Antimicrobial resistant bacteria in animal products are of major public health concern. The concerns about prudent use of antimicrobials have largely focused on feed grade antibiotic products used both for growth promotion and for routine prevention. Resistance to antibiotics among the normal swine commensal bacterium, Staphylococcus aureus, is of public health significance because of its zoonotic potential as an opportunistic pathogen of humans. Methicillin-resistant Staphylococcus aureus (MRSA) is widely recognized as hospital acquired, community associated zoonotic pathogen, which is of major concern in both humans and animals. The majority of nosocomial infections in intensive settings occur largely due to hospital acquired and community associated MRSA strains; very little evidence exists of common livestock-associated strains causing clinical illness in humans. Studies from the European Union have hypothesized that the emergence and dissemination of MRSA among swine production system is influenced by the use of in-feed antimicrobials, particularly tetracycline. The primary objective of our research was to evaluate the potential association of high levels of in-feed zinc supplementation (as an alternative to tetracycline), versus in-feed subtherapeutic or therapeutic levels of chlortetracycline (CTC), on the prevalence of methicillin resistant Staphylococcus aureus (MRSA) in post-weaned pigs.
A field experiment was conducted using weaned piglets, which were randomized to pens by weight and allowed to acclimatize to their surroundings and diet. All piglets received normal dietary rations as per NRC recommendations. The pens were assigned randomly to six treatment groups (incomplete factorial design with low CTC, high CTC, high Zn being the factors; low and high CTC could not be interacted per FDA regulations). Swab samples (nasal, skin, and tonsils) were collected on Days 0, 21 and 42. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered after streaking swabs onto a selective medium, CHROMTM MRSA agar(Hardy Diagnostics) at 37ºC for 24 h. Bacterial isolation was performed by picking three distinct colonies from each plate and re-streaking onto blood agar plates. We isolated 720 bacterial colonies for each sampling site. All isolates were subjected to species confirmation by PCR followed by detection of mecA (methicillin resistance) and czrC (zinc resistance) genes. All MRSA isolates were also subjected for spa typing, sequencing the spa gene repeat region, to further study the molecular epidemiology (this added step resulted in a request for a one-year no-cost extension to the original grant).
Overall, the prevalence of mecA-positive MRSA was 42.8% (308/720), 37.2% (268/720), and 42.6% (307/720) among nasal, skin, and tonsillar samples, respectively. The prevalence of mecA- and czrC-positive MRSA was affected by Zn (P < 0.05) but not CTC supplementation (P > 0.05). The prevalence of czrC-positive MRSA was 20% (144/720), 21.1% (152/720), and 14.3% (103/720) in nasal, skin, and tonsillar swabs, respectively. The occurrence of the czrC gene was strongly associated with mecA-positive MRSA isolates (P < 0.0001). The median minimum inhibitory concentrations (MICs) of Zn for zinc resistant and susceptible isolates were 8 and 4 mM, respectively (P < 0.001). We conducted spa gene repeat sequence analysis on all MRSA isolates originating from the three anatomic pig collection sites. The preliminary results from several isolates are presented here as the sequence analysis is still in progress (July 2015). Among the nasal isolates (n=121), the majority of them belong to t034 (50; 41.3%), unknown spa type (42; 34.7%), and t007 (29; 23.9%) spa types. In case of tonsillar MRSA isolates (n=109), the major spa types were t034 (41; 37.6%), t007 (37; 33.9%), and 28.4% (31) of unknown spa types. In the case of skin isolates (n=105), the major spa types were t034 (89; 84.7%), t127 (8; 7.6%), t007 (7; 6.6%), with only a single isolate not belonging to any known spa types.
There was a clear epidemic spread in the first 21 days of the feeding trial which provided most of the increase in the prevalence of MRSA in nasal, tonsillar, and skin regions of piglets and across all treatment groups. This suggests that either: 1) the MRSA strains were endemic in the barn environments before pigs were introduced, or 2) the MRSA strains were present in a very few piglets upon arrival and the stress of transport, mixing, and new environs triggered epidemic spread of the organism. Since our study was longitudinal in nature with repeated sampling it helped us to identify infectious disease dynamics and a clearly discernable epidemic spread of MRSA. In contrast, most previous studies of MRSA in swine in North America have been cross-sectional in design; or, with spacing of visits and animal sampling such that infectious disease dynamics could not be observed.
The genetic link between czrC and mecA genes points to the potential importance of elevated Zn supplementation in co-selection and propagation of antibiotic resistance; most notably, under non-epidemic situations. Importantly, Zn is a required micro-nutrient in swine diets and so a truly negative Zn diet is impractical, at best, and obviously dangerous to swine health and well-being at worst. As well, it is very unlikely that Zn supplemented at baseline nutrient requirement levels will select for those strains of Staphylococcus aureus harboring the czr gene. Based on our spa typing results, spa type t034 is the most predominant from all three collection sites. This spa type belongs to the clonal complex398 (CC398) and these are the most predominant livestock associated-MRSA clonal lineage, primarily in pigs, that have been reported from many countries, including the United States. The reason for the emergence of CC398 in pigs is not well understood and might involve a complex interplay between management and dietary factors. Both t007 and t127 also form a part of LA-MRSA complex and also have been reported from many countries including the United States. Although these clones or sequence types are reported from pigs, farmers, and veterinarians there are rarely cases of human infection (as opposed to colonization) reported. Importantly, if this situation changes and these strains become associated with human illness, they may pose a serious therapeutic challenge in the case of invasive infections since most of these isolates possess several virulence factors and toxins beside antimicrobial resistance determinants.
The data generated from our study will help to clarify the link between zinc and chlortetracycline use in swine production to other antibiotic resistance in a major pathogen of public health significance. It is imperative that the speculated associations between the use of these antibiotic and non-antibiotic antimicrobials and MRSA be examined in realistic field settings so as to more quantitatively evaluate the potential risks to public health. These data are essential for conducting meaningful quantitative risks assessments (QRA) and to draw effective intervention strategies with the help of more epidemiological surveillance. More importantly, our research findings will help swine producers to make informed decisions in optimizing existing management practices (such as mineral supplementation and the use of feed grade antimicrobials) in order to best manage public health risks in their settings.