Methicillin-resistant Staphylococcus aureus isolated from dogs and cats in Switzerland

Twenty-two methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from various infected locations in domestic cats and dogs between June 2008 and September 2014 were analyzed for their genotype, genetic fingerprint, virulence and antibiotic resistance profile. Eighteen strains belonged to the clonal complex (CC) 22 [ST22(MLST)-A(PFGE)-t032(spa)-IV(SCCmec) and ST22-A-t1214-IV], 2 strains to the livestock-associated MRSA ST398-t011-IV and two were individual strains of ST5-t002-II and ST1-t001-IV. They contained virulence factors such as γ-hemolysins, β-hemolysin converting phage genes, leukocidins and enterotoxins. Most widespread resistances were observed against β-lactams, trimethoprim and fluoroquinolones, but single strains also exhibited resistance to macrolides, lincosamides, aminoglycosides, tetracycline, chloramphenicol and/or mupirocin. The predominant presence of CC22 MRSA strongly indicates clonal spread of a human associated lineage in Swiss companion animals. It is therefore of public health importance to maintain a low level of MRSA infections in animals to avoid uncontrolled dissemination of MRSA clones in humans and animals.


Methicillin-resistente Staphylococcus aureus Stämme isoliert aus Hunden und Katzen in der Schweiz
Antibiotic Resistance (ZOBA) of the Institute of Veterinary Bacteriology, University of Bern, for confirmation and strain collection establishment.These strains were further characterized to determine which types of MRSA are associated with infections in Swiss pets and determine their virulence and resistance profile.

Sample collection, species identification and growth conditions
A total of 22 MRSA strains were obtained from routine diagnostic procedures from the ZOBA, University of Bern, the IDEXX Diavet Laboratory, Bäch, Switzerland and the Laboratory Laupeneck AG, Bern, Switzerland.

Antimicrobial susceptibility testing and virulence profile determination Introduction
Staphylococcus aureus can cause a wide range of purulent and toxic infections in humans and animals (Lowy, 1998).Methicillin-resistant Staphylococcus aureus (MRSA) are characterized by the acquisition of staphylococcal cassette chromosome mec (SCCmec) element carrying the mecA gene, a penicillin-binding protein conferring resistance to practically all β-lactams with the exception of ceftaroline (Chambers, 1997;Steed and Rybak, 2010).In addition, MRSA often carries further antibiotic traits which are limiting options for therapy (de Lencastre et al., 2007).During the last decades, MRSA infections have not only been relevant in humans, but also increased in companion animals (David and Daum, 2010;Weese and van Duijkeren, 2010;Vincze et al., 2014b).The most commonly reported MRSA infections in dogs and cats are associated with wounds or surgical sites, the skin, ears and the urinary tract (Weese and van Duijkeren, 2010).Additionally, healthy companion animals can be colonized with MRSA, but prevalence of colonization is significantly lower than in the human population and of transient nature (Weese, 2010).Molecular typing and whole-genome sequencing showed that MRSA strains isolated from household pets are identical to types spreading in the regional human population, suggesting that pets acquire MRSA through their colonized owner (Baptiste et al., 2005;O'Mahony et al., 2005;Moodley et al., 2006;Strommenger et al., 2006;Weese et al., 2006;Loeffler et al., 2013).For instance, clones belonging to the clonal complexes (CC) CC5, CC8, and CC22 are the most frequently isolated strains from canine and feline MRSA colonization and infection in Europe (van Duijkeren et al., 2004;Loeffler et al., 2005;Moodley et al., 2006;Strommenger et al., 2006;Coelho et al., 2011;Monecke et al., 2011;Vincze et al., 2014b).Those MRSA of CC5, CC8, and CC22 belong to the so called hospital-acquired (HA-MRSA) and community-acquired (CA-MRSA) lineages which spread in human health care settings or communities (Monecke et al., 2011).CA-MRSA have emerged since the mid-1990s and are frequently associated with the acquired Panton-Valentine leucocidin (PVL), a cytotoxin which causes severe skin and soft tissue damages (David and Daum, 2010).CC398 or livestock-associated MRSA (LA-MRSA) which is mainly associated with pigs, horses, farm personnel and veterinarians has also been reported to cause infection in companion animals, but less frequently (Nienhoff et al., 2009;Monecke et al., 2011;Sieber et al., 2011;Haenni et al., 2012;Wettstein et al., 2014;Lekkerkerk et al., 2015).
Since 2008, some of the MRSA isolated from companion animals in Switzerland have been referred to the Centre for Zoonoses, Bacterial Animal Diseases and Band 158, Heft 6, Juni 2016, 443-450, © GST | SVS  Table 1: Schwendener et al., 2011).Antibiotic resistance genes were detected with a custom-made microarray (AMR+ ve-5 array tubes; Alere GmbH, Jena, Germany) (Strauss et al., 2015).Mutations in the quinolone resistance determining region (QRDR) of the GyrA and GrlA topoisomerase subunits, as well as in the mupirocin resistance region of the isoleucyl-tRNA synthetase were identified by amino acid sequence analysis of translated PCR products as described previously (Schmitz et al., 1998;Fujimura et al., 2003).Mutations affecting trimethoprim susceptibility of S. aureus were detected by amino acid sequence analysis obtained from the entire dihydrofolate reductase gene of S. aureus amplified by PCR using primers dhfr-F (5'-AGGAATTACATGAATGTTGT-TTGCTTC) and dhfr-R (5'-GCAAAATCATTATTTC-TATCACACTTTATG) (Vickers et al., 2009).

