MRSA emerged in the early 1960s and remained largely restricted to the hospital environment (43
). Not until resistance occurred in other lineages 30 years later did it rapidly spread in the community (43
). CC5 is often the first lineage in which new antibiotic resistance genes appear (10
). Our work provides quantitative support for an earlier proposition, based largely on PFGE (15
), that each occurrence of Tn1546-
conferred vancomycin resistance in S. aureus
represents an independent acquisition, rather than patient-to-patient spread. Modeling evolutionary distances on a time scale () shows that the last common ancestor of all VRSA strains occurred at about the time of methicillin introduction, about 1960, 40 years earlier than vancomycin resistance was found to have entered the species. The early 1960s also is the approximate time when the Japanese CC5 isolates diverged. The modeling analysis employed was based on a calculated mutation rate of 3.46 × 10−6
per site per year and employed conservative, relaxed-clock assumptions. This rate is in excellent agreement with the 3.3 × 10−6
± 0.7 × 10−6
mutation rate calculated independently by others in a rigorous application of second-generation sequencing in a study of the global spread of the MRSA lineage ST239 that is prominent in Asia (48
). Only in the case of strains VRS11a and VRS11b, which were isolated from the same patient, does the uncertainty with respect to the time of strain divergence extend beyond the date of isolation of that VRSA strain. The 1960 time point represents the time when the most divergent VRSA strain, VRS3b (PFGE type USA800), branched from the rest, most being USA100 PFGE type (). Within the USA100 group, extensive diversification occurred next in about 1978.
FIG 6 Consensus tree for CC5 under relaxed clock conditions. North American CC5 strains are shown in black, while other strains are shown in gray. Blue bars indicate range of 95% highest posterior density interval (95% confidence interval, 1.76 × 10 (more ...)
In contrast to expectations, VRSA restriction barriers appear to be largely intact. Strain VRS3a possesses a defect in the Sau1 endonuclease that is likely to be of functional consequence. However, most other polymorphisms in the Sau1 system were limited to the νSaα-encoded copy of a modification gene, with the νSaβ-encoded copy fully intact. Another strain, VRS10, possesses change in the type III-like restriction, shortening the predicted primary translation product from 953 amino acids to 856 amino acids by removal of the amino terminus. The functional consequence of this truncation is currently unknown. It may be important that all VRSA strains, except for the phylogenetic outlier strain VRS3a, possess a nonsense mutation early in the dprA
gene that is predicted to truncate a majority of the polypeptide. DprA (also known as Smf [49
]), is highly conserved and contributes to efficient DNA transformation in naturally competent bacteria (29
). Transformation efficiency of plasmids in a B. subtilis dprA
mutant is decreased 60-fold (29
). Experiments with Escherichia coli dprA
mutants do not show an obvious role in transformation or conjugation (55
). Its function in S. aureus
remains to be explored.
The most variable feature of the VRSA genome is plasmid content. In all cases, Tn1546 resides on a plasmid, even though it clearly transposed upon entry into some strains, and because of size, the chromosome would seem to be the most probable target for transposon insertion. The basis for the insertion site preference for plasmids over the S. aureus chromosome, and also for an apparent incompatibility between the enterococcal Inc18 plasmid that played a major role in the Michigan outbreak and an endogenous S. aureus pSK41 plasmid present in several recipients, is unknown. VRSA genomes are replete with plasmids of enterococcal origin, highlighting their cooccurrence in polymicrobic infections and possibly in other ecologies. The multiplicity of plasmid structures conveying Tn1546, including S. aureus/enterococcal cointegrate plasmids, increases the odds of future transfers, possibly into staphylococcal lineages or species where a lower fitness cost is incurred.
The genomic island νSaβ is a distinguishing feature of CC5. Of potential ecological importance, the bsa
operon usually encoded within this island is absent in VRSA and other CC5 strains. Frequent application of antibiotics in the hospital environment may select for strains with an enhanced ability to comingle with potential resistance donors of other species. Interestingly, this trait is also lacking in otherwise highly divergent strains that are prevalent in hospitals in the United Kingdom. The fact that United Kingdom and U.S. hospital strains with widely different chromosomal backgrounds share very similar νSaβ islands suggests that there may be active selection for this configuration in the hospital environment. Multidrug-resistant enterococci are much more likely to lack clustered regularly interspaced short palindromic repeat (CRISPR) defenses of the genome than commensal strains (56
), indicating that the widespread use of antibiotics has selected for hospital-adapted bacteria that have enhanced abilities to exist in mixed communities and exchange resistance determinants. Loss of bacteriocin production as well as immunity may also explain why, for 30 years, CC5 MRSA strains were not able to establish methicillin resistance in the community at a high level. They may have been inhibited by the functional bacteriocin loci of S. aureus
strains of other sequence types (such as CC8 and CC1) already present in the community niche. This may also be limiting the spread of vancomycin resistance from CC5 strains to other clades.
Staphylococcal enterotoxins, T cell mitogen superantigens (57
) that dysregulate the host response by stimulating CD8+
regulatory T cells (Tregs
) at low concentrations (58
) and other mechanisms, are particularly abundant in the CC5 νSaβ element, as well as that of the United Kingdom clones that are prevalent in hospitals. Enterotoxins, together with lipoproteins (59
) and leukocidin (41
) may facilitate overgrowth of mixed populations of bacteria at a site of infection, contributing to the creation of conditions favorable for resistance transfer. Notably, most VRSA strains occurred in mixed infections of plantar ulcers of diabetic patients (2
), infections that are known to be highly polymicrobic (60
The hospital is a unique environment, where colonization and patient-to-patient propagation of a strain may depend less on bacterial virulence traits associated with transmission (62
) than on transmission vectors in the form of hospital staff and environmental surfaces and antibiotics (63
). Strains that are prevalent in hospitals are under continuous antibiotic selection pressure and are exposed to an ever rotating arsenal. CC5 isolates appear to be very well adapted for surviving and evolving in this environment by acquiring resistance to new antibiotics.