D. Ashley Robinson

The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclature exists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from different countries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clones than has previously been possible. Using multilocus sequence typing and an algorithm, burst, we analyzed an international collection of 912 MRSA and methicillin-susceptible S. aureus (MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotype of each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using burst, which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains, and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these major MRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful S. aureus genotypes.

Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex.

Vancomycin-resistant enterococci (VRE) have caused hospital outbreaks worldwide, and the vancomycin-resistance gene (vanA) has crossed genus boundaries to methicillin-resistant Staphylococcus aureus. Spread of VRE, therefore, represents an immediate threat for patient care and creates a reservoir of mobile resistance genes for other, more virulent pathogens. Evolutionary genetics, population structure, and geographic distribution of 411 VRE and vancomycin-susceptible Enterococcus faecium isolates, recovered from human and nonhuman sources and community and hospital reservoirs in 5 continents, identified a genetic lineage of E. faecium (complex-17) that has spread globally. This lineage is characterized by 1) ampicillin resistance, 2) a pathogenicity island, and 3) an association with hospital outbreaks. Complex-17 is an example of cumulative evolutionary processes that improved the relative fitness of bacteria in hospital environments. Preventing further spread of this epidemic E. faecium subpopulation is critical, and efforts should focus on the early disclosure of ampicillin-resistant complex-17 strains.

Evolutionary Models of the Emergence of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Five major lineages of methicillin-resistant Staphylococcus aureus (MRSA) have evolved since the introduction of methicillin for the treatment of infections caused by penicillin-resistant S. aureus in 1959. The clones of these lineages are responsible for the vast majority of hospital-acquired MRSA disease globally. We have constructed high-resolution evolutionary models for each lineage using a parsimony approach with 15 partial gene sequences from 147 geographically diverse isolates. On the basis of these models, we infer that MRSA has emerged at least 20 times upon acquisition of the methicillin resistance determinant, which is carried on a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec). The acquisition of SCCmec by sensitive clones was four times more common than the replacement of one SCCmec with another. Notably, SCCmec type IV was found in twice as many clones as any other SCCmec type, and it is this SCCmec type which is commonly found in clones from patients with community-acquired MRSA disease. Our findings suggest that most clones of MRSA arise by the acquisition of SCCmec type IV by methicillin-sensitive isolates.

A Novel Staphylococcus aureus Biofilm Phenotype Mediated by the Fibronectin-Binding Proteins, FnBPA and FnBPB

Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligand-binding activities of these multifunctional surface proteins.

Multilocus Sequence Typing Scheme for Enterococcus faecalis Reveals Hospital-Adapted Genetic Complexes in a Background of High Rates of Recombination

ABSTRACT A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

Re-emergence of early pandemic Staphylococcus aureus as a community-acquired meticillin-resistant clone

During the 1950s, the notorious penicillin-resistant clone of Staphylococcus aureus known as phage type 80/81 emerged and caused serious hospital-acquired and community-acquired infections worldwide. This clone was largely eliminated in the 1960s, concurrent with the widespread use of penicillinase-resistant beta lactams. We investigated whether early 80/81 isolates had the genes for Panton-Valentine leucocidin, a toxin associated with virulence in healthy young people. Multilocus sequence analysis suggested that descendants of 80/81 have acquired meticillin resistance, are re-emerging as a community-acquired meticillin-resistant S aureus (MRSA) clone, and represent a sister lineage to pandemic hospital-acquired MRSA.

Evolution of Staphylococcus aureus by Large Chromosomal Replacements

Conjugative transfer and replacement of hundreds of kilobases of a bacterial chromosome can occur in vitro, but replacements in nature are either an order of magnitude smaller or involve the movement of mobile genetic elements. We discovered that two lineages of Staphylococcus aureus, including a pandemic methicillin-resistant lineage, were founded by single chromosomal replacements of at least approximately 244 and approximately 557 kb representing approximately 10 and approximately 20% of the chromosome, respectively, without the obvious involvement of mobile genetic elements. The replacements are unprecedented in natural populations of bacteria because of their large size and unique structure and may have a dramatic impact on bacterial evolution.

Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections

ABSTRACT Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolates genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

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.

Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus

Hospital-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health burden dominated by a small number of bacterial clones. The pandemic EMRSA-16 clone (ST36-II) has been widespread in UK hospitals for 20 y, but its evolutionary origin and the molecular basis for its hospital association are unclear. We carried out a Bayesian phylogenetic reconstruction on the basis of the genome sequences of 87 S. aureus isolates including 60 EMRSA-16 and 27 additional clonal complex 30 (CC30) isolates, collected from patients in three continents over a 53-y period. The three major pandemic clones to originate from the CC30 lineage, including phage type 80/81, Southwest Pacific, and EMRSA-16, shared a most recent common ancestor that existed over 100 y ago, whereas the hospital-associated EMRSA-16 clone is estimated to have emerged about 35 y ago. Our CC30 genome-wide analysis revealed striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements and nonsynonymous mutations affecting antibiotic resistance and virulence. Importantly, phylogeographic analysis indicates that EMRSA-16 spread within the United Kingdom by transmission from hospitals in large population centers in London and Glasgow to regional health-care settings, implicating patient referrals as an important cause of nationwide transmission. Taken together, the high-resolution phylogenomic approach used resulted in a unique understanding of the emergence and transmission of a major MRSA clone and provided molecular correlates of its hospital adaptation. Similar approaches for hospital-associated clones of other bacterial pathogens may inform appropriate measures for controlling their intra- and interhospital spread.