasive

PFESA2059

ERS365811

MRSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Other

PFESA2062

ERS365827

MRSA

8

t008

IVa

USA300

2010

HC7

Texas

Inpatient

Wound

PFESA2063

ERS365740

MRSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Wound

PFESA2067

ERS365772

MSSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Other

PFESA2074

ERS365828

MRSA

8

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Wound

PFESA2075

ERS410365

MRSA

8

t008

IVa

USA300

2010

HC7

Texas

Outpatient

Invasive

PFESA2077

ERS365749

MRSA

8

t008

IVa

USA300

2010

HC1

Florida

Inpatient

Wound

PFESA2090

ERS365758

MRSA

8

t008

IVa

USA300

2010

HC1

Florida

Inpatient

Wound

PFESA2098

ERS365822

MRSA

8

t008

IVa

USA300

2010

HC1

Florida

Inpatient

Wound

PFESA2103

ERS365767

MRSA

8

t008

IVa

USA300

2010

HC1

Florida

Inpatient

Respiratory

PFESA2104

ERS365775

MRSA

8

t008

IVa

USA300

2010

HC1

Florida

Inpatient

Respiratory

PFESA2153

ERS410377

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Outpatient

Other

PFESA2157

ERS365791

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Inpatient

Wound

PFESA2158

ERS410401

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Outpatient

Other

PFESA2163

ERS410409

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Outpatient

Wound

PFESA2164

ERS365831

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Inpatient

Wound

PFESA2166

ERS410417

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Outpatient

Other

PFESA2169

ERS410441

MRSA

8

t008

IVa

USA300

2010

HC2

Michigan

Outpatient

Wound

PFESA2188

ERS410387

MRSA

8

t008

IVa

USA300

2009

HC11

Florida

Inpatient

Respiratory

PFESA2192

ERS410419

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Outpatient

Wound


Table S1. Description of 191 CC8 S. aureus isolates analysed in this study. For spa and SCCmec columns NT refers to non-typeable. Public name

ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA2193

ERS410427

MRSA

8

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Wound

PFESA2272

ERS410276

MRSA

8

t008

IVa

USA300

2010

HC6

Tennessee

Outpatient

Wound

PFESA2273

ERS410284

MRSA

8

t008

IVa

USA300

2010

HC6

Tennessee

Outpatient

Other

PFESA2282

ERS365745

MRSA

8

t008

IVa

USA300

2010

HC6

Tennessee

Inpatient

Respiratory

PFESA2283

ERS410300

MRSA

8

t008

IVa

USA300

2010

HC6

Tennessee

Outpatient

Other

PFESA2285

ERS410308

MRSA

8

t008

IVa

USA300

2010

HC6

Tennessee

Outpatient

Other

PFESA2329

ERS410318

MRSA

8

t008

IVa

USA300

2010

HC8

Wisconsin

Outpatient

Other

PFESA2431

ERS410207

MRSA

8

t008

IVa

USA300

2010

HC5

Pennsylvania

Inpatient

Respiratory

PFESA2435

ERS410239

MRSA

8

t008

IVa

USA300

2010

HC5

Pennsylvania

Inpatient

Respiratory

PFESA2293

ERS365809

MRSA

8

t008

IVg

-

2010

HC2

Michigan

Inpatient

Invasive

PFESA2056

ERS365787

MRSA

8

t008

NT

USA300

2009

HC7

Texas

Inpatient

Respiratory

PFESA1884

ERS410621

MSSA

8

t024

-

-

2009

HC3

Missouri

Inpatient

Other

PFESA2176

ERS410386

MSSA

8

t024

-

USA300

2009

HC11

Florida

Inpatient

Wound

PFESA1304

ERS411131

MRSA

8

t024

IVa

USA300

2004

HC7

Texas

Inpatient

Invasive

PFESA1337

ERS410961

MRSA

8

t024

IVa

USA300

2004

HC1

Florida

Outpatient

Other

PFESA1346

ERS411189

MRSA

8

t024

IVa

USA300

2004

HC1

Florida

Inpatient

Wound

PFESA1388

ERS410996

MRSA

8

t024

IVa

USA300

2004

HC6

Tennessee

Outpatient

Invasive

PFESA1598

ERS410756

MRSA

8

t024

IVa

USA300

2009

HC6

Tennessee

Outpatient

Other

PFESA2156

ERS410393

MRSA

8

t024

IVa

USA300

2010

HC2

Michigan

Outpatient

Other

PFESA2469

ERS432172

MRSA

8

t024

IVa

USA300

2010

HC9

Wisconsin

Inpatient

Wound

PFESA1795

ERS410677

MSSA

8

t064

-

-

2009

HC5

Pennsylvania

Inpatient

Respiratory

PFESA2439

ERS432122

MSSA

8

t064

-

-

2010

HC5

Pennsylvania

Inpatient

Respiratory

PFESA1318

ERS411140

MRSA

8

t064

IVd

USA500

2004

HC7

Texas

Inpatient

Wound

PFESA1351

ERS410938

MRSA

8

t064

IVd

USA500

2004

HC10

Georgia

Outpatient

Wound

PFESA1361

ERS411165

MRSA

8

t064

IVd

USA500

2004

HC10

Georgia

Inpatient

Invasive



ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA1987

ERS365739

MRSA

8

t064

IVd

USA500

2010

HC10

Georgia

Inpatient

Invasive

PFESA1974

ERS411168

MRSA

8

t064

IVg

USA500-like

2009

HC10

Georgia

Inpatient

Respiratory

PFESA1980

ERS432077

MRSA

8

t064

IVg

USA500-like

2010

HC10

Georgia

Outpatient

Other

PFESA1984

ERS411200

MRSA

8

t064

IVg

USA500-like

2010

HC10

Georgia

Inpatient

Respiratory

PFESA1985

ERS411208

MRSA

8

t064

IVg

USA500-like

2010

HC10

Georgia

Inpatient

Invasive

PFESA2086

ERS365821

MRSA

8

t064

IVg

USA500-like

2010

HC1

Florida

Inpatient

Wound

PFESA2199

ERS410380

MRSA

8

t064

IVg

USA500-like

2010

HC12

Pennsylvania

Inpatient

Respiratory

PFESA1327

ERS411120

MRSA

8

t068

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1367

ERS410947

MRSA

8

t121

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA2281

ERS234133

MRSA

8

t121

IVa

USA300

2010

HC6

Tennessee

Inpatient

Respiratory

PFESA2277

ERS365808

MSSA

8

t1348

-

-

2010

HC6

Tennessee

Inpatient

Wound

PFESA2087

ERS365829

MRSA

8

t1578

IVa

USA300

2010

HC1

Florida

Inpatient

Wound

PFESA1989

ERS365755

MSSA

8

t1705

-

-

2010

HC10

Georgia

Inpatient

Other

PFESA2094

ERS365790

MSSA

8

t1882

-

-

2010

HC1

Florida

Inpatient

Respiratory

PFESA2170

ERS410449

MRSA

8

t211

IVa

USA300

2010

HC2

Michigan

Outpatient

Other

PFESA2187

ERS410379

MSSA

8

t2179

-

USA300

2009

HC11

Florida

Inpatient

Wound

PFESA1390

ERS411142

MSSA

8

t2558

-

-

2004

HC6

Tennessee

Inpatient

Other

PFESA2331

ERS410334

MSSA

8

t3240

-

-

2010

HC8

Wisconsin

Outpatient

Wound

PFESA1478

ERS410906

MSSA

8

t334

-

-

2009

HC8

Wisconsin

Inpatient

Respiratory

PFESA2280

ERS365824

MSSA

8

t334

-

-

2010

HC6

Tennessee

Inpatient

Respiratory

PFESA1370

ERS410963

MRSA

8

t4166

IVa

USA300

2004

HC10

Georgia

Outpatient

Other

PFESA1890

ERS410574

MRSA

8

t622

IVa

USA300

2010

HC3

Missouri

Inpatient

Wound

PFESA1982

ERS432085

MRSA

8

t622

IVa

USA300

2010

HC10

Georgia

Outpatient

Other

PFESA1981

ERS411192

MRSA

8

t681

IVa

USA300

2010

HC10

Georgia

Inpatient

Respiratory

PFESA1990

ERS365763

MRSA

8

t681

IVa

USA300

2010

HC10

Georgia

Inpatient

Other


Table S1. Description of 191 CC8 S. aureus isolates analysed in this study. For spa and SCCmec columns NT refers to non-typeable. Public name

