Genetic Lineages,Antimicrobial Resistance,and Virulence in Staphylococcus aureus of Meat Samples in Spain: Analysis of Immune Evasion Cluster (IEC) Genes

Introduction

S taphylococcus aureus is a cause of food poisoning due to its capacity to produce enterotoxins that can contaminate food products (Argudín et al., 2010), and its surveillance is an issue of special growing interest (Wendlandt et al., 2013). Methicillin-resistant S. aureus (MRSA) of the sequence type ST398 is now a global problem due, in part, to its great ubiquity in different environments (Wendlandt et al., 2013). MRSA ST398 was initially detected in farm animals, especially in pigs, and was considered LA-MRSA (livestock-associated MRSA); nevertheless, MRSA ST398 has been increasingly detected in other animals and in humans, either as colonizer or causing infections (Lozano et al., 2011), as well as in food products (Wendlandt et al., 2013).

In recent years, there has been special interest to know the genetic lineages of S. aureus that are circulating in different environments in order to track its evolution in the different niches. Genetic lineages adapted to humans frequently contain the genes of the immune evasion cluster (IEC) system; however these genes are generally nondetected among lineages mostly adapted to animals. Each S. aureus lineage seems to evade the host immune responses differently, but they can adapt to new host environments by the acquisition of the IEC genes (McCarthy and Lindsay, 2013). The IEC system contains the scn, chp, sak, and sea or sep genes, showing different functions to achieve the survival of S. aureus within human innate defenses (van Wamel et al., 2006).

A previous study performed in our group in 2007–2009 detected MRSA in 1.6% of food samples in La Rioja (Northern Spain), but the prevalence of methicillin-susceptible S. aureus (MSSA) or the presence of virulence or IEC genes was not evaluated (Lozano et al., 2009). The aim of the present study was to analyze the rate of meat contamination by MRSA and/or MSSA in food samples from La Rioja during 2011–2012, as well as to perform the molecular characterization of detected isolates in order to check the circulating lineages and their possible human or animal origins.

Materials and Methods

One hundred meat samples (pork, 34; chicken, 31; turkey, 15; beef, 15; and lamb, 5) were collected in large supermarkets (47 samples) or small butcheries (53 samples) in the La Rioja region. Samples (0.3 g) were suspended in 3 mL of saline solution (0.9%) and 100 μL were inoculated in brain-heart-infusion (BHI) broth (Becton-Dickinson, France) containing 6.5% NaCl, and incubated at 37°C for 24–36 h. Then, 200 μL of the suspension was seeded on mannitol–salt–agar plates (Becton-Dickinson) and ORSAB plates (Oxoid, England) supplemented with 2 mg/L oxacillin to recover S. aureus and MRSA, respectively. Both types of plates were incubated at 37°C for 24–48 h. Two presumptive S. aureus or MRSA colonies per sample were selected and identified by DNase and by multiplex polymerase chain reaction (PCR) of nuc and mecA genes (Benito et al., 2013), although only one isolate/sample was further studied when a similar resistance profile was detected.

Recovered S. aureus isolates (both MRSA and MSSA) were typed by (1) spa (www.ridom.com), (2) agr, (3) multilocus sequence typing (saureus.mlst.net), and (4) staphylococcal-cassette-chromosome mec (SCCmec) (Lozano et al., 2011). Resistance phenotype to 17 antibiotics was determined by the disk-diffusion method (CLSI, 2012; CA-SFM, 2011), and the presence of 21 resistance genes was analyzed by PCR (Benito et al., 2013). Moreover, 14 virulence genes (including lukF/lukS-PV, tsst-1, eta, etb, lukD-lukE, lukM, aur, bap, cna, hla, hlb, hld, hlg, and hlgv) and 18 staphylococcal enterotoxins genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, and seu) were tested by multiplex PCR (Jarraud et al., 2001; Hwang et al., 2007). The five genes of the IEC cluster (scn, chp, sak, sea, and sep) were studied by PCR (Van Wamel et al., 2006). Positive and negative controls for each PCR were used.

Results and Discussion

S. aureus was detected in 7 of the 100 meat samples collected (7%), of which 6 samples contained MSSA and only 1 sample harbored MRSA (1%). Five of the contaminated samples were from small butcheries and two were obtained from large supermarkets. One isolate/positive-sample was further studied. Table 1 shows the characteristics of the seven S. aureus isolated in this study.

The MRSA strain was recovered from a pork meat sample, and was typed as SCCmec typeV, spa-type-t011, agr-I, and sequence-type ST398 (included in CC398 clonal complex). This strain exhibited a multidrug-resistance phenotype and genotype, lacked the egc-cluster genes, and was negative for IEC genes, presenting the typical LA-MRSA profile.

The 6 MSSA isolates were detected from pork (n=3) or chicken (n=3) samples. Three of them were typed as agr-type I, spa-type t015, and ST45/CC45, showed penicillin resistance, contained the egc-like cluster (seg, sei, sem, sen, seo, and seu) and the cna toxin gene, as well as the IEC system (type B). These ST45/CC45 isolates were recovered from two pork and one chicken samples from two different butcheries. The other three MSSA isolates were typed as ST5/CC5, ST109/CC9, and ST617/CC45, with different antibiotic-resistance profiles (Table 1). The strains CC5 and CC9 harbored all the enterotoxin genes included in the egc-cluster (seg, sei, sem, sen, and seo), and the IEC-type B system. Moreover, the strain typed as ST109/CC9 carried the eta gene, encoding exfoliatin A. The strain ST617/CC45 lacked egc-cluster, and presented the IEC type D. The presence of the egc cluster among our MSSA strains is relevant due to the potential role of egc-encoded toxins SEG and SEI in staphylococcal food poisoning (Jarraud et al., 2001; Argudín et al., 2010).

All S. aureus recovered in this study (both MRSA and MSSA) lacked the genes of Panton-Valentine leukocidin (lukF/lukS-PV) and of shock syndrome toxin 1.

The carriage rate of S. aureus and MRSA among the pork samples in our study was 11.7% and 2.9%, respectively. Regarding chicken samples, the carriage rate of S. aureus was 10%, while no MRSA-positive sample was identified. S. aureus was not recovered among the food samples of other origins tested. Other studies have shown higher S. aureus rates in meat samples (16%–45%) (Wendlandt et al., 2013). In the case of MRSA, percentages of MRSA-positive samples varied with the sample collections investigated in different countries (0%–37%) (Wendlandt et al., 2013; de Boer et al., 2009). The low rate of MRSA detected in meat samples in our study was similar to that found some years before in the same region (Lozano et al., 2009).

The presence of the genes of the IEC system in our MSSA strains belonging to CC5, CC9, and CC45 clonal complexes points to a food contamination by an undefined human source. Instead, our MRSA ST398 strain presented the typical characteristics of an animal-related lineage, as suggested by its broad antibiotic-resistance profile, including tetracycline (agent widely used in animal husbandry, especially in pigs), and also due to the absence of IEC genes. The IEC system could be a good marker to follow the S. aureus contamination source in food products.