Nationwide molecular epidemiology of methicillin-resistant Staphylococcus aureus responsible for horse infections in France

Background The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in horse infections is not well documented, especially in France. The aim of the study was to evaluate the prevalence of MRSA isolates in horse infections from 2007 to 2013 in France and to characterize phenotypically and genotypically this collection. Results Out of 1393 S. aureus horse isolates, 85 (6.1%) were confirmed to be MRSA. Interestingly, the prevalence of MRSA significantly increased from 2007–2009 to 2010–2013 (0.7 vs. 9.5%, P <0.0001). Resistance to methicillin was due to the presence of the mecA gene in 84 strains (98.8%) while one strain (1.2%) possessed the mecC gene. The vast majority of the strains (83/85, 97.6%) was resistant to at least three different classes of antibiotics. Multi-locus sequence typing (MLST) showed that MRSA strains belonged mainly since not all belong to two sequence types (STs): ST398 (53/85, 62.4%) and ST8 (28/85, 32.9%). It is worth to note that all ST398 MRSA isolates were detected in the period 2010–2013. Other molecular typing methods were also used, such SCCmec analysis, spa typing and rep-PCR (Diversilab, bioMérieux). All these four techniques were in good agreement, with spa typing and rep-PCR being more discriminative than MLST and SCCmec typing. Conclusions This study is the first epidemiological study in France with extensive characterization of MRSA isolates associated with horse infections in stud farms. It shows that there is a significant increase of MRSA prevalence between 2007 and 2013, which mainly results from the spread of ST398 clones. It also highlights the importance of horses as a potential reservoir of important antimicrobial resistance genes.


Background
Initially reported as a major cause of hospital-acquired infections in humans, methicillin-resistant Staphylococcus aureus (MRSA) has increasingly been reported as responsible for community-acquired infections as well as for infections in animals. Possible transmission of MRSA between humans and animals has raised concern about the role of animals as major reservoirs of MRSA clones involved in human infections [1][2][3][4]. Although MRSA strains are usually resistant to β-lactams through the acquisition of the mecA gene, a homolog gene (called mecC) has been recently reported both in animal and human populations [5][6][7]. It is well known that MRSA is responsible for a large variety of infections in numerous animals; however, specific studies in horses are scarce [6][7][8][9]. Indeed, some studies have demonstrated that horses are colonized and infected by MRSA clones that commonly belong to the sequence type (ST)8 and related STs within the clonal complex (CC)8 [6,9]. More recently, studies from Europe and Canada reported horses to be colonized by MRSA clones belonging to ST398, designated livestock-associated (LA)-MRSA, which is primarily recognized as a colonizer of pigs and pig farmers [6]. LA-MRSA ST398 can be responsible for infections in humans in close contact with animals. Phenotypically, LA-MRSA ST398 is generally susceptible to antibiotics other than β-lactams even if it is characteristically resistant to tetracyclines [6]. Note that almost all equine MRSA isolates carry mecA while mecC has been rarely detected so far [6,7]. From an epidemiological point of view, the prevalence of MRSA in horse infections has been poorly investigated in France [7,10] and most importantly there is no data on nationwide molecular epidemiology.
The aim of the study was then 1) to evaluate the prevalence of MRSA isolated from horse clinical samples recovered between 2007 and 2013 in France, and 2) to extensively characterize phenotypically and genotypically this large collection of equine MRSA strains.

Prevalence of MRSA isolates
From 2007 to 2013, the laboratory received 226,878 horse clinical samples with the recovery of 17,651 different bacterial isolates. S. aureus was the third most frequent bacterial species isolated (n = 1393; 7.9%) after group C streptococcus (n = 4510; 25.6%) and Escherichia coli (n = 3481; 19.7%). Out of the 1393 S. aureus horse isolates, 85 (6.1%) were categorized as MRSA (Table 1). Interestingly, the prevalence of MRSA significantly increased from 2007-2009 to 2010-2013 (0.7 vs. 9.5%, P <0.0001) ( Table 1). They were recovered from different sources of infection: skin and soft-tissue (n = 39), genital tract (n = 20), respiratory tract (n = 8), bone and joint (n = 8) and others (n = 10). Note that MRSA isolates were collected from 56 different stud farms located in 24 different French departments (1 to 24 strains by department), mainly representing the Northwestern parts of France (Fig. 1). This roughly corresponds to the actual geographical distribution of stud farms with a high number of them in Normandy. No isolate harboured pvl and tst toxin genes (data not shown).