ENA accession

Organism

ST

spa

SCCmec

Clone

Year

HC ID

State

Patient type

Infection type

PFESA2763

ERS409806

MRSA

8

t681

IVa

USA300

2010

HC2

Michigan

unknown

Other

PFESA2742

ERS409790

MSSA

8

t8286

-

-

2010

HC2

Michigan

unknown

Other

PFESA1344

ERS411001

MRSA

8

NT

IVa

USA300

2004

HC1

Florida

Outpatient

Wound

PFESA1988

ERS365747

MRSA

8

NT

IVa

USA300

2009

HC10

Georgia

Inpatient

Invasive

PFESA2330

ERS410326

MSSA

976

t008

-

-

2010

HC8

Wisconsin

Outpatient

Other

PFESA2279

ERS365816

MSSA

1150

NT

-

-

2010

HC6

Tennessee

Inpatient

Wound

PFESA1548

ERS410896

MRSA

2253

t008

IVg

-

2010

HC12

Pennsylvania

Inpatient

Invasive

PFESA2189

ERS410395

MRSA

2253

t008

IVg

-

2010

HC12

Pennsylvania

Inpatient

Wound

PFESA2203

ERS410412

MRSA

2253

t008

IVg

-

2010

HC12

Pennsylvania

Inpatient

Wound

PFESA2073

ERS365820

MRSA

2319

t008

IVa

USA300

2009

HC7

Texas

Inpatient

Wound

PFESA1617

ERS410789

MRSA

3000

t008

IVa

USA300

2010

HC4

New York

Inpatient

Wound

PFESA2473

ERS432204

MSSA

3001

t121

-

-

2010

HC9

Wisconsin

Inpatient

Wound

PFESA2278

ERS410292

MRSA

3007

t008

IVa

USA300

2010

HC6

Tennessee

Outpatient

Wound

PFESA1983

ERS432093

MRSA

3008

t008

IVa

USA300

2010

HC10

Georgia

Outpatient

Other

PFESA2054

ERS409812

MRSA

3010

t008

IVa

USA300

2009

HC7

Texas

Outpatient

Wound

PFESA1542

ERS410848

MRSA

3015

t008

IVa

USA300

2009

HC12

Pennsylvania

Inpatient

Other


Figure S1. Midpoint-rooted phylogenetic tree of analysed S. aureus CC8 isolates with distribution of MGE-associated virulence and resistance genes. Four reference genomes were included (corresponding nodes marked with a star). Branches of USA300, USA500 and USA500-like clades have been highlighted. The tree has been annotated to show the variable distribution of virulence genes associated with prophages φSa2 (pvl) and φSa3 (sak, scn, chp, seK, seQ, seA) as well as the distribution

of

antimicrobial

resistance

genes

associated

with

plasmids

pUSA300HOUMR-like (blaZ, smrA, mphBM, aphA-3, bcrA, sat) and pUSA03-like

0.01

Reference genom es COL USA500_2395 USA300_ISMMS1 USA300_FPR3757

MGE present

VIR/AMR gene present 0.01

Clades USA300 USA500 USA500-like

0.01

MS/MR Reference genom es MSSA COL MRSA USA500_2395

ST USA300_ISMMS1 8USA300_FPR3757 976

MGE 1150

present 2253

VIR/AMR 2319 gene present 3000

3001 3007

0.01 3008substitutions/site 3010 3015

Year of isolat ion 2004 2009 2010

Healt hcare cent re HC1 HC2 HC3 HC4 HC5 HC6 HC7 HC8 HC9 HC10 HC11 HC12


pU S ile A03 S2 ,li aa $ ke ! dD $

pU S bl A30 aZ 0 m $ HO UM sr m A$ R, ph lik e$ ap BM h $ bc A23 $ rA sa $ t$

φS a sa 3 k$ sc n ch p$ se K$ se Q se $ A$

φS a p v 2! l$

(ileS, aadD).

Figure S2. Comparative alignment of recombinant variants of SCCmec and ACME elements identified in USA300 isolates. (a) Alignment of SCCmec-ACME hybrid identified in isolate PFESA2056 to SCCmecIVa-ACME region of S. aureus USA300_FPR3757 strain. (b) Alignment of SCCmec hybrid identified in isolate PFESA2090 to sequence of SCCmec IVa from USA300_FPR3757 and SCCmec V (5&5C2) from S. aureus JCSC6944 strains. (c) Alignment of region containing remnant SCC and ACME element identified in isolate PFESA2064 to SCCmecIVaACME region of USA300_FPR3757 strain and SCCpbp4 from S. epidermidis ATCC 12228. Alignment figures were constructed using Easyfig software v 2.1

