Skin Lesion by Methicillin-Resistant Staphylococcus aureus ST398-t1451 in a Spanish Pig Farmer: Possible Transmission from Animals to Humans

Introduction

M ethicillin-resistant Staphylococcus aureus (MRSA) is a global problem that affects both hospitalized and community population. In the last years, a livestock-associated clonal lineage of MRSA, of sequence-type ST398, has emerged first in animals, and later either colonizing or causing infections in humans, especially in people in contact with pigs (van Loo et al. 2007, Lewis et al. 2008). Since its first detection in 2005 (Voss et al. 2005), the number of human infections caused by MRSA ST398 has been increasing steadily, being associated in most cases to skin and soft-tissue infections, although serious infections by this microorganism have been also reported (Witte et al. 2007, Lewis et al. 2008, Mammina et al. 2010, Lozano et al. 2011). The aim of this study was to perform the genetic characterization of the MRSA strains recovered from a patient with folliculitis, from his brother, and from pigs of the farm where both individuals worked, to determine the possible MRSA transmission from animals to humans.

Materials and Methods

A 30-year-old patient, who worked in a pig-farm in Spain, presented a severe folliculitis on his face. He had been previously treated with tetracycline due to an adverse evolution of acne. Samples were taken from the lesions of the patient (March 4th, 2010) by skin scraping and by follicle puncturing and they were cultured on oxacillin-resistant S. aureus agar media (Oxoid), colistin nalidixic acid (bioMérieux), and blood agar media (Oxoid). The presence of MRSA was confirmed in the samples by classical microbiological methods as well as by detection of nuc and mecA genes by polymerase chain reaction (PCR) (Gómez-Sanz et al. 2010). In addition, a mycological study was performed with the patient samples. New samples of the patient (from lesions and nasal swabs) and nasal swabs of his brother (a healthy person who worked in the same pig-farm) and of his parents (who did not work in the farm) were obtained 1 week later (March 11th, 2010), and were cultured as previously indicated. One isolate per positive sample was further characterized. Topical and oral treatments of the patient with fusidic acid and nasal treatment with mupirocin were administered.

Four weeks later, the patient's clinical situation improved and new nasal swabs and samples from skin (by skin scraping) were collected (April 8th, 2010). Nasal samples from nine pigs of the farm were randomly screened (March 11th, 2010) and tested for MRSA recovery. Animal nasal swabs were inoculated into brain heart infusion broth (Difco) containing 6.5% NaCl and incubated at 35°C for 24 h. Then, 100 μL was seeded on oxacillin-resistance screening agar base plates (Oxoid) with oxacillin (2 mg/L), and incubated at 35°C for 36 h. One MRSA colony per sample was selected and identified by biochemical and molecular methods (Gómez-Sanz et al. 2010).

All human and animal MRSA strains were tested for susceptibility to 20 antimicrobial agents (penicillin, oxacillin, cefoxitin, erythromycin, clindamycin, quinupristin-dalfopristin, gentamicin, tobramycin, tetracycline, ciprofloxacin, levofloxacin, cotrimoxazole, vancomycin, teicoplanin, mupirocin, linezolid, fosfomycin, nitrofurantoin, fusidic acid, and rifampin) by using the VITEK-2 system (bioMérieux), except for kanamycin for which the disk-diffusion method was used (CLSI 2008).

The presence of genes that confer resistance to beta-lactams (mecA), tetracycline (tetK, tetL, tetM, and tetO), and macrolide/lincosamides (ermA, ermB, ermC, and msrA) was analyzed by PCR (Gómez-Sanz et al. 2010). All MRSA strains were characterized by multilocus-sequence-typing, pulsed-field gel electrophoresis (PFGE, with ApaI enzyme), determination of SCCmec-type, agr-type, and spa-type. Presence of the genetic determinants of Panton-Valentine-Leukocidin (lukS/lukF), Toxic-Shock-Syndrome-Toxin 1 (tst), and Exfoliative-Toxin A (eta), B (etb), and D (etd) was analyzed by PCR (Gómez-Sanz et al. 2010).

Results

Gram-positive cocci in the presence of polymorphonuclear leukocytes were detected in samples of skin lesions of the patient that were taking in March 4th and 11th, and they were identified as MRSA. The mycological study of these samples was negative. MRSA strains were also obtained from nasal samples of the patient and of his brother but not from those of their parents (March 11th). Four MRSA strains were kept for further study: two strains from positive skin samples of the patient (obtained in March 4th and 11th), one from the nasal sample of the patient and the other from the nasal sample of his brother.

