Zinc-resistance gene czrC identified in methicillin-resistant Staphylococcus hyicus isolated from pigs with exudative epidermitis

Abstract

Methicillin-resistant Staphylococcus hyicus (MRSH) was investigated for czrC, a gene conferring zinc-resistance. The czrC gene was identified in 50% (14/28) of MRSH isolates, representing 14 pigs with exudative epidermitis from 8 farms. Newly weaned pigs, which are particularly susceptible to exudative epidermitis, are commonly fed high levels of zinc oxide.

Staphylococcus hyicus is the causative agent of mild to severe skin lesions in swine, a disease commonly known as greasy pig disease or exudative epidermitis. Infections due to S. hyicus often arise from wounds caused by fighting amongst pigs, particularly when pigs are first mixed together after weaning. The disease is common and generally of minor concern but severe outbreaks of the disease can result in significant morbidity and mortality (1).

Exudative epidermitis is typically treated with topical antimicrobials but multidrug-resistant S. hyicus has been identified (2), including methicillin-resistant strains possessing the mecA gene (3). The mecA gene resides within the staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element within the genome of methicillin-resistant staphylococci. The SCCmec has been described as a mobile “resistance island” due to the tendency for multiple antimicrobial-resistance genes to accumulate within this region (4). Previous research has shown that the SCCmec is capable of horizontal gene transfer between different staphylococci species (5,6).

One resistance gene of increasing interest in methicillin-resistant S. aureus (MRSA) of swine origin is the czrC gene, which confers zinc and cadmium resistance by a cellular mechanism that has not been characterized (7). The czrC gene is located within the SCCmec of MRSA (7), but it is unknown whether methicillin-resistant S. hyicus (MRSH) also carries this gene within its SCCmec. This is of concern because of the common use of high levels of zinc oxide in swine feed (≥ 2500 ppm) in some countries (8), leading to a potential cross-selection of methicillin-resistance through zinc oxide exposure in pigs. The objective of this study was to determine whether the czrC gene is found within the genome of MRSH isolated from pigs with exudative epidermitis.

Staphylococcus hyicus isolates were collected in a previous study of 186 pigs that had clinical signs of exudative epidermitis on 30 farms in southern Ontario (9). Briefly, 124 isolates of S. hyicus were screened for phenotypic β-lactam-resistance by disk diffusion and the mecA gene using PCR (3). Typing of SCCmec was completed on methicillin-resistant isolates as described by Zhang et al (10). Overall, 28 MRSH isolates were identified and SCCmec typing showed that 64% (18/28) of these isolates were SCCmec type V. The mecC gene was not detected in any of the S. hyicus isolates. The 28 isolates of MRSH were from 28 pigs divided across 15 farms (3). The mean age of the pigs carrying MRSH was 28 ± 3.4 d.

In the present investigation, the 28 isolates of MRSH were tested for the czrC gene using PCR amplification of genomic DNA. For each MRSH isolate, 2.0 μL of extracted DNA was used in combination with 12.5 μL of Taq Ready Mix KAPA 2G FHS (D-Mark Biosciences, Toronto, Ontario), 8.5 μL of DNase-free water, and 1.0 μL each of the forward and reverse primers, designed by Cavaco et al (7). Total reaction volume was 25 μL and final concentration of each primer was 400 nM. The temperatures for denaturation, annealing, and amplification were 94°C, 55°C, and 72°C, respectively. After 30 cycles of PCR amplification, gel electrophoresis was used to detect the amplified fragment (655 bp) of the czrC gene. Statistical analyses were completed in STATA 10.0 (Stata Corporation, College Station, Texas, USA), and P < 0.05 was considered statistically significant.

Fourteen (50%) pigs were colonized with MRSH isolates that carried czrC. These 14 pigs came from 8 (53%) of the 15 farms from which MRSH had been isolated. Furthermore, 100% (14/14) of the MRSH isolates carrying the czrC gene were SCCmec type V, compared to 29% (4/14) that were czrC negative (P < 0.01, Fisher’s Exact Test). This is consistent with previous studies that reported czrC is exclusive to SCCmec type V in MRSA (7,11), and suggests there may be a similar association in MRSH.

Of the pigs carrying czrC positive MRSH, 64% (9/14) were reported to be raised without exposure to antibiotics. The association between zinc-resistant MRSH and the absence of antibiotic usage was statistically significant (P < 0.05, Fisher’s Exact Test). The risk difference between groups was 56% [95% confidence interval (CI): 32% to 81%], indicating that pigs raised without antibiotic exposure were at a greater risk of carrying czrC positive MRSH. Since high levels of in-feed zinc oxide are used as an alternative to in-feed antibiotics (12), one would expect exposure and subsequent tolerance to zinc oxide to be greater on farms that do not expose pigs to antibiotics. However, since the sample size is limited in this study, future research should confirm and explore this association further.

The finding that czrC is common among MRSH and distributed across multiple Ontario farms raises concern because of the common practice of supplementation of swine starter ration with high levels of zinc oxide (≥ 2500 ppm) to prevent post-weaning colibacillosis (8). Exposure to zinc oxide in the starter ration may be selecting for MRSH in newly weaned pigs which are particularly susceptible to exudative epidermitis. Furthermore, in some countries the practice of zinc oxide supplementation is a permissible alternative to in-feed antibiotics and it is becoming increasingly common as there is a movement towards decreasing antibiotic use in swine production. However, antimicrobial-resistant staphylococci have emerged in swine production systems regardless of antibiotic usage (13), and factors influencing the emergence and persistence of antimicrobial resistance are complex, multifactorial, and poorly understood. The use of in-feed zinc oxide, combined with the presence of zinc-resistant staphylococci may provide a possible explanation for the emergence and dissemination of resistant bacteria on antimicrobial-free farms. The association between czrC and SCCmec, particularily SCCmec type V, which is common among livestock associated MRSA, indicates that zinc exposure could maintain selection pressure for methicillin-resistant staphylococci in the absence of traditional antimicrobial agents. This claim is supported by the finding that pigs raised without exposure to antibiotics are at a greater risk of carrying czrC positive MRSH. The importance of this finding for swine practitioners is that an outbreak of exudative epidermitis among newly weaned pigs being fed high levels of zinc oxide may be very difficult to treat successfully because of antimicrobial resistance. This study warrants further investigation into the effects of in-feed zinc oxide on the emergence and persistence of antimicrobial-resistant staphylococci in swine production systems.