Results
MRSA were isolated from surgical intervention sites, skin infections/purulences or the urinary and pulmonary tract of dogs and cats from different veterinary clinics in Switzerland (Fig. 1).Four different clonal complexes were found (CC1, CC5, CC22, CC398).MRSA of CC22 were the most frequent, with 18 strains detected in 6 different veterinary clinics (Fig. 1).All the strains belonging to CC22 also displayed a similar PFGE pattern gathering into PFGE cluster A (Fig. 1).Fourteen strains even exhibited the exact same PFGE profile.Differences in the PFGE profiles of the remaining 4 CC22 strains KM1224/08, IMD1271/11, KM894/09 and KM1341/12 resulted from variations in 2 to 4 fragments in the PFGE pattern (Fig. 1).The strains also diverged from those of the larger CC22 cluster by containing less or additional virulence or resistance genes (Tab.1).Otherwise, all CC22 strains contained the virulence factors hlgA, hlgB (lukF), hlgC (lukS), which are γ-hemolysin associated genes, the enterotoxin genes seg, sei, selm, seln, selo and selu, as well as the β-hemolysin converting phage genes sak, chp and scn.Furthermore, this larger group of CC22 strains exhibited the same resistance pattern being resistant to the β-lactams penicillin [mecA, blaZ], oxacillin and cefoxitin [mecA], to trimethoprim (amino acid substitution F99K in the dihydrofolatreductase) and to fluoroquinolones [amino acid substitutions in the gyrase subunit A GyrA (S84L) and in the DNA topoisomerase IV GrlA (S80F)].Other less frequent clonal lineages of MRSA consisted of single strains of CC1 and CC5 MRSA, as well as two strains belonging to MRSA of CC398.These groups of strains also exhibited a specific PFGE pattern (Fig. 1).The two strains belonging to the CC1 and CC5 contained additional enterotoxins (sed, sej and ser) and/or leukocidin genes (lukD, lukE) compared to those from CC22, as well as additional resistance genes.The additional resistances of the CC5 strain (IMD39/12) were associated with chloramphenicol and kanamycin resistance genes, as well as with mutations causing mupirocin resistance.Those of the CC1 strain (KM1460/14) were associated with genes conferring resistance to tetracycline, gentamicin, erythromycin, clindamycin and kanamycin (Tab.1).The livestock-associated ST398-t011-IV MRSA strains contained only γ-hemolysin associated genes, but exhibited additional resistance such as to aminoglyco-sides, macrolides, lincosamides and tetracyclines.Overall, the analyzed CC1, CC5 and CC398 MRSA strains were more resistant to antibiotics than the CC22 MRSA (Tab.1).All the analyzed strains were Panton-Valentine leucocidin (PVL) negative.

Discussion
Molecular typing of MRSA recovered from infection sites of Swiss companion animals identified clonal lineages similar to those associated with MRSA colonization and infection in companion animals in Europe.CC22 MRSA is the most frequent clonal lineage causing infection in companion animals in Switzerland and among the most predominant lineages in pets from Germany, Portugal, Ireland, the UK and Sweden (van Duijkeren et al., 2004;Loeffler et al., 2005;Moodley et al., 2006;Coelho et al., 2011;Grönlund Andersson et al., 2014;Vincze et al., 2014b ).CC22 MRSA belong to HA-MRSA pandemic in humans all over the world and the specific ST22-IV MRSA clone is the second most isolated lineage reported in human hospitals in Switzerland (Monecke et al., 2011;Senn et al., 2013).Pets may acquire such HA-MRSA or CA-MRSA through their owners or caretakers, but colonization is usually of transient nature (Weese, 2010;Weese and van Duijkeren, 2010;Loeffler et al., 2013).In cases of surgical interventions, accidental wounding or skin diseases, this transient colonization period highly increases the risk of infection development in animals.Furthermore, isolation of a CC22 clone with the exact same fingerprint, virulence and resistance profile over a long time period in veterinary clinics and practices of different regions in Switzerland suggests that these CC22 clones can persist and disseminate in the local veterinary environment.Admission to veterinary clinics, especially if they have more than 10 employees, antimicrobial courses and having received surgical implants, are among the reported risk factors of MRSA colonization and infection development (Loeffler and Lloyd, 2010;Soares Magalhaes et al., 2010;Vincze et al., 2014a).Whether animal owners or the veterinary setting play a role as a source for MRSA infection in dogs and cats in Switzerland was not investigated in our study.However, a study conducted in 2012 on the prevalence of MRSA in Swiss veterinarians revealed that small animal practitioners were colonized with MRSA clones CC398, CC5, CC8 and CC88 lineages, but no CC22 clones were detected at this time (Wettstein et al., 2014).The two CC398 LA-MRSA from pets in this study exhibited the exact same spa-type, resistance gene pattern and virulence gene pattern as the MRSA ST398-t011-IV lineages previously isolated from Swiss large animal veterinarians, horse clinic personnel and horses indicating the broad potential for dissemination of this clonal lineage in the veterinary setting (Huber et al., 2010;Sieber et al., 2011;Wettstein et al., 2014).ST5-t002-II MRSA (IMD39/12) of which we only had a single isolate was already detected in Swiss small animal practitioners in 2012 and is a worldwide pandemic clone and the most prevalent clone in Swiss hospitals (Monecke et al., 2011;Senn et al., 2013;Wettstein et al., 2014).The presence of human MRSA clones like those of CC22, CC5 and CC1 in small companion animals is of public health concern and emphasizes the need for infection control in both human and veterinary settings.

Conclusion
This study shows that Swiss cats and dogs may get infected by MRSA which are resistant to clinically impor-tant antibiotics and belong to known clonal lineages with large potential for nosocomial dissemination in both animal and human hospitals.It is therefore necessary to maintain a low level of MRSA infection in companion animals keeping continuous and strict infection control strategies following guidelines for prudent use of antimicrobials, such as the guidelines recently released by the European Union (European Commission, 2015).