Among the many MRSA isolates, only a few MRSA clones were widespread as major clones over a long period of time. In order to understand the pandemic MRSA clones and the molecular evolution of MRSA in PUMCH over a long period, we investigated the molecular characteristics of 302 MRSA and 164 MSSA isolates by SCCmec-spa-MLST typing methods from 1994 to 2008.
Between 1994 and 2008, the Brazilian clone ST239-MRSA-III was the most prevalent clone determined by the molecular typing of 302 MRSA isolates from PUMCH. The Brazilian MRSA clone was first discovered in Brazil and widely disseminated in various hospitals (29
). Subsequently, this clone spread to neighboring South American countries (Argentina [8
] and Uruguay and Chile [4
]) and to Europe (Portugal [3
], the Czech Republic [23
], and Greece [1
)]), where it displaced the local major clones. Recently, ST239 also became the epidemic clone in most Asian countries (12
). Our previous study demonstrated that this clone was also the dominant one in most of the cities in China from 2005 to 2006 (21
). ST239 represented a distinct branch within CC8 in the evolutionary model of the emergence of MRSA. The previous study proposed that ST239 arose from a single recombination event that involved the exchange of >200 kb of contiguous DNA between ST30 and ST8 (26
ST239 still presented as the most predominant ST of MRSA over all the years of the study period, but by the higher-resolution spa typing method, the epidemic clone had a rapid change. Our data demonstrated that spa t037, which was prevalent before 2000, was rapidly replaced by the t030 MRSA clone, which emerged in 2000. Since then, t030 has become the most popular MRSA clone. Surprisingly, it was after 2000 that the prevalence of MRSA increased rapidly in PUMCH, which was coincident with the introduction of t030. It seemed that ST239-MRSA-III-t030 had a stronger survival advantage and could easily transmit in our hospital. The most obvious difference between these two clones was that most t030 isolates were resistant to rifampin, while only a few t037 isolates were resistant.
In order to investigate whether t030 evolved from the former epidemic clone or was an alien clone, we reviewed the medical records of the patients from whom the t030 MRSA isolates were first isolated in 2000. Most of these patients were transferred to PUMCH from other hospitals. So we speculated that t030 was introduced in this hospital from outside and replaced the previous epidemic clone t037. In addition, we randomly selected 15 t030 MRSA isolates between 2000 and 2008 for PFGE typing. The results showed that the 15 isolates had identical or similar PFGE types, which suggested that they belonged to the same ST239-MRSA-III-t030 clone that had spread in this hospital since 2000. Besides, there were some minor evolutionary events related to the epidemic clone ST239. Four MRSA isolates evolved from ST239 to ST158 by a single point mutation of yqiL; another two isolates belonged to ST585, which evolved from ST239 by a new single-locus variant (aroE) mutation; and another isolate belonged to ST1097, which evolved from ST239 by a single point mutation of glpF (Fig. ).
Proposed evolutionary pathways of different MRSA clones in this study. The model was based on eBURST analysis combined with spa typing and acquisitions of the SCCmec type. Arrows indicate the directions of changes between clones.
ST5-MRSA-II was the second epidemic MRSA clone in PUMCH which belonged to the New York/Japan international clone. This clone was initially described as the main clone in the United States (25
) and Japan (2
) and subsequently detected in several European (6
) and Asian (18
) countries. The previous data also showed that ST5-MRSA-II was the prevalent clone in several cities (such as Shenyang and Dalian) in China (21
). In this study, ST965 (a single-locus variant of pta
of ST5) and ST5 MSSA were found as early as 1996. Then as time went by, ST5-MRSA-II isolates emerged in 2002, and ST965-MRSA-II was detected in 2008. Another clone, ST641-MRSA-II, evolved from ST5-MRSA-II by aroE
mutation (Fig. ).
Frequent conversion of MSSA to MRSA by horizontal transfer of SCCmec
has been described (13
), suggesting that MSSA was the origin of MRSA and that MRSA independently developed by multiple evolutionary events in the accessory genome within a single ST. Thus, to elucidate the origin of MRSA, 164 MSSA isolates recovered between 1994 and 2008 were characterized by spa
and MLST. Interestingly, the genotypes of MSSA isolates were much more diverse than those of MRSA. The majority of MRSA isolates typically belonged to one or a few different clonal types. In comparison, MSSA isolates were more often genetically variable. In this study, ST5, ST59, and ST965 coexisted in both MRSA and MSSA isolates, and ST5-MSSA-t002 and ST59-MSSA-t437 had already existed before the emergence of ST5-MRSA-t002 and ST59-MRSA-t437, which might indicate that several MRSA clones had evolved from MSSA in PUMCH.
Another interesting finding was that some high-frequency MSSA genotypes closely resembled those of the well-known epidemic CA-MRSA clones. Of the types determined for the 164 MSSA isolates, ST398 was the most popular type, but this ST was not found with MRSA isolates. Recently, ST398-V MRSA was found to cause community infections in pigs, pig farmers, and the patients who had the pig contact experience (19
). Another popular ST was ST1, which bears the same ST as MW2. MW2 was the first CA-MRSA strain reported in the United States and is characterized as PFT USA400, SCCmec
IV, and pvl
). Another ST that has been reported to cause skin and soft tissue infections in the community is ST121 (22
), and 6 isolates belonged to this ST in this study. Currently, none of the isolates belonging to this ST have been reported as MRSA, but given the opportunity, this type might be converted to CA-MRSA. Therefore, the findings above suggest that these MSSA clones, once they acquire some accessory genes, like, SCCmec
, or the pathogenicity island, would cause serious CA-MRSA infections or even outbreaks; thus, we should pay more attention to these clones.
Through the eBURST analysis of MSSA, ST121 was the predicted founder of CC121, five isolates belonged to ST837, and two isolates belonging to ST95 evolved from this founder by yqiL mutation. Another four isolates belonged to ST965, which is a single-locus variant of ST5, and six isolates belonged to ST188, which was a double-locus variant (DLV) of ST1, which was the predicted founder of CC1. One MSSA strain belonged to ST195, which was a DLV of ST630, and the two STs belonged to CC8. Therefore, MSSA also presented evolutionary events.
In this study, the number of MSSA isolates carrying the pvl
= 9) was higher than that of MRSA isolates (n
= 2), which seemed to indicate that toxic gene deletion occurs during the evolution of MSSA to MRSA. We found two pvl
-positive MRSA isolates from the outpatients that belonged to ST59-MRSA-IV, and the clinical data confirmed them to be CA-MRSA. ST59-MRSA-IV was reported to be prevalent in skin and soft tissue infections in children in Taiwan (16
), Hong Kong (15
), and mainland China (33
), but in this study, except for these two isolates, the rest that belonged to ST59 were MSSA, which might indicate that ST59-MRSA-IV was not popular in community-acquired infections in adults.
In summary, we have described for the first time the long-term molecular evolution of MRSA in a tertiary care hospital in China. The epidemiology of MRSA has been evolving rapidly. A new clone (ST239-MRSA-III-spa t030) was introduced and replaced the previously predominant clone (ST239-MRSA-III-spa t037). Long-term multicenter MRSA evolution and the genetic evidence for the survival advantage of t030 need further study.