The evolution of methicillin resistance in Staphylococcus aureus: Similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones

Results

The preservation of all S. aureus blood isolates in Denmark since the early 1950s has allowed genetic analysis of the unique epidemiological scenario surrounding the emergence of MRSA in that country. (i) All but 9 of the 646 MRSA identified among a total of 3,704 S. aureus blood isolates collected between 1957 and 1970 showed a special multiresistance pattern that included (in addition to M) resistance to penicillin (P), streptomycin (S), tetracycline (T), and often to erythromycin (E) as well. (ii) The great majority of MRSA (616 of 646 or 95%) belonged to either phage group III (40 isolates) or the closely related 83A complex (576 isolates). These two phage types represented about half of all S. aureus blood isolates (including both MRSA and MSSA strains) identified in Denmark between 1957 and 1970. (iii) The same multiresistant antibiotype and phage group was also characteristic of the first MRSA isolates from the U.K. (3, 4). (iv) Interestingly, the P-S-T or P-S-T-E antibiotype also was identified in about 20% (627 strains) of the 3,058 MSSA strains collected during the same period, and the majority of these isolates (519 of 627 or 82%) also belonged to phage group III and/or the related 83A complex. (v) Yet another interesting feature of the epidemiology of early MRSA strains in Denmark was that the “wave” of MSSA strains with the P-S-T-(E) phenotype and belonging to phage groups III and or 83A preceded in time the appearance of the wave of MRSA strains with the same phenotype and phage types (Fig. 1).

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Figure 1

Sequential appearance of M-susceptible and M-resistant blood isolates of S. aureus belonging to phage group III and the related 83A complex (in Denmark). ■, P-S-T-M isolates; ♦, P-S-T isolates. The numbers plotted represent all S. aureus blood isolates identified in Denmark during the particular period.

These data strongly suggested that in our search for the original recipient of the mec gene complex we should focus on MSSA strains belonging to phage group/type III/83A with the P-S-T or P-S-T-E antibiotype. All but one of the 25 MSSA and all 24 MRSA strains selected for the genetic analysis shared this phage group and antibiotype.

Early MSSA and MRSA and Contemporary MRSA Clones.

The early (“archaic”) MRSA isolates from Denmark and the U.K., which shared similar genetic backgrounds with early MSSA isolates, also were compared with the genetic profiles of several contemporary MRSA clones. Data in Table 3 show the remarkable similarities between properties of the archaic MRSA isolates and those of one of the most widely spread contemporary MRSA, the “Iberian” clone (19–21). Additional, close similarities became apparent between the genetic profile of the phage nontypable MSSA strain E1410 and “EMRSA 16”—another widely dispersed MRSA clone (22). The genetic background (PFGE pattern, spaA type, and MLS type) of the MSSA strains E2104 and 3001 was found to be very similar to the genetic background of two distinct MRSA lineages, the “Pediatric” clone (23) and the “New York” clone (refs. 24–26; see Table 3). Computer analysis of the MLST data followed by the construction of unweighted pair group method with arithmetic mean (UPGMA) trees (data not shown) confirmed the close genetic relatedness of the MRSA contemporary clones and their postulated evolutionary relatives (maximum genetic distance around 0.15).

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Table 3

Postulated evolutionary relationships among historically early isolates of MSSA, MRSA, and contemporary epidemic clones

The PFGE patterns in Fig. 2A and the corresponding dendrogram (Fig. 2B) demonstrate the similarities between early MSSA and MRSA isolates and several contemporary MRSA clones such as the Iberian, Pediatric, and New York clones.

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Figure 2

(A) Comparison of PFGE patterns from historically early MSSA and MRSA isolates from Denmark and the U.K. and representatives of the Iberian, Pediatric, and New York clones of MRSA. Lanes 2–12 show early MSSA strains (Table 1). Lanes 13 and 15–22 show early MRSA strains (Table 2). Lanes 23–25 show contemporary MRSA (Table 3). Strains used in A are underlined in Tables 1, 2, and 3. Lane 2, E213; lane 3, E228; lane 4, E306; lane 5, E803; lane 6, E1215; lane 7, E1907; lane 8, E2104; lane 9, E1410; lane 10, E3008; lane 11, E2260; lane 12, E2251; lane 13, E2672; lane 15, E2125; lane 16, E2213; lane 17, E3015; lane 18, E3150; lane 19, E3520; lane 20, E4278; lane 21, ST61/6219; lane 22, ST63/458; lane 23, Iberian clone HPV107 (19); lane 24, Pediatric clone HDE288 (23); lane 25, New York clone BK2464 (24). Lanes 1, 14, and 26 show strain NCTC8325 used as molecular weight standard. (B) Computer-generated dendrogram of the PFGE patterns shown in A. Results are from analysis of similarity by Jacquard's coefficient; clustering was done by the minimum linkage method.

Discussion

The acquisition of M resistance has provided S. aureus with a mechanism that has made all members of the largest and most useful family of antimicrobial agents, the β-lactam antibiotics, obsolete as therapeutic agents against these bacteria. The emergence and global spread of MRSA may be viewed as a process of accelerated evolution (27), which took place in the contemporary clinical environment, condensed into a remarkably short time frame of 4 decades during which MRSA has emerged and spread to every corner of the world, driven by the selective pressure of immense quantities of antimicrobial agents introduced into the clinical environment. The starting point of this process is the entry of the resistance gene mecA and associated mec element into the species of S. aureus from a heterologous source. Although the exact timing of this event is not known, it is reasonable to assume that it is linked to the time of introduction of the penicillinase-resistant oxazolidine class of antibiotics, such as M, into therapy at the end of the 1950s and/or early 1960s. The very first MRSA in Europe was isolated in a British hospital in 1961 (3) and the first Danish blood isolates of MRSA were recovered in 1963 (2).

The early MRSA isolates may represent the progeny of MSSA that was the original recipient of the heterologous mec element. If this hypothesis is true, then one expects to identify the postulated recipient among MSSA strains originating in the same geographic area and same era. To test this hypothesis, we compared the genetic backgrounds of historically early MSSA as well as MRSA isolates from Denmark and from the U.K. recovered shortly before and after the introduction of M into clinical practice, i.e., around the year 1960.

The results of the experiments described in this communication confirm this hypothesis by demonstrating the striking similarities between MRSA strains and most of the MSSA isolated in Denmark and the U.K. during this era. Most early MSSA examined shared a common phage group and multiresistance (P-S-T) pattern with the early MRSA strains and showed close resemblance if not identity in genetic profiles. The simplest interpretation of these findings is that the largest group of the early MSSA characterized here indeed represented the progeny of an S. aureus strain that must have been one of the first recipients of the mec element during the evolutionary history of MRSA. The postulated heterologous donor of the mec element and its mode of introduction into S. aureus remain unknown at the present time.

The presence of resistance determinants to P, S, and T, and occasionally to E as well, in the earliest MRSA isolates most likely mirrors the sequential introduction of these different types of antimicrobial agents into therapeutic practice. Data from the records of the Danish Health Board indicate that the years of introduction of these antimicrobial agents were 1945/46 (P), 1948 (S), 1950 (T), 1953 (E), and 1960 (M) (8). The accumulation of multidrug-resistance traits in MRSA strains has remained the hallmark of antibiotic-resistant S. aureus throughout much of the subsequent history of this bacterial pathogen, most likely as the consequence of the preferential use of new antimicrobial agents against bacteria that were already resistant to all previously used therapeutic agents, thus presenting a scenario of built-in obsolescence for new antimicrobial agents (28). The introduction of radical and effective infection-control measures in Denmark in the 1970s caused a virtual disappearance of MRSA from Danish hospitals, including the archaic MRSA strains (29). The few MRSA isolates that were identified in 1986 in clinical specimens with properties of the archaic clone seem to have survived only in the unique and protected milieu of osteomyelitis (1).

Extending the genetic profiling (PFGE, spaA type, and MLS type) to some of the widely spread contemporary MRSA clones also has allowed us to identify among the historically early MSSA several strains with genetic backgrounds matching those of contemporary epidemic clones of MRSA. The most important one of these is the Iberian clone, which shares similar PFGE type, spaA type, and MLST pattern with the majority of early MSSA and MRSA and also carries a mec element similar in genetic structure to that of the mec element (type I SCCmec) identified in early MRSA by Hiramatsu and colleagues (30); the only difference being the insertion of linearized plasmid pUB110 in the case of the Iberian clone downstream of mecA (31).

Two of the early MSSA strains, E2104 and 3001, exhibiting resistance only to P, showed a PFGE pattern and spaA and MLS types that were very similar to the properties of two internationally spread MRSA: the Pediatric clone, which most often shows resistance only to β-lactam antibiotics (23), and another contemporary multiresistant MRSA widely spread in the northeastern part of the United States, the New York clone (25–26). Bacteria belonging to the New York clone carry a mec element that seems to correspond to what Hiramatsu and colleagues (30) named the type II SCCmec (ref. 30 and D.C.O., A.T., and H.d.L., unpublished data). The nature of the mec element in the Pediatric clone is currently under investigation.

A single isolate, strain E1410, among the 25 early MSSA, was nontypable by the phages used and was resistant to P-S, possessed a unique PFGE type g, and also showed spaA and MLS type, which was different from that shown in the rest of the 24 MSSA tested. A search through contemporary MRSA clones with the molecular typing techniques identified the MLS type seen in strain E1410, as typical of yet another major contemporary MRSA clone, the “EMRSA 16”, widely spread in hospitals in the U.K. (13). Interestingly, MSSA strains with the same genetic background are still frequent among MSSA isolates from the U.K. (13). Analysis of a representative of this clone suggests that it carries a type II SCCmec (30).

These observations indicate that MSSA strains ready to participate as recipients in the horizontal spread of mecA were already present among MSSA isolates recovered around the early 1960s. These strains may have remained in circulation to acquire various forms of the mec element and provide the genetic backgrounds for some of the contemporary MRSA clones.

The number of times the mec element entered the species of S. aureus from heterologous sources is not known and may be limited (11). Nevertheless, examination of MRSA isolates from the 1980s has demonstrated clearly the extensive horizontal spread of the mec gene throughout the entire genetic breath of S. aureus, possibly by homologous gene transfer (32). The role of such sporadic isolates in the epidemiology of MRSA is not clear.

Molecular epidemiological studies conducted in several countries since the late 1980s clearly indicate that the major factor responsible for the massive geographic spread of MRSA is the dissemination of a relatively few highly epidemic clones. The archaic MRSA carrying the P-S-T multiresistance traits and phage group 83A/III was clearly such a successful lineage, as judged by the rapid spread of this clone to many Danish as well as British hospitals within a few years of its appearance. The results described in this article indicate that one of the most successful contemporary MRSA clones, the Iberian MRSA, detected as the dominant MRSA in many hospitals in western and southern Europe and in at least one hospital in New York City (33), is a direct descendant of the archaic MRSA. These observations strongly suggest that the genetic background of this particular MRSA lineage, extending from MSSA identified in disease-causing strains since the late 1950s through the early MRSA isolates to one of the major contemporary MRSA clones, carries genetic traits essential for the superior epidemicity and virulence of these bacteria.