Molecular epidemiology of