Molecular epidemiology of MRSA in Singapore
At the time of the study, Singapore had a population of over 5 million people served by six acute care public sector hospitals. This study focused on MRSA collected between 2000 and 2010 from the three main general hospitals: Singapore General Hospital, Tan Tock Seng Hospital and Changi General Hospital, which will subsequently be referred to as Hospital 1, Hospital 2 and Hospital 3, respectively (Figure S1A in Additional file 1). During this period all of the hospitals experienced a replacement of the MRSA clones associated with clinical infection. Early indications of distinct MRSA populations were based on observed differences in antibiotic resistance profiles; a clone resistant to multiple antibiotics (including trimethoprim-sulfamethoxazole, gentamicin, tetracycline, erythromycin with either inducible or constitutive resistance to clindamycin, and fluoroquinolones) [6] was being replaced by a clone resistant to fewer antibiotics (erythromycin with inducible resistance to clindamycin, and fluoroquinolones). Molecular typing using MLST confirmed the presence of two MRSA clones, and showed that clonal replacement of ST239 by ST22 was occurring. In Hospital 1, the first appearance of ST22 was in 2002, followed by a rise in incidence over the following years such that by 2010 it had become dominant (Figure 1).
MLST and other common molecular typing techniques used to distinguish S. aureus lacked the resolution to investigate the fine-scale epidemiological changes occurring in the population. To establish an accurate MRSA population structure in Singapore, we sequenced the whole genomes of 205 randomly selected clinical isolates (Figure S1B in Additional file 1) from 2000 to 2010. Hospitals 2 and 3 randomly selected five to six MRSA isolates each year from a systematic collection of all bacteremia isolates, which was established in 2001 and 2004, respectively. Hospital 1 stored a proportion of MRSA associated with clinical infection each year since 2000, from which isolates were randomly selected from each year. To provide historical genetic context we also sequenced the genomes of all stored and available MRSA isolated in the 1980s (n = 10) and 1990s (n = 28) from Hospital 1 (SGH), the only hospital of the study hospitals to have a historic collection of MRSA for the preceding two decades. To further supplement the historic collection, 17 isolates from a fourth hospital (the National University Hospital; Hospital 4) from 1997 were also sequenced. This fourth hospital was formed in 1985 as an offshoot of Hospital 1, taking with it some of its members of staff and patients.
The 205 isolates from 2000 to 2010 were assigned using sequence data to five MLST sequence types (ST239, ST22, ST78, ST45 and ST5; Figure S1B in Additional file 1; Additional file 2), although the majority were ST239 (n = 110; 54.4%) followed by ST22 (n = 87; 42.6%). Six ST45 strains were isolated in 2009 and 2010 (Additional file 2), and single ST78 and ST5 strains were both isolated in 2004. Analysis of historical isolates from the 1980s and 1990s also revealed a predominance of ST239; all isolates (n = 50) were ST239, with the exception of the very earliest isolates that were ST8 (n = 5). Notably, no other ST8 isolates were identified in the study after 1982. The first ST22 isolates were found within the sample from Hospital 1 in 2003, after which ST22 increased in prevalence and spread to other hospitals (Figure S1B in Additional file 1). Correspondingly, the number of ST22 isolates in our random sample increased from 2003 onwards.
Phylogenetic reconstruction of the ST239 and ST22 populations
Genome sequence data were used to generate phylogenies based on core single nucleotide polymorphisms (SNPs) for ST239 and ST22. Preliminary phylogenetic analysis revealed that two isolates in the ST22 phylogenies were found on very long branches in comparison to contemporaneous isolates (isolates B11318/05 and DB055850/09). Both of these isolates were obtained from blood cultures, and a review of the clinical records revealed that they were obtained from patients who had been heavily treated with antibiotics for persistent HA-MRSA infection. Neither of the patients had received treatment in an overseas hospital nor had any significant travel history of note, suggesting that greater diversity of the isolates was not likely to be the result of the importation of a genetically divergent strain. Analysis of the genomes of these isolates revealed a deletion of a single guanine residue within the DNA repair gene radA for DB055850/09, resulting in a frame-shift mutation, and in isolate B11318/05 a non-synonymous SNP was found in the mutS gene encoding a DNA mismatch repair protein. It is likely that these mutations occurred during prolonged antibiotic pressure, resulting in these isolates becoming hyper-mutators. As a result these were excluded from the subsequent temporal analysis.
