Early MRSA belong to a diverse clone

Preserved in the culture collection of the Staphylococcal Reference Laboratory at Public Health England are representatives of the very first MRSA identified. These original isolates have been preserved as freeze-dried cultures, and have not been repeatedly passaged over the years. One hundred and eighty eight isolates that represented the earliest MRSA were recovered from the ampoules and their genomes sequenced (Additional file 1: Table S1). All the isolates belonged to CC8 MRSA and were originally isolated between 1960 and the late 1970s, and included eight isolates from the original study describing MRSA in 1961 [3]. In addition, 21 CC8 MRSA isolated between 1964 and 1989 in Denmark [8, 11] were sequenced, as representatives of the earliest MRSA detected elsewhere in Europe. We also included early methicillin-sensitive isolates of ST250 or ST247 (n = 11); however, only a limited number of these were found in the reference laboratory collection.

Analysis of the MLST of the isolates identified two main groups, ST250 (n = 126) and a single locus variant (SLV), ST247 (n = 78), plus two novel SLVs of ST247 (n = 4) (Additional file 1: Table S1). A supplementary isolate from the Public Health England collection was included to provide an outgroup for the analysis; RH12000692_7401696 is an MRSA which was collected in 1967 and is a triple locus variant of ST250 (Additional file 1: Table S1).

The S. aureus isolate COL, a representative member of this early MRSA lineage first identified in the 1960s [12], had previously been fully sequenced, and the chromosome was used as a reference for mapping. Following exclusion of mobile genetic elements (MGEs) and predicted recombination events in the collection, a total of 4220 SNPs were identified and used to construct a phylogeny (Fig. 1a). The population framework revealed a diverse population structure containing several distinct clades. The mapping of the ST information on to the phylogeny reveals that the ST250 population is basal to the ST247, suggesting that ST247 emerged from ST250, which is consistent with the epidemiological evidence, and supports the hypothesis that this pandemic multidrug-resistant MRSA clone emerged out of the ancestral MRSA genotype [8, 9].

Fig. 1 Population structure of historic MRSA isolates. a Maximum likelihood tree of historic MRSA isolates. The tree was built using a maximum likelihood method using SNPs from the core genome of 209 isolates. Included in the phylogeny is the COL reference isolate to which the sequence reads were mapped. The tree is rooted with RH12000692_7401696 as an outgroup; this is a CC8 isolate and is a triple locus variant of ST250. Tree branches are coloured according to their ancestral sequence type population; blue branches indicate the ST250 population and green branches the ST247 population. Isolates from Denmark are highlighted in blue shading and isolates described in the Jevons study are outlined in the dashed box, and a zoomed in view of the phylogeny is displayed in b. The coloured branch labels indicate the three individuals who supplied the original isolates in the Jevons study Full size image

Highlighted in the expanded view (Fig. 1b) are the isolates from the Jevons study, derived from three individuals at the same hospital in the South London area between July and November 1960 [3]. The isolation source and resistance profiles of these isolates are shown in Additional file 2: Table S2. These isolates are genetically very closely related, differing by seven SNPs only. Present within this cluster are additional isolates from the Public Health England collection originating between 1960 and 1961. Full epidemiological data are not available for these, but two of these isolates were identified in the same region as the hospital where the original Jevons study isolates originated. The genetic distance between isolates and their phylogenetic relationships suggests there was transmission within the hospital between patients A and C and nurse B, and that they were also transmitted beyond the hospital as part of a local outbreak.

Although all of the Jevons isolates are confined to a single clade, other isolates from the early 1960s are distributed throughout the entire phylogeny (Fig. 2). This suggests that the earliest MRSA circulating in the UK were not from a single recently emerged clone, but were representatives of an established population. In addition to the UK isolates, there were 21 from Denmark, which represent the earliest MRSA detected outside the UK. These derive from 1964 onwards, and include the youngest isolates within the collection from the late 1980s. The Danish isolates are found in three clusters distributed throughout the phylogeny (Fig. 1a), suggesting that, like the early UK MRSA, they originated from an established and diverse population.

