Veterinary Microbiology 158 (2012) 123–128 Contents lists available at SciVerse ScienceDirect Veterinary Microbiology jo urnal homepage: www.elsevier.com/locate/vetmic Species and staphylococcal cassette chromosome mec (SCCmec) diversity among methicillin-resistant non-Staphylococcus aureus staphylococci isolated from pigs a,b, a,b,c a,b a Wannes Vanderhaeghen *, Stien Vandendriessche , Florence Crombe´ , Marc Dispas , c b b a,b Olivier Denis , Katleen Hermans , Freddy Haesebrouck , Patrick Butaye a Department of General Bacteriology, Veterinary and Agrochemical Research Centre, Groeselenbergstraat 99, B-1180 Ukkel, Belgium b Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium c Laboratoire de Re´fe´rence MRSA-Staphylocoques, Department of Microbiology, Hoˆpital Erasme, Universite´ Libre de Bruxelles, 1070 Anderlecht, Belgium A R T I C L E I N F O A B S T R A C T While methicillin-resistant Staphylococcus aureus (MRSA) ST398 is known to be Article history: Received 6 September 2011 widespread in pig farms, few studies have investigated the species diversity and SCCmec Received in revised form 16 January 2012 types of methicillin-resistant non-S. aureus staphylococci (MRNAS) residing in the nose of Accepted 19 January 2012 pigs. We examined nasal swab samples of 200 pigs originating from 10 Belgian pig farms previously found positive for MRSA ST398. Suspected staphylococcal isolates were Keywords: subjected to a 16S rRNA-mecA-nuc PCR. Confirmed MRNAS were genotypically identified Coagulase-negative staphylococci to the species level and investigated with a SCCmec typing PCR. MRNAS (n = 72) were Methicillin resistance detected on all 10 farms and were carried by 29.5% of the pigs. Seven MRNAS species were Pigs found: Staphylococcus epidermidis (38.9%), Staphylococcus sciuri (18.1%), Staphylococcus Reservoir SCCmec pasteuri (18.1%), Staphylococcus rostri (12.5%), Staphylococcus warneri (8.3%), Staphylo- coccus haemolyticus (2.7%) and Staphylococcus hominis (1.4%). SCCmec cassettes were of Methicillin-resistant Staphylococcus epider- midis type IVa (29.2%), type IVc (25%), type III (22.2%), type V (5.6%) or could not be assigned to any of the known types (NT types) (18.1%). Five distinct NT types were found. The predominance of methicillin-resistant S. epidermidis (MRSE) in our samples is remarkable, as MRSE is mainly associated with humans. The finding of three different SCCmec elements (IVa, V, NT type 1) in MRNAS that also prevail or predominate in MRSA ST398 shows that MRNAS might be an important SCCmec reservoir for MRSA in pigs. Yet, the occurrence of multiple other SCCmec types illustrates that further studies are required to understand the presence and spread of SCCmec in methicillin-resistant staphylococci from animals. ß 2012 Elsevier B.V. All rights reserved. 1. Introduction some mec (SCCmec). Based on structural composition, 11 different types (I–XI) and numerous subtypes of SCCmec Methicillin-resistant Staphylococcus aureus (MRSA) have been recognized in MRSA so far (website of the originates from methicillin-susceptible S. aureus (MSSA) International Working Group on the Classification of by acquisition of mecA, a gene carried within a mobile Staphylococcal Cassette Chromosome Elements, IWG- genetic element called staphylococcal cassette chromo- SCC; www.sccmec.org). In non-S. aureus staphylococci (NAS), mecA/SCCmec has been demonstrated to prevail as well (Ibrahem et al., 2009). In human clinical samples, the * Corresponding author at: Department of General Bacteriology, presence of methicillin-resistant NAS (MRNAS) has been Veterinary and Agrochemical Research Centre, Groeselenbergstraat 99, well-studied, and the MRNAS species most frequently B-1180 Ukkel, Belgium. Tel.: +32 2 379 04 35; fax: +32 2 379 06 70. encountered are methicillin-resistant Staphylococcus epi- E-mail addresses: [email protected], dermidis (MRSE) and methicillin-resistant Staphylococcus [email protected] (W. Vanderhaeghen). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.01.020 124 W. Vanderhaeghen et al. / Veterinary Microbiology 158 (2012) 123–128 haemolyticus (Petinaki et al., 2001; Ibrahem et al., 2009). In colony of each different suspected morphology was selected veterinary medicine, MRNAS have mostly been studied as and purified on Columbia agar with 5% sheep blood (Bio- pathogens in ruminant mastitis (Rajala-Schultz et al., Rad, Belgium). Isolates were grown for 48–72 h at 37 8C 2009). Only a few studies also investigated carriage of followed by another 24–48 h at room temperature, to be MRNAS in livestock (Bagcigil et al., 2007; Zhang et al., able to fully judge their morphotype (Kloos and Bannerman, 2009; Huber et al., 2011) and companion animals (van 1994). Afterwards, for each sample, the purified isolates Duijkeren et al., 2004; Bagcigil et al., 2007). However, little from the four different agar plates were compared. Isolates is yet known on the carriage of MRNAS in pigs or on the showing identical morphotypes were judged as being MRNAS species involved. identical to each other (Kloos and Bannerman, 1994) and It has been suggested that MRNAS function as a mecA/ only one such isolate was further included in the study. If SCCmec reservoir associated with the formation of MRSA identical morphotypes were found on (one of) the agar from MSSA (Barbier et al., 2010; Bloemendaal et al., 2010). plates with cefoxitin (ChromID MRSA, blood agar + cefox- Although this has mainly been studied in human medicine, itin) and (one of) the plates without cefoxitin (ChromID S. it could also be relevant for animals. Indeed, in the past aureus, colistin-aztreonam agar), an isolate originating from years, the emergence of a specific MRSA lineage, sequence (one of) the cefoxitin-containing agar plates was selected. type (ST) 398, was reported in domestic animals (Voss Hereafter, an ‘isolate’ refers to a pure culture showing a et al., 2005; Van den Eede et al., 2009; Vanderhaeghen colony morphotype unique for a given sample. Isolates et al., 2010b). While MRSA ST398 strains have been shown were kept at À80 8C in 50% glycerol until further use. to carry predominantly SCCmec cassettes of type IV(a) and V (Vanderhaeghen et al., 2010b), little is known on the 2.3. Detection of mecA and identification of MRNAS types of SCCmec elements that are present in MRNAS from animals. From all isolates, DNA was extracted as previously The objectives of this study were to assess the carriage described (Vanderhaeghen et al., 2010a). Then, all isolates and species distribution of MRNAS on pig farms previously were examined with a 16S rRNA-mecA-nuc triplex PCR found positive for MRSA ST398 and to investigate the (Maes et al., 2002). MRNAS isolates, recognized as showing SCCmec elements that they possess. amplification of 16S rRNA and mecA, were further identified to the species level using tRNA intergenic spacer PCR combined with capillary gel electrophoresis (Baele 2. Materials and methods et al., 2000). It was taken into consideration that certain staphylococcal species, such as S. simulans and Staphylo- 2.1. Sampling coccus sciuri, give poor or no amplification of 16S rRNA in Ten Belgian pig farms were visited between August and the triplex PCR (Maes et al., 2002); therefore, also isolates December 2009. Farms were selected based on the that were only mecA positive in the triplex PCR but had a presence of MRSA ST398 positive pigs as determined in clear staphylococcal morphology were included in a tRNA a 2007 survey (Crombe´ et al., 2011). Four farms harboured identification assay. only fattening pigs, two farms were breeding farms, with In case tRNA intergenic spacer PCR was not sufficient to sows and piglets, and four farms were farrow-to-finish identify an isolate, rpoB sequencing was performed, with farms, with sows, piglets and fattening pigs. On each farm, the primers and conditions reported by Drancourt and a convenience sample of ten pigs of each represented age Raoult (2002). An isolate was identified when there was group was sampled, resulting in a total of 200 pigs (60 98% or more sequence similarity with GenBank sequences sows, 60 piglets and 80 fattening pigs). All pigs were of one species. healthy at the moment of sampling. A dry cotton swab was used to collect a sample from 2.4. Cefoxitin disk diffusion testing both nostrils of each animal. Samples were stored in Stuart transporter medium (Meus s.r.l., Italy) for transport to the The correspondence between presence of mecA and laboratory, and laboratory processing of the samples was phenotypic cefoxitin resistance was investigated for all commenced three to six hours after sampling. MRNAS isolates with the disk diffusion test, using 30 mg cefoxitin disks (Rosco Diagnostics, Denmark) and follow- 2.2. Bacterial isolates ing CLSI recommendations M31-A3 (Clinical and Labora- tory Standards Institute, 2008). S. aureus strains ATCC Each sample was grown overnight at 37 8C in 7.5% NaCl 25923 and ATCC 43300 were used as internal quality Brain Heart Infusion (BHI) broth (Becton Dickinson, US) controls. The strength of the correlation was assessed by and then subcultured on four different agars: ChromID estimating the occurrence of resistance to cefoxitin in the MRSA agar (BioMe´rieux, France), Columbia Agar (Oxoid, mecA-positive population, using logistic regression analy- Germany) supplemented with 5% sheep blood (BioMe´r- sis with significance level of 5%. ieux, France) and 3.5 mg/l cefoxitin (Sigma-Aldrich, US), ChromID S. aureus agar (BioMe´rieux, France), and Colum- 2.5. Determination of oxacillin and cefoxitin minimum bia colistin-aztreonam agar with 5% sheep blood (Oxoid, inhibitory concentration (MIC) France). Plates were incubated at 37 8C. After 24–36 h, each of the four plates was examined for colonies showing a For those MRNAS isolates that had discordant results staphylococcal morphology; per plate, one representative for mecA PCR and cefoxitin resistance in the disk diffusion W.