ACME

SCCmec IVa

(a) S. aureus USA300_FPR3757,, PFESA2056,

SCCmec IVa

(b) S. aureus USA300_FPR3757,, PFESA2090, S. aureus JCSC6944,

SCCmec V (5&5C2)

ACME

SCCmec IVa

(c) S.,aureus,USA300_FPR3757,, PFESA2064, S.,epidermidis,ATCC,12228,

SCCpbp4


Figure S3. Midpoint-rooted phylogenetic tree of analysed S. aureus CC8 isolates showing distribution of the ICE6013 mobile genetic element. Four reference genomes were included (corresponding nodes marked with a star). Branches of USA300, USA500 and USA500-like clades have been highlighted. Tree has been annotated to show correlation between phylogenetic distribution and, starting from the innermost annotation circle, ICE6013 carriage, ICE6013 insertion type (intergenic or intragenic) and ICE6013 insertion site. For the latter, name of the coding sequence from USA300_FPR3757 reference genome was used, for intergenic location sites names of the flanking coding sequences are provided. 0.01 0.01

0.1

Reference genom Reference genom eses

Clades

COL COL

USA300

USA500_2395 USA500_2395

USA500

USA300_ISMMS1 USA300_ISMMS1

USA500-like

USA300_FPR3757 USA300_FPR3757

Year of isolat ion 2004

ICE6013 carriage ICE6013 carriage

2009

Present Present

2010

ICE6013 insert t ype ICE6013 insert ionion t ype Int ergenic Int ergenic

ST 8

Int ragenic Int ragenic

3007

ICE6013 inset ICE6013 inset ionion sit sit e e

3008

SAUSA300_0606 SAUSA300_0606

SAUSA300_2502/SAUSA300_2503 SAUSA300_2502/SAUSA300_2503 MS/MR MSSA SAUSA300_2368 SAUSA300_2368 MRSA SAUSA300_2440/SAUSA300_244 SAUSA300_2440/SAUSA300_244

SAUSA300_2431 SAUSA300_2431 SAUSA300_0110 SAUSA300_0110

Healt hcare cent re

SAUSA300_2145 SAUSA300_2145

HC1 HC6

SAUSA300_1213/SAUSA300_1214 SAUSA300_1213/SAUSA300_1214 HC7 SAUSA300_0771/SAUSA300_0772 SAUSA300_0771/SAUSA300_0772 HC10 SAUSA300_2145/SAUSA300_2146 SAUSA300_2145/SAUSA300_2146 SAUSA300_1207 SAUSA300_1207 SAUSA300_2438/SAUSA300_2439 SAUSA300_2438/SAUSA300_2439 SAUSA300_1476/SAUSA300_1490 SAUSA300_1476/SAUSA300_1490

0.01 substitutions/site


Figure S4. Mid-point rooted phylogenetic tree of 483 S. aureus USA300 isolates. The phylogeny consists of 154 USA300 isolates analysed in this study together with 329 previously published USA300 genomes derived from New York City (Uhlemann, Dordel et al. 2014). Two most closely related non-USA300 isolates were included as an out-group (isolates PFESA2332 and PFESA1492, branches coloured in red). Branches were coloured to highlight the phylogeny’s nested clade structure. Clades were labelled from 1 to 6, to illustrate a parallel population structure between USA300 isolates analysed in this study and this expanded collection. SNPs that define each labelled clade correspond to previously described polymorphisms (Table 1), except for clade 5 where mutation at site corresponding to 1374405 position in USA300_FPR3757, was detected as C ! T transition (5a) followed by T ! A transversion (5b), in contrast to previously described single step C ! A transversion. Two reference genomes were included and corresponding nodes are marked with a star. 0.01

0.01 Reference genom es

COL USA500_2395 Reference genom es

COL USA300_ISMMS1

1

USA500_2395 USA300_FPR3757

ST

USA300_ISMMS1 8USA300_FPR3757

2

0.002 250 substitutions/site

ST976 8

1150

250 2253

3

976 2319 3000 1150 3001 2253 3007

2319

3008

3000

3010

3001

3015

3007 Year3008 of isolat ion 2004

3010 2009

4 5a

3015 2010

Healt cention re Year hcare of isolat HC1 2004

5b

HC2

2009

HC3

2010

HC4 HC5

Healt hcare cent re HC6 HC1 HC7

HC2

6

HC8

HC3

HC9

HC4

HC10

HC5 HC11 HC6 HC12 HC7 MGE HC8 present HC9 HC10 HC11 HC12

MGE

0.002

present
