Partial Excision of the Chromosomal Cassette Containing the Methicillin Resistance Determinant Results in Methicillin-Susceptible Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) organisms are among the most important nosocomial pathogens, being responsible for a wide range of infections, some of which are associated with high mortality (2). Although the first MRSA strain was isolated in 1961, MRSA is still considered as an emerging pathogen, and public health threats result from the spread of hospital-acquired as well as community-acquired MRSA (1, 17, 20). Thus, important efforts have been made during the past decades in order to get a detailed knowledge of MRSA epidemiology and to improve infection control strategies (9). For instance, the enigma of acquisition of methicillin resistance has been partially solved with the discovery of the Staphylococcus aureus cassette chromosome mec (SCCmec): susceptible staphylococci acquire methicillin resistance by means of transfer of these mobile elements carrying mecA, the central element of methicillin resistance in staphylococci, and the ccrAB locus, which encodes its integration and excision (13).

In French hospitals, the MRSA isolation rate in blood cultures reached 33% in 2002, and this value has remained stable over the past years (25). Nevertheless, since 1992 MRSA epidemiology in France has changed with the emergence of gentamicin-susceptible MRSA strains, which are also more susceptible to other antibiotic classes when compared to previous dominant strains (4). These strains belong to clonal complex 8, which comprises most of the epidemic MRSA strains in Europe since 1960, and they derive directly from the methicillin-susceptible Staphylococcus aureus (MSSA) with sequence type 8 (ST8) after the acquisition of SCCmec type IV (22). An intriguing feature of these strains is the high frequency of the loss-of-methicillin-resistance phenotype due to the excision of SCCmec. This excision phenomenon originates in MSSA strains that have no SCCmec but that are still resistant to erythromycin, lincomycin, and fluoroquinolones (3). However, between 1999 and 2002, we have collected MSSA isolates from several French hospitals with a similar resistance profile except for the unusual addition of the resistance to tobramycin. Since resistance to tobramycin in French MRSA clone isolates is due to the aadD gene localized within the SCCmec type IV (linearized pUB110 in the mecA downstream vicinity) and encoding a nucleotidyl transferase (20), it was of interest to check for the presence of mecA and other SCCmec-associated elements in these isolates.

The relevant characteristics of the nine strains used in this study are summarized in Table 1 together with results of the detailed PCR analysis of the SCC structures. All primers used for PCRs were described elsewhere (12, 13, 16, 17, 18, 19, 20). Amplification of the attB site, the site of integration of the SCCmec into the S. aureus chromosome, was performed using cL1 and cR2 primers as described by Katayama et al. (13) and using the MSSA strain RO791 as a positive control. The allelic characterization of the ccrAB locus by PCR was performed as described by Ma et al. (16) with four sets of primers: (i) αc/βc, control for the presence of the ccrAB locus; (ii) α1/βc, specific for ccrAB allele 1; (iii) α2/βc, specific for ccrAB allele 2; and (iv) α3/βc, specific for ccrAB allele 3. The presumptive assignment of SCCmec types was performed by a multiplex PCR strategy according the method of Oliveira and de Lencastre (20). MRSA strains used as controls were COL for type I, PM64 (EMRSA-16) for type II, AR1239 for type III, and PM60 (EMRSA-15) for type IV. Then, six “uniplex” PCRs were performed as previously described (18, 19) to scan for the presence of the structure HVR-IS431-pUB110-IS431-dcs-orfx, which may be found in SCCmec type II and in some variants of SCCmec types I and IV (subtypes I-A and IV-A, respectively) (Fig. 1). Strain N315, which is the prototype strain for SCCmec type II and positive for the HVR-IS431-pUB110-IS431-dcs-orfx structure, was used as positive control. Finally, spa typing and multilocus sequence typing (MLST) were performed as previously described (6, 14, 23).

By SCCmec multiplex PCR all these isolates were negative for mecA. Nevertheless, positive signals for the dcs and the IS431::pUB110 junction were detected for tobramycin-resistant MSSA isolates but not for the tobramycin-susceptible strain 660804. To investigate if SCCmec derivative elements were still integrated at the attB site (the conserved insertion site for SCCmec within the putative open reading frame orfX), a PCR was performed. A positive result was obtained only for MSSA strain 660804 but not for any of the tobramycin-resistant MSSA isolates. This suggests that SCCmec element derivatives are integrated at the attB site like the complete SCCmec in MRSA. By “uniplex” PCR, all tobramycin-resistant isolates were confirmed to be positive for the IS431::pUB110::IS431::dcs structure, which, except for isolate 1060728, closely maps to the orfX (Fig. 1). The more parsimonious hypothesis is that these MSSA strains are derived from a French MRSA clone which is characterized by a SCCmec type IV variant (subtype A) harboring the HVR::IS431::pUB110::IS431::dcs structure at its 3′ end (19). The likelihood of this hypothesis is strengthened by the fact that both MSSA 479968 and MRSA 761641 have the same sequence type, ST8, by MLST and the same spaA type, YHGFMBQBLO. Since all these MSSA strains were found to be negative by PCR for the ccrAB locus, the presence of a SCC-like structure may be excluded. Therefore, the presence of the IS431::pUB110::IS431::dcs structure is likely due to a partial excision of the SCCmec from MRSA, which involved the deletion of the mecA gene and its upstream vicinity. Like the diversity in geographic origin, the variability found in the organization of the SCCmec element derivatives argues strongly for frequent and independent excision events rather than dissemination of an epidemic MSSA strain due to the clonal expansion of a single isolate. Nevertheless, since isolates have been collected over a 3-year period, we cannot exclude rearrangements of SCCmec occurring in an epidemic strain.

Recovery of methicillin susceptibility in resistant isolates has been described a few years after emergence of MRSA, first in laboratory studies (5, 8, 13) and then in clinical strains (3, 11, 15). The recent description of the staphylococcal cassettes allows a better understanding of this phenomenon. SCCmec carries not only the mecA gene but also ccrAB genes, which encode recombinases acting for the integration into or the excision from the chromosome (13). There are four main types of SCCmec and probably many variants, especially for type IV (12, 24). This last type is present in many more genetic backgrounds, including hospital-acquired and community-acquired MRSA epidemic lineages, than other types, suggesting an enhanced mobility (4, 7, 19, 21, 22). An indirect argument for this mobility would be the high frequency of excision observed among isolates belonging to the French MRSA clone.

To our knowledge this is the first detailed report of clinical MSSA isolates containing SCCmec elements. Recently Hutlesky et al. have reported amplification of the junction SCCmec-orfX in 4.6% of isolates from a worldwide collection of MSSA strains, but, as stated by the authors, it was not clear if these isolates retained SCCmec elements after excision or possessed another staphylococcal cassette (10). Recognition of these MSSA isolates may pose some problems in clinical microbiology laboratories which perform detection of MRSA carriage by real-time amplification of the dcs::orfX junction for SCCmec (10, 26). The presence of dcs in MSSA strains, as described in this study, might compromise the efficiency of this test.

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FIG. 1.

Putative organization of the IS431::pUB110::IS431::dcs structure. The relative positions of the primers used in the confirmation PCRs are indicated; the arrowheads indicate orientation (5′ to 3′).

• Copyright © 2005 American Society for Microbiology