Jonathan C. Thomas

Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection

ABSTRACT We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.

Inferring a Population Structure for Staphylococcus epidermidis from Multilocus Sequence Typing Data

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCCmec) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCCmec was acquired at least 56 times by S. epidermidis. Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 (P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCCmec.

Improved Multilocus Sequence Typing Scheme for Staphylococcus epidermidis

ABSTRACT We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.

Comparison of Molecular Typing Methods for Characterization of Staphylococcus epidermidis: Proposal for Clone Definition

ABSTRACT In the present study we give some direction on the selection of the most appropriate typing method(s) to be used for the characterization of Staphylococcus epidermidis, in view of the most recent findings on the evolution, population structure, and epidemiology of this species. In order to achieve this aim, quantitative assessment of the correlation of the results of three typing methods—pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing, which target different regions of the chromosome that evolve at different rates—was performed. In order to evaluate the discriminatory ability and the strength and direction of the correlation of the different typing methods, Simpsons index of diversity (SID), the adjusted Rand coefficient (AR), and the Wallace coefficient (W) were calculated. PFGE was the most discriminatory method (SID = 99%), followed by MLST (SID = 90%) and SCCmec typing (SID = 75%). The values of AR and W (0.10 < AR < 0.30; 0.50 < W < 0.75) indicated that the partition of the same isolate collection by PFGE, MLST, and SCCmec typing provided results that had only a poor correlation with each other. However, the information provided by the combination of PFGE and SCCmec enabled the prediction of the results obtained by MLST at the level of the clonal complex with a high degree of precision (W > 0.90). We propose that clones of S. epidermidis be defined by the combination of the PFGE type followed by the SCCmec type, which provides reliable information on the short-term epidemiology and the ability to predict with consistency long-term clonal evolution.

Candidate Targets of Balancing Selection in the Genome of Staphylococcus aureus

Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajimas D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen.

Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island

Staphylococcus aureus is a major pathogen of humans and animals. The capacity of S. aureus to adapt to different host species and tissue types is strongly influenced by the acquisition of mobile genetic elements encoding determinants involved in niche adaptation. The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content. However the basis for the diversity and the mechanism underlying mobilization of the genomic islands between strains are unexplained. Here, we demonstrated that the genomic island, νSaβ, encoding an array of virulence factors including staphylococcal superantigens, proteases, and leukotoxins, in addition to bacteriocins, was transferrable in vitro to human and animal strains of multiple S. aureus clones via a resident prophage. The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ. Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus.

Differing lifestyles of Staphylococcus epidermidis as revealed through Bayesian clustering of multilocus sequence types

Staphylococcus epidermidis is part of the normal bacterial flora of human skin and a leading cause of infections associated with indwelling medical devices. Previous phylogenetic analyses of subgenomic data have been unable to distinguish between S. epidermidis strains with nosocomial or commensal lifestyles, despite the identification of specific phenotypes and accessory genes that may contribute to such lifestyles. To attempt to better define the population structure of this species, the international S. epidermidis multilocus sequence typing database was analyzed with the Bayesian clustering programs STRUCTURE and BAPS. A total of six genetic clusters (GCs) were identified. A local population of S. epidermidis from clinical specimens was classified according to these six GCs, and further characterized for antibiotic susceptibilities, biofilm, and various genetic markers. GC5 was abundant and significantly enriched for isolates that were resistant to four classes of antibiotics, high biofilm production, and positive for the virulence markers icaA, IS256, and sesD/bhp, indicating its potential clinical relevance. In contrast, GC2 was rare and contained the only isolates positive for the putative commensal marker, fdh. GC1 and GC6 were abundant but not significantly associated with any of the examined characteristics, except for sesF/aap and GC6. GC3 was rare and identified as a potential genetic sink that received, but did not donate, core genetic material from other GCs. In conclusion, population genetics analyses were essential for identifying clusters of strains that may differ in their adaptation to nosocomial or commensal lifestyles. These results provide a new, population genetics framework for studying S. epidermidis.

Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen, Staphylococcus hominis

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.

Draft Genome Sequences of Staphylococcus aureus Sequence Type 34 (ST34) and ST42 Hybrids

Staphylococcus aureus is a major cause of antimicrobial-resistant infections of humans. Hybrids of S. aureus, which originate from large-scale chromosomal recombinations between parents of distinct genetic backgrounds, are of interest from clinical and evolutionary perspectives. We present draft genome sequences of two S. aureus hybrids of sequence type 34 (ST34) and ST42.

Molecular Signatures Identify a Candidate Target of Balancing Selection in an arcD-Like Gene of Staphylococcus epidermidis

A comparative population genetics study revealed high levels of nucleotide polymorphism and intermediate-frequency alleles in an arcC gene of Staphylococcus epidermidis, but not in a homologous gene of the more aggressive human pathogen, Staphylococcus aureus. Further investigation showed that the arcC genes used in the multilocus sequence typing schemes of these two species were paralogs. Phylogenetic analyses of arcC-containing loci, including the arginine catabolic mobile element, from both species, suggested that these loci had an eventful history involving gene duplications, rearrangements, deletions, and horizontal transfers. The peak signatures in the polymorphic S. epidermidis locus were traced to an arcD-like gene adjacent to arcC; these signatures consisted of unusually elevated Tajima’s D and π/K ratios, which were robust to assumptions about recombination and species divergence time and among the most elevated in the S. epidermidis genome. Amino acid polymorphisms, including one that differed in polarity and hydropathy, were located in the peak signatures and defined two allelic lineages. Recombination events were detected between these allelic lineages and potential donors and recipients of S. epidermidis were identified in each case. By comparison, the orthologous gene of S. aureus showed no unusual signatures. The ArcD-like protein belonged to the unknown ion transporter 3 family and appeared to be unrelated to ArcD from the arginine deiminase pathway. These studies report the first comparative population genetics results for staphylococci and the first statistical evidence for a candidate target of balancing selection in S. epidermidis.