MRSA Harboring mecA Variant Gene mecC, France

We describe human cases and clustered animal cases of mecALGA251–positive methicillin-resistant Staphylococcus aureus in France. Our report confirms that this new variant has a large distribution in Europe. It may represent a public health threat because phenotypic and genotypic tests seem unable to detect this new resistance mechanism.

S ince its fi rst description in the early 1960s, methicillinresistant Staphylococcus aureus (MRSA) has become a major public health issue because of worldwide spread of several clones. More than 20 years later, the specifi c genetic mechanism of its resistance has been identifi ed as a mobile genetic element (staphylococcal cassette chromosome mec) integrated into the S. aureus chromosome, within which the mecA gene encodes a specifi c methicillin-resistant transpeptidase (penicillin-binding protein 2a) [PBP2a] (1). This protein has a low affi nity for β-lactam antimicrobial drugs. Thus, bacteria expressing this protein are resistant to all types of these drugs.
A new divergent mecA homolog (mecC or mecA LGA251 , in reference to LGA251 isolates from which it was characterized) (2,3) was recently described in a novel staphylococcal cassette chromosome mec named type XI (2). This newly identifi ed protein has <63% aa identity with PBP2a encoded by mecA and was described in S. aureus or coagulase-negative staphylococci. This new mecA homolog has been detected in bacteria from dairy cattle in England and humans in England, Scotland, and Denmark (3). We report the emergence of human and animal cases of mecA-variant MRSA identifi ed in France.

The Study
The fi rst case was detected in a 67-year-old man admitted to Aix-en-Provence Hospital in southern France on November 8, 2007, because of suspected joint infection of his left knee 3 years after total knee joint replacement. He reported gonalgia that had been present for 6 months. The patient was apyretic but asthenic and reported a 10kg weight loss. He had voluminous knee effusion. The synovial fl uid leukocyte count was >1,000 cells/mm 3 with 86% neutrophils, fi ndings consistent with a prosthetic knee infection (4). The patient had a deep and hyperkeratosic lesion on his left heel that was considered to be the source of infection.
Direct microscopic examination of the fl uid identifi ed gram-positive cocci in grape-like clusters. S. aureus was identifi ed in pure culture. Susceptibility tests performed by using the disk diffusion method as recommended by the Société Française de Microbiologie (Paris, France) (www. sfm-microbiologie.fr) showed methicillin resistance. The presence of the mecA gene was investigated by using 2 methods, an-in house PCR (5) and the GenoType Staphylococcus test (Hain Lifescience GmbH, Nehren, Germany), but results were negative.
A 2-stage revision of arthroplasty of the infected knee was performed. Initial treatment was rifampin (1,800 mg/ day) and ofl oxacin (400 mg/day) for 5 months. Ofl oxacin was then replaced with clindamycin (1,800 mg/day) for 2 months because of quinolone-induced tendinitis. The unusual length of treatment was required because of an abnormal delay in healing, persistence of knee infl ammation, and subclinical infl ammatory syndrome. The clinical outcome was favorable at a 3-year follow-up.
The second case was detected in 2 cows with clinical mastitis on the same farm in the Meurthe-et-Moselle District of northeastern France in December 2008. Two S. aureus strains were isolated from milk samples from these 2 cows. Drug susceptibility profi les determined by using the disk diffusion method were identical; both isolates showed methicillin resistance and susceptibility to other antimicrobial drugs, an unusual profi le in veterinary microbiology. Only 1 strain was stored and sent to the French Agency for Food Environmental and Occupational Health and Safety (Lyon, France) where a mecA PCR (6) result was negative.
After failure of empiric treatment with cefalonium, a fi rst-generation cephalosporin, the 2 cows were successfully treated with neomycin/spiramycin. Because these mastitis cases occurred in a context of recurrent bacterial infections (clinical and subclinical mastitis caused by S. aureus in dairy cows, diarrhea caused by Escherichia coli in veal calves), hygienic measures were instituted, including decontamination of milking machines, disinfection of teats before milking, and application of standard practices for infection control. These measures were successful, and MRSA was not detected again on this farm. Using primers and the protocol reported by García-Álvarez et al. (3), we detected a mecA variant (mecA LGA251 ) in both isolates. Sequencing of a PCR-amplifi ed fragment confi rmed >99% homology with the sequence obtained for the LGA251 strain. Molecular typing of both isolates showed that they harbored an agr allele 3, were spa type t843, and belonged to clonal complex 130 (sequence type [ST] 130 for the cow isolate and ST1945, a single-locus variant of ST130 that differs by only 1 nucleotide within the pta gene, for the human isolate). These characteristics matched those of the most prevalent clones described by García-Álvarez et al. (3).
These isolates were also subjected to DNA microarray analysis by using the StaphyType Kit (Alere Technologies GmbH, Jena, Germany). Results confi rmed assignment of the isolates to clonal complex 130, and showed that both isolates had hybridization profi les identical with those of 2 mecA variant isolates described by Shore et al. (2), except that the isolates were negative for ccr-A3 and ccr-B3. Both isolates were misidentifi ed as methicillin-susceptible S. aureus by use of this microarray genotyping approach and the real-time PCR (GeneOhm Staph SR; BD Diagnostics, San Diego, CA, USA) because of mecA was not amplifi ed.
The human isolate was determined to be methicillin sensitive by using the BD Phoenix PMIC/ID60 panel (BD Diagnostics) (oxacillin MIC 0.5 mg/L, cefoxitin MIC 4 mg/L, moxalactam MIC 16 mg/L). In addition to the inability of the Xpert MRSA/SA SSTI assay (Cepheid, Sunnyvale, CA, USA) reported by Shore et al. (2) to detect mecA variant isolates, these data confi rm the inability of commercial molecular methods and some phenotypic panels currently available to identify all mecA variant isolates. These fi ndings raise fears that such MRSA isolates are misidentifi ed and that their prevalence is underestimated.

