Cloning and Occurrence of czrC,a Gene Conferring Cadmium and Zinc Resistance in Methicillin-Resistant Staphylococcus aureus CC398 Isolates

We recently reported a phenotypic association between reduced susceptibility to zinc and methicillin resistance in Staphylococcus aureus CC398 isolates from Danish swine (F. M. Aarestrup, L. M. Cavaco, and H. Hasman, Vet. Microbiol. 142:455-457, 2009). The aim of this study was to identify the genetic determinant causing zinc resistance in CC398 and examine its prevalence in isolates of animal and human origin. Based on the sequence of the staphylococcal cassette chromosome mec (SCCmec) element from methicillin-resistant S. aureus (MRSA) CC398 strain SO385, a putative metal resistance gene was identified in strain 171 and cloned in S. aureus RN4220. Furthermore, 81 MRSA and 48 methicillin-susceptible S. aureus (MSSA) strains, isolated from pigs (31 and 28) and from humans (50 and 20) in Denmark, were tested for susceptibility to zinc chloride and for the presence of a putative resistance determinant, czrC, by PCR. The cloning of czrC confirmed that the zinc chloride and cadmium acetate MICs for isogenic constructs carrying this gene were increased compared to those for S. aureus RN4220. No difference in susceptibility to sodium arsenate, copper sulfate, or silver nitrate was observed. Seventy-four percent (n = 23) of the animal isolates and 48% (n = 24) of the human MRSA isolates of CC398 were resistant to zinc chloride and positive for czrC. All 48 MSSA strains from both human and pig origins were found to be susceptible to zinc chloride and negative for czrC. Our findings showed that czrC is encoding zinc and cadmium resistance in CC398 MRSA isolates, and that it is widespread both in humans and animals. Thus, resistance to heavy metals such as zinc and cadmium may play a role in the coselection of methicillin resistance in S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) isolates mainly belonging to CC (clonal complex) 398 have emerged recently in livestock production around the world (7, 10, 20, 22). This clonal lineage is able to persist in the farm environment and therefore constitutes a large reservoir for transmission to humans working in such environments, which raises occupational health concerns (14, 22, 27). Resistance to antimicrobials, as well as other factors, may have contributed to the success of MRSA CC398 and its emergence in the animal reservoirs (1).Metal-containing compounds are widely used as a feed supplement or for the prevention of gastrointestinal diseases in food animals. Resistance to metals is widely disseminated among bacterial species, and we have shown previously that the use of copper in feed can coselect for resistance to macrolides and glycopeptides in Enterococcus faecium (5). Heavy-metal resistance, i.e., resistance to arsenic, mercury, and cadmium, has been described among S. aureus isolates, including MRSA (2, 17-19). We recently described a phenotypic association between reduced susceptibility to zinc and MRSA CC398 from pigs in Denmark (1). It was suspected that the genetic background for the metal resistance was genetically linked to the methicillin resistance, and it was hypothesized to be present in the staphylococcal cassette chromosome mec (SCCmec) element carried by these MRSA isolates, as metal resistance genes had been described previously in the SCCmec element type III of MRSA isolate ST239 (9).The purpose of this study was to identify the genetic determinant(s) involved in the observed zinc resistance phenotype among the CC398 MRSA isolates from animals in Denmark and to investigate its effects on susceptibility to several metallic compounds. Furthermore, the prevalence of resistance to zinc and the putative resistance gene were determined among a larger collection of swine and human S. aureus CC398 isolates from Denmark.