Antimicrobial resistance profiles
Resistance to methicillin was associated with the presence of the mecA gene in 84 isolates (98.8%) while the mecC gene was detected in only one strain isolated in 2012 (1.2%) ( Table 2). Note that this mecC-positive strain was susceptible to all other antibiotics ( Table 2). A vast majority (83/85, 97.6%) were resistant to aminoglycosides with a KTG (kanamycin-tobramycin-gentamicin) phenotype, which was due to the presence of the aac(6′)aph(2″) gene in all cases (  Table 2). Finally, all strains remained susceptible to glycopeptides, linezolid and fusidic acid. Note that the vast majority of the strains (83/85, 97.6%) was multiply resistant to at least three different classes of antibiotics.

Discussion
In this study, we have showed that the prevalence of MRSA strains isolated from horse infections before 2011 was low (~2%) in France, with ST8 being predominant during this period. The prevalence has since increased from 2011 to reach about 10%, and ST398 has become the predominant MRSA lineage. These data on MRSA prevalence in equine infections are in agreement with those observed in other European veterinary hospitals: 22% in Switzerland [12] and 19% in Germany [13]. However, it is important to note that MRSA isolates in our study were recovered in stud farms and not in veterinary hospitals. Indeed, the lower prevalence in France observed in our study may be explained by the fact that these samples are from stud farms that are exposed to different environmental conditions and risk factors as compared to veterinary hospitals. Although horses were generally hosted in French stud farms geographically distant, these results suggest a national diffusion of clonally related MRSA ST8 and ST398 isolates.
The mecA-carrying ST8 and ST398 have been reported as the major strains that infected or colonized horses [13,14]. Colonization and infection of horses in the USA and Canada generally involve ST8-type MRSA isolates, classified by PFGE as Canadian MRSA-5 or USA500 [6]. ST8 is infrequently found in humans and is among STs isolated from horses of the clonal complex (CC) 8. ST8-type MRSA has also been recently isolated in Australian horses [15]. In France, only one molecular study has been performed on MRSA isolated in horses [10]. In this study, only three MRSA were identified among 59 S. aureus equine isolates and all belonged to the ST8 [10]. ST398-type MRSA is a non-CC8 clone initially report in France in pigs [6], which is currently spreading in horse clinics in Europe and North America [10,12,16]. Even if it is characteristically resistant to tetracyclines, this emerging ST is generally susceptible to other antibiotics, which was quite different from what we observed in our study. Indeed, we found that 52 out of 53 (98.1%) ST398 strains were multiply resistant to at least three classes of antibiotics. Even though multidrug resistance in MRSA is de facto defined as MDR [17], it is important to highlight the very high proportion (97.6%) of strains exhibiting resistant to three-toeight antimicrobial categories. Although ST398 is Fig. 1 Map of France representing the distribution of stud farms and the number of MRSA isolates recovered from horses by department. The figure was obtained using the online map tool available at www.lion1906.com/Pages/francais/utile/outils.html# associated with livestock, sporadic cases, and outbreaks in equine hospitals, colonization of horses and associated personnel have been reported in Europe [6,11,18,19]. Recently, a suspected transmission of MRSA ST398 from a horse to a Dutch girl, which resulted in a foot infection, has been reported [20].
Among other STs, ST612, which belong to the CC8, was recently described in horses and seems to be strongly associated with equine practice veterinarians [21] while ST5, described in pets, swine and poultry [6], was recently reported in horses in Japan [22]. Finally, some authors have recently characterized in animal MRSA isolates belonging to CC130 (ST1245), the most prevalent CC among mecC-positive strains [7,23,24].

Conclusions
In conclusion, this first epidemiological survey conducted in France has shown an increase in the prevalence of MRSA isolates associated with horse infections since 2010 in stud farms, which is in part related to the emergence of  clonally-related ST398 MRSA isolates. Since this new ST398 type is known to cause outbreaks in horses and to colonize/infect humans, hygiene measures and appropriate antimicrobial use should be maintained and reinforced in order to limit the transmission of S. aureus between horses as well as between horses and humans.

Methods
Bacterial isolates and antimicrobial susceptibility testing The regional veterinary laboratory of Normandy (LABÉO) is specialized in the analysis of specimens from infected horses and receives clinical samples from numerous stud farms located in various regions of France. From January 2007 to December 2013, all non-duplicate clinical isolates of S. aureus were prospectively studied.
For molecular typing, four different techniques were used for all the strains. The MLST was performed as previously described [34] using the MLST database (http://saureus.mlst.net/). The spa typing was carried out as previously described [35] using the Ridom StaphType software (Ridom GmbH, Würzburg, Germany). The typing of staphylococcal cassette chromosome mec element (SCCmec) was performed according to mec and ccr complexes previously defined [36]. Genetic relatedness was determined by rep-PCR using the semi-automated Diversilab system (bioMérieux, Marcy l'étoile, France) [14,37].

Statistical analysis
The Fisher's exact test was used to compare categorical variables. P values <0.05 were considered to be statistically significant. All tests were 2 tailed. Statistical tests were performed using GraphPad Prism, version 6.