Conclusions
Our results provide information on global distribution of the mecA variant gene. Geographic and temporal diversity of the isolates suggest that such strains are widely distributed and were not recently introduced in France. Moreover, animal cases were described only in the United Kingdom, and no cluster of clinical cases (2 cases of cow mastitis) was reported. Our data demonstrate the ability of such strains to cause clustered cases.
This new methicillin-resistance mechanism in S. aureus may be a new public health threat. Global dissemination of mecA LGA251 S. aureus should be investigated and controlled in humans and animals. Control measures should include rational use of antimicrobial drugs, accurate and rapid microbiological laboratory services, and specifi c infectioncontrol measures.
In addition, the epidemiologic situation should be carefully monitored. However, such monitoring is made diffi cult by a combination of 3 issues. The fi rst issue is the inability to detect mecA variant MRSA by using commercial (used for screening) molecular approaches or in-house (used for confi rmation) amplifi cation tests. The second issue is lack of sensitivity of some commercial phenotypic panels used for routine drug susceptibility testing. The third issue is the need for specifi c and easy-to-read phenotypic tests to confi rm methicillin resistance caused by additional PBP (i.e., absence of synergy between oxacillin and amoxicillin/clavulanic acid disks to exclude borderline oxacillin-resistant S. aureus without being able to exclude a modifi ed PBP-resistant S. aureus phenotype). The fi rst step in overcoming this diffi culty would be inclusion in the surveillance system systematic characterization of clinical strains harboring methicillin-resistance genes associated with susceptibility to all other antimicrobial drugs, a profi le typical of mecA-variant MRSA isolates.
This report of new mecA variants in France confi rms their wide geographic range, but many questions remain. The prevalence of mecA LGA251 -positive isolates in France and other countries should be evaluated in livestock and humans. The origin, evolutionary mechanisms, potential animal reservoirs, and mode of dissemination of mecAvariant clones over large areas remain unknown. The clinical effect of expression of the PBP2a variant has not been defi nitively established in patients and should be explored in animal models.