Fig. 2 Distribution of antibiotic resistance determinants in the archetypal MRSA clone. A maximum likelihood tree of historic MRSA isolates (n = 209) plus the COL reference is displayed on the left, and the panels on the right indicate dates of isolation (coloured according to year; see key below for years), and the presence (purple boxes) and absence (white space) of genetic determinants responsible for antibiotic resistance in the genomes of the isolates. The identity of genetic determinants are listed at the top and divided into acquired genes (red text; left hand side), and core mutations (green text; right hand side). The antibiotics linked to the genetic determinants for the acquired genes are: β-lactams, blaZ and mecA; chloramphenicol, catA1, catA2, and catA3; erythromycin, ermA; clindamycin, linA; aminoglycosides, aacA-aphD, aad9, aadD, aph3A, and str; tetracycline, tetM and tetK; disinfectants, qacA and qacC. And for the core gene mutations are: streptomycin substitution of arginine for a lysine at residue 56 (K56R) of the ribosomal protein rpsL; fusidic acid, substitution of a proline for a leucine at residue 406 (P404L) of the transcription elongation factor fusA; trimethoprim, substitution of an tyrosine for a phenylalanine at residue 99 (F99Y) of the dihydrofolate reductase dfrA. Sixteen isolates lacked complete type I SCCmec elements, 4 of which contained internal deletions in the SCCmec element but retained the mecA gene Full size image

Genetic basis of resistance to methicillin and other antibiotics in the archetypal MRSA population

Previous studies have shown that the archaic MRSA clone carried a type I SCCmec element, which was the first type of this MGE family to be classified [5, 13]. Notably, the description of the type I element was based upon the SCCmec derived from S. aureus strain NCTC10442 identified in the 1960 Jevons study (Fig. 1b; Additional file 2: Table S2) [13]. The type I element carries mecA as its only resistance gene in combination with a truncated gene encoding the MecRI regulatory proteins (together known as a class B mec gene complex) with type 1 chromosomal recombinases (ccrA1 and ccrB1). The original description of SCCmec type I identified the presence of a frameshift mutation in ccrB1 which disrupts the translation of this site-specific recombinase [13]; the mutation occurs after codon 321 and is caused by a single base deletion in a poly-A hexamer resulting in a pentamer sequence. In the collection, 193 of the isolates contained intact SCCmec elements carrying the mecA gene (Fig. 2). Of these, 192 were SCCmec type I elements, all of which contained the pentamer sequence and the same frameshift mutation in ccrB1 as the NCTC10442 reference. The only non-type I element identified in the collection was in the outgroup isolate RH12000692_7401696, which contained a type IVh SCCmec element. The remaining 16 isolates lacking complete SCCmec elements were distributed throughout the phylogeny, suggesting that these represent methicillin-sensitive S. aureus (MSSA) arising from the loss of the type I SCCmec element, rather than forming an ancestral MSSA population.

In addition to methicillin resistance, the first MRSA described were also resistant to penicillin, streptomycin and tetracycline [3]. Analysis of the genomes of these isolates identified blaZ and tetK genes conferring resistance to penicillin and tetracycline, respectively, but failed to identify the str, aadE or aad9 genes associated with streptomycin resistance in S. aureus. In the absence of an acquired resistance gene, the core genome was examined for mutations potentially responsible for resistance to streptomycin. In Mycobacterium tuberculosis, mutations in the ribosomal protein RpsL were shown to confer streptomycin resistance, including the substitution of an arginine for a lysine residue at residue 43 [14]. Alignment of the M. tuberculosis and S. aureus sequences revealed that RpsL in the Jevons isolates contained an arginine in the equivalent position, residue 56. Comparison with RpsL sequences in the public sequence databases showed that in S. aureus the frequent amino acid residue at position 56 was lysine. Examining the whole collection, all but one of the sequenced isolates contained the arginine residue at position 56, the exception being the outgroup isolate RH12000692_7401696 (Fig. 2). This demonstrates that the non-synonymous substitution resulting in an arginine for a lysine residue at residue 56 (K56R) occurred most likely very early during emergence of the archetypal MRSA population.

In silico analysis of the resistomes of the isolates revealed genetic resistance determinants to numerous other antibiotics, including penicillin (blaZ), erythromycin (ermA and linA), kanamycin (aadD), gentamicin and kanamycin (aacA-aphD), spectinomycin and streptomycin (aad9), and chloramphenicol (catA1, catA2 and catA3), fusidic acid (fusA P404L) and trimethoprim (dfrA F99Y), as well as genes associated with decreased susceptibility to disinfectants (qacA and qacC). The frequency and widespread dispersal of these determinants reveal the strong selective pressure exerted by antibiotics on the archetypal MRSA clone over an extensive period. Examining their distribution in the context of the phylogeny shows that some of these traits have been co-acquired (Fig. 2), such as ermA and aad9, which are carried on Tn554, and that these acquisition events can be mapped on to the phylogeny [15].

Evolution and emergence of methicillin resistance

To determine if the methicillin resistance emerged once or multiple times in the archetypal MRSA population, we examined the variation within the SCCmec type I elements. In total, 194 variant sites were identified in 192 elements present in the collection. Analysis of the distribution of the variation within the elements suggested that some could be attributed to homologous recombination. Two regions contained the majority of the variation: 124 SNP sites were identified in the gene encoding the LPxTG surface protein pls, and 31 SNP sites within a 549-bp intergenic region between a hypothetical protein (SACOL0030) and a glycerophosphoryl diester phosphodiesterase (SACOL0031). Excluding these predicted recombination regions, 39 core variants sites across 28.6 kb distinguished the 192 elements, with half of the isolates (n = 96) carrying an identical element. The maximum SNP distance distinguishing any two elements was eight SNPs, and phylogenetic analysis revealed that the elements present in the historic MRSA clone were closely related (Additional file 3: Figure S1) and shared a common evolutionary origin.

Our analysis of the evolutionary events surrounding the emergence of methicillin resistance in the archetypal MRSA lineage focused on a subset of 122 isolates that had precise dates and places of origin which could be linked to original submission records (Additional file 2: Table S1). This enabled us to generate a robust Bayesian phylogeny and temporal calibration. Examining the distribution of the type I SCCmec variants (Fig. 3a) within the context of a core genome phylogeny generated with BEAST (Fig. 3b) revealed congruence between the phylogenetic relationships of the two. All of the canonical SNPs associated with the SCCmec genotypes could be singularly mapped onto nodes of the core phylogeny, suggesting that the variation observed in the SCCmec elements had occurred during expansion of the ST250 and ST247 populations. On the basis of this, we propose that a type I SCCmec element was acquired once in a single primordial development of methicillin resistance (Fig. 3b) that could be dated back to the emergence of this clone.

Fig. 3 Diversity and distribution of SCCmec type I elements in the archetypal MRSA population. a Parsimonious minimal spanning tree of SCCmec type I elements present in the archetypal MRSA isolates present in the clade credibility tree in b. The tree is built with core SNPs identified in the SCCmec type I elements, and excludes SNP in the pls gene that were predicted to have arisen by recombination. In total, ten genotypes were observed, and the genetic events that distinguish each genotype from the founder genotype are indicated. The tree is centred on the majority genotype inferred as the founder population, and colour-coded according to their genotype. Black asterisks indicate isolates that lack the type I SCCmec element. The sizes of the circles illustrate the relative sizes of the genotype populations. The key below the tree describes the canonical SNPs differentiating SCCmec type I genotypes and the number of variants with that genotype. b Maximum clade credibility tree of the archetypal MRSA clone population based on BEAST analysis. Tips of the tree are constrained by isolation dates; the time scale is shown below the tree. The tree is built with core genome SNPs from a subset of the total collection’s isolates (n = 122), which had precise dates of isolation, and whose origins could be linked back to the original submission documentation. The branches of the tree are coloured according to the genotype of the SCCmec type I element present in that strain (illustrated in a). Internal branches are coloured according to parsimonious reconstruction of the predicted genotype. Where terminal branches are black and highlighted by a black asterisk, this indicates the absence of an SCCmec element, which is predicted to reflect loss of the element. An arrow indicates the point in the phylogenetic reconstruction where an ancestral type I SCCmec element was acquired. The root of the tree corresponds to the basal node of the ST250/ST247 population in Fig. 1 rooted by the RH12000692_7401696 outgroup. From the analysis the estimated mutation rate of population is 1.8 × 10−6 SNPs/site/year. This substitution rate falls within the reported ranges of multiple successful S. aureus lineages [31] and therefore it is unlikely likely that long-term storage of the isolates has created any temporal artefacts Full size image

In our Bayesian phylogenetic analysis of the core genome SNPs we utilized a range of population and clock model combinations. The combination of an exponential population and relaxed-log normal clock model was found to be the best fit to our data based on Bayes factors using the harmonic mean estimator. This indicated the time to most recent common ancestor (TMRCA) of the ST250/ST247 population was 1946 (95% highest posterior density (HPD) 1938–1952) (Additional file 3: Figure S2), and therefore the time of acquisition of SCCmec was likely around, or before, this date. Notably, the TMRCA of the type I SCCmec elements in these isolates based on a linear regression of a core SNP phylogeny was predicted to be early 1941 (Additional file 3: Figure S3).

To ensure that the Bayesian result was not an artefact of the clock or population models used in the analysis, we calculated the TMRCA for a range of model combinations and found that our chosen model exhibited a predicted TMRCA that was encompassed by the 95% HPDs of all other model combinations (Fig. 4).