Bayesian phylogenetic reconstruction was used to explore the population structure and temporal spread of the two major Singaporean MRSA clones (Figure 2; Additional file 3). Comparison of the two phylogenies showed that the Singaporean ST239 exhibited a greater overall phylogenetic diversity than Singaporean ST22 (Figure 2), consistent with the larger temporal spread of the ST239 isolates. Examination of the ST239 population revealed that the oldest isolates in the collection from the 1980s and 1990s shared a common ancestor dating back to approximately 1964 (Figure 2A; 95% highest posterior density [HPD], 1951 to 1974), around the time of the most recent common ancestor (tMRCA) of the ST239 lineage (tMRCA mid 1960s) [14]. The majority of ST239 isolates from 2000 to 2010 originated from a single lineage that emerged in approximately 1994, and subdivided into three distinct clades shortly after (indicated as clades A, B and C in Figure 2A). In comparison, the ST22 population was predicted to have a tMRCA of July 2001 (Figure 2B; 95% HPD, June 2000 to April 2002), which is contemporaneous with the first epidemiological description of ST22 in Singapore in 2002 [7,15].
During the early to mid-2000s, shortly after the introduction of ST22 into Singapore, the ST239 population was composed of isolates that belonged to the three main sub-clades (colored in Figure 2A) and also some more distantly related isolates, possibly remnants of the early Singaporean ST239 MRSA population, or imports into Singapore from a related population in the surrounding region (Figure 2A). As the ST22 population became increasingly well established, there was a shift in the ST239 population such that by the end our study period, only isolates belonging to two of the three sub-clades were sampled.
In both the ST239 and ST22 phylogenetic trees, isolates from different hospitals were intermingled, indicating that frequent exchanges had occurred between hospitals. This phylogenetic dispersal is consistent with regular inter-hospital transfer of patients and medical staff, which occurred throughout the study period. An exception to this genetic dispersal was noted for ST239 isolates from Hospital 3, which with one exception all belonged to a single sub-clade of the ST239 population (Figure 2A). This clustering contrasted with the ST22 from this hospital, which were distributed throughout the phylogenetic tree (Figure 2B).
Singaporean isolates in a global context
We next placed the Singaporean isolates into a global genetic context by expanding both datasets with genomes from global isolate collections sequenced and used previously to reconstruct the origins of both lineages (Figure 3) [10,14,16]. This demonstrated that all of the ST22 isolates from Singapore belonged to EMRSA-15 (Figure 3B), a healthcare-associated MRSA first identified in 1991, and originally defined by phage typing [10]. Recent phylogenomic analysis of a global collection of ST22 revealed that the pandemic clone of EMRSA-15 emerged in the UK in the late 1980s, and quickly spread throughout the UK and beyond [10]. Examining the Singapore isolates in the context of a global collection of ST22 isolates that correspond to the EMRSA-15 pandemic population (referred to as ST22-A2 by Holden et al. [10]) showed that they all emanated from a single ancestral node, consistent with the single introduction of ST22 into Singapore, followed by clonal expansion. The isolate immediately ancestral to the Singaporean ST22 clade was isolated in the UK, suggesting a possible origin for this introduction.
By contrast, the Singaporean ST239 population was interspersed with isolates from other regions (Figure 3A). The main clade containing Singaporean isolates also contained two European isolates (UK and Demark) together with other Asian isolates from Thailand and China, suggesting several transmissions to and from Singapore over the 28-year sampling timeframe. One of the European isolates in the Singapore clade was TW20, which was isolated in October 2003 as part of an outbreak on an ICU in a London hospital [17]. A previous study of a global collection of ST239 genomes found that TW20 clustered most closely with isolates from Thailand (85 SNPs to the last common ancestor with a Thai isolate) [14], and it was speculated that TW20 was carried to the UK through intercontinental transmission from Asia. Based on the genetic relatedness of TW20 to the Singaporean isolates (22 SNPs to the last common ancestor with a Singaporean isolate, 4045-41), and the tMRCA of TW20 and the Singaporean isolates of 21st October 2002 (95% HPD 26th April 2001 to 3rd December 2003), it is possible that, within Asia, the origin of this transmission is closer to Singapore than Thailand.
Competition between MRSA lineages generated selection that has shaped the ST239 population structure
The topologies of the phylogenetic trees belonging to the two clones were very distinct. The ST22 tree was composed of branches that radiated out from a central node to produce a star-like phylogeny, whereas the ST239 population had more unbalanced phylogeny. These features suggested that selection has acted differentially on the two populations, and that in the case of ST239 the population had experienced some form of directional selection. Based on the differences in the phylogenies, we hypothesized that competition between ST22 and ST239 for the common niches within hospitals had resulted in selection, which led to differential effects on the two populations. In order to look in more detail at the evolutionary impact, we conducted demographic analysis on the ST239 and ST22 populations (Figure 4). Bayesian skyline analysis [18] provided an estimate of the genetic diversity of the ST239 and ST22 populations. The success of ST22 following its introduction into Singapore was associated with an increase in the genetic diversity of the population from 2002 until 2004, after which this plateaued. During the same period, the genetic diversity of the ST239 population started to decline. Around 2005, when the genetic diversity of ST22 had stabilized, the genetic diversity of ST239 continued to fall until 2007, when, surprisingly, it rebounded and started to increase. At this point the overall clinical prevalence of ST239 in comparison to ST22 in Singapore was falling, and therefore the apparent increase in genetic diversity was likely due to the emergence of successful subpopulations of ST239.
Having observed the signature of an adaptive response in the ST239 population, we conducted comparative genomic analysis to investigate the genetic basis of the apparent success. From the phylogeny it was clear that no single ST239 sub-clone emerged at the time of the increase in diversity (Figure 2); therefore, it was unlikely that mutations in the core genome and clonal expansion were responsible for the observed effect. We then examined the population for evidence of homologous recombination that could be associated with an increase in genetic diversity. This revealed that across the whole ST239 population (isolates from 1988 to 2010) the rate of homoplasy was low; 48 of the 4,349 core SNP sites were homoplasic (1.1%), consistent with previous studies examining ST239 [14]. Analysis of the ST239 chromosomal sequences identified 18 putative recombination regions that corresponded to approximately 7 kb of the chromosome (Additional file 4). However, the distribution of these regions displayed phylogenetic structure, suggesting that, like the SNPs, recombination was not contributing to the increase in genetic diversity.
A possible explanation for the observed increase in the genetic diversity of the population was the emergence of multiple ST239 variants, following the acquisition of mobile genetic elements (MGEs). Analysis of the accessory genome of the ST22 and ST239 populations revealed that, for both lineages, the accessory genomes were relatively stable (Figure 5A); the accessory regions of both the reference genomes (TW20 and HO 5096 0412) were conserved in the majority of isolates. Analysis of the ST22-ST239 pan-genome revealed limited evidence of genetic exchange between ST239 and ST22 populations. In both of these lineages, the prophage component of the genomes appeared to be relatively conserved (Figure 5A; ϕSa1(TW20) and ϕSPβ-like(TW20), and ϕSa2(HO 5096 0412) and ϕSa3(HO 5096 0412), from the ST239 and ST22 reference genomes, respectively); however, we found one of the ST239 isolates (352_06) had acquired a ϕSa2(HO 5096 0412)-like phage, and one of the ST22 isolates (491_05) had acquired a ϕSPβ-like(TW20) phage. This later prophage encodes SasX, a cell wall-anchored surface protein, which is important for colonization and pathogenicity and linked to the success of ST239 in Asia [19]. In the case of the ST22 isolate 491_05, infection by the ϕSPβ-like(TW20) phage has led to lysogenic conversion, introducing the sasX into a genetic background where it has previously been unseen. This observation highlights the potential for co-circulating lineages of S. aureus to exchange phage and transfer virulence determinants across the population, thereby generating new variants of epidemic clones shaped to the competing population.
The most widely exchanged MGE between the two clones was a small plasmid encoding erythromycin resistance which had been acquired by ST239 isolates. In ST239, erythromycin resistance is typically encoded by an ermA Tn554 transposon associated with the SCCmer element [14]; in our collection, 100% of the ST239 isolates contained ermA. In contrast, erythromycin resistance in ST22 is frequently mediated by ermC encoded on a small 2.4-kb plasmid [10,20]; in our collection, 84% of the ST22 isolates contained ermC. Analysis of the accessory genomes of the ST239 isolates revealed that 10 isolates (6%) had augmented their resistome by acquiring the ermC plasmid (Figure 5A), to add to the ermA they already possessed in their chromosomes.
Independent evolution of new variants of ST239 carrying the ACME
One of the most abundant novel MGEs in the ST22-ST239 pan-genome (that is, an MGE not present in the reference genomes) was an approximately 12 kb fragment in the ST239 isolates (Figure 5A) that encodes a type II arginine catabolic mobile element (ACME-II; arc+ opp-3-; Figure 5B) [21]; 14 isolates were identified which contained ACME-II. Comparative genomic analysis of the ST239 isolates from the period when the genetic diversity of the population increased (2006 to 2010), with isolates from earlier in the decade (2000 to 2005), identified that the later isolates were more likely to contain the ACME-II compared with those from the preceding 6 years (28.9% versus 1.5%, respectively). The earliest ST239 strain in the collection that contained an ACME region was isolated from Hospital 1 in 2001 (DB014329/01). This strain was found outside the three main Singaporean ST239 sub-clades, and from the global population context it was most closely related to isolates from Australia and North America.
In contrast, the phylogenetic position of the ACME-positive isolates from 2006 to 2010 indicated that these isolates originated from the Singaporean ST239 population (Figure 4A). Parsimony analysis suggested that there had been a number of potential acquisition events of ACME during this period rather than the expansion of a single clone (Additional file 5). ACME elements were detected in isolates belonging to two of the three ST239 sub-clades. In sub-clade B ACME were present in isolates 097_08 and TTSH1_10, which shared the same phylogenetic node on the tree. Comparative analysis of their ACME regions revealed that the DNA upstream flanking the elements was distinct (Additional file 2). Additionally, we also found that the ACME regions in these two isolates were distinguished by a SNP (in 097_08 there was a SNP in the intragenic region 81 bp upstream of the arcR translational start site), suggesting that although the isolates were phylogenetically linked, they may have acquired separate ACME elements. The only other variation in the ACME regions was found in the sub-clade C isolates. The ACME of four of the isolates (CGH4_10, CGH6_06, 158_08 and 006_10) contained a single base deletion in a poly-T octomer 592 bp upstream of the arcR translational start. All of these isolates were phylogenetically linked, suggesting a common ancestry for this ACME element variant. The rest of the ACME regions in the sub-clade C isolates were identical and interleaved in the phylogeny with isolates that lacked the element (Figure 4A). From the data it is not possible to identify if the ACME distribution in sub-clade C was the result of multiple acquisitions, or the result of a single acquisition and subsequent loss on multiple occasions.
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