Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology.

Full characterization of methicillin-resistant Staphylococcus aureus (MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous mec element these bacteria carry, which is associated with drug resistance determinant mecA. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the mec element types based on the structural features that were shown to be typical of mec elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated mec elements was previously determined by hybridization and PCR screenings and also by DNA sequencing. The method was tested together with multilocus sequence typing and other typing methods for the characterization of 18 isolates representative of the MRSA clones recovered during a hospital outbreak in Barcelona, Spain. The multiplex PCR was shown to be rapid, robust, and capable in a single assay of identifying five structural types of the mec element among these strains, three major and two minor variants, each one of which has been already been seen among MRSA characterized earlier. This technique should be a useful addition to the armamentarium of molecular typing tools for the characterization of MRSA clonal types and for the rapid tentative identification of structural variants of the mec element.Molecular typing techniques have been used with increasing frequency in studies of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and also for a better understanding of the evolutionary relationships among MRSA clones (3,7,9,26). One of the conclusions emerging from these studies was that a complete characterization of MRSA lineages requires not only identification of the genetic background of the bacteria but also identification of the structural types of the large and heterologous mec element, which carries methicillin resistance determinant mecA (11,26,27).Studies by Ito et al. have elucidated the complete structure of three major mec elements, also referred to as the staphylococcal chromosomal cassette (SCCmec). Type I (34 kb) was identified in the first MRSA strain isolated in 1961 in the United Kingdom (strain NCTC10442), type II (52 kb) was identified in an MRSA strain isolated in 1982 in Japan (strain N315), and type III (66 kb) was identified in an MRSA strain isolated in 1985 in New Zealand (strain 82/2082) (12, 13). More recently, a smaller fourth mec element, SCCmec type IV (20 to 24 kb), was independently identified among representatives of the Pediatric clone (26) and in two community-acquired MRSA strains (18).In a recent report we described the characterization of the structural types of the mec elements carried by 28 MRSA isolates belonging to five widespread epidemic clones, which were all characterized by multilocus sequence typing (MLST) as single-locus or double-locus variants of two completel...

Since the 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has emerged, disseminated globally and become a leading cause of bacterial infections in both health-care and community settings. However, there is marked geographical variation in MRSA burden owing to several factors, including differences in local infection control practices and pathogen-specific characteristics of the circulating clones. Different MRSA clones have resulted from the independent acquisition of staphylococcal cassette chromosome mec (SCCmec), which contains genes encoding proteins that render the bacterium resistant to most β-lactam antibiotics (such as methicillin), by several S. aureus clones. The success of MRSA is a consequence of the extensive arsenal of virulence factors produced by S. aureus combined with β-lactam resistance and, for most clones, resistance to other antibiotic classes. Clinical manifestations of MRSA range from asymptomatic colonization of the nasal mucosa to mild skin and soft tissue infections to fulminant invasive disease with high mortality. Although treatment options for MRSA are limited, several new antimicrobials are under development. An understanding of colonization dynamics, routes of transmission, risk factors for progression to infection and conditions that promote the emergence of resistance will enable optimization of strategies to effectively control MRSA. Vaccine candidates are also under development and could become an effective prevention measure.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drugresistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

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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.