No MRSA strains were recovered from skin lesion or nasal samples of the patient after treatment with mupirocin and fusidic acid (April 8th).

In addition, MRSA were obtained from nasal samples of the nine tested pigs and one strain per animal was further studied. All four human and nine animal MRSA strains presented identical phenotypic and genotypic characteristics. All strains showed resistance to erythromycin, clindamycin, and tetracycline and diminished susceptibility to levofloxacin; they showed susceptibility to the other tested antimicrobials. The mecA, tetK, tetM, and ermC resistance genes were detected in all MRSA strains.

The 13 MRSA strains of human and animal origin were typed as ST398, spa-type t1451, SCCmec-type V, and agr-type I. They were negative for all tested toxin genes (lukS/lukF, tst, eta, etb, and etd). Moreover, all MRSA isolates were analyzed by PFGE using ApaI restriction enzyme and all of them showed closely related patterns, with five subtypes (A1–A5) that differed in only one to three bands (Fig. 1).

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FIG. 1.

Pulsed-field gel electrophoresis patterns of chromosomal DNA digested with ApaI enzyme of methicillin-resistant Staphylococcus aureus isolates recovered from humans and animals in this study. M, lambda pulse marker; lanes 1 and 2, isolates from the skin lesions in two samples taken with 1 week of difference (subtype A1); lane 3, isolate from the nasal sample of the patient (subtype A2); lane 4, isolate from the nasal sample of the brother (subtype A3); lanes 5–8 and 13, isolates from the nasal samples of the pigs (subtype A4); lanes 9–12, isolates from the nasal samples of the pigs (subtype A5).

Discussion

The phenotypic and genotypic similarity of the MRSA strains of human and animal origin detected in this study indicates the wide dissemination of this clone in the studied pig-farm given that all nine tested pigs and two workers were colonized by the same MRSA clonal lineage. MRSA ST398-t1451 lineage had been previously detected in samples from pigs or from people in contact with animals (EFSA 2009, Köck et al. 2009, Lozano et al. 2011). The spa-types most frequently identified among MRSA ST398 had been t011, t034, and t108 (Witte et al. 2007, van Belkum et al. 2008), although it seems that other spa-types may be spreading (EFSA 2009, Gómez-Sanz et al. 2010). Although the spa-type t1451 has been detected in some patients (Köck et al. 2009) it does not seem to be more virulent than other ST398 strains since we have not detected lukS/lukF, tst, eta, etb, and etd toxin genes among isolates of this lineage. Moreover, the brother of the patient was positive for MRSA ST398-t1451, but did not show any infection.

The acne is a multifactorial skin disease and sometimes the antimicrobial therapy is not enough for the improvement of the patient. In this case the clinical picture was notably improved with the therapy chosen: fusidic acid (topical and oral) and decolonization with nasal mupirocin. Notably, the patient had been previously treated with tetracycline when a severe folliculitis on his face was detected. It is possible that another microorganism could have caused the infection, after which a superinfection by MRSA ST398 t1451 happened, being stimulated by the tetracycline treatment. The MRSA strains isolated from the lesions after this treatment showed resistance to this antibiotic. The strains presented two different tetracycline resistance genes, tetK, and tetM. The tetK gene confers resistance to tetracycline but not to minocycline; tetM gene confers resistance to all antibiotics of the tetracycline group (Schmitz et al. 2001). The combination of these genes has been previously reported and has been shown to be more commonly found in MRSA strains than in methicillin-resistent s. aureus strains (Schmitz et al. 2001).

All our MRSA strains were resistant to macrolides and clindamycin and they harbored the ermC gene. The ermA and ermC genes are the most prevalent MLS_B resistance genes detected in S. aureus. High prevalence of ermC gene among MRSA ST398 has also been found in other studies (Denis et al. 2009).

In summary, MRSA ST398-t1451 has been associated with severe folliculitis lesions, and strains with identical phenotypic and genotypic characteristics and with closely related PFGE profiles have been identified in nasal swabs of the patient and of his brother, and in nasal swabs of all the studied pigs of the farm. These results suggest the animal-to-human transmission of MRSA ST398 strains and indicate the capacity of spread of this microorganism among individuals with close contact with these MRSA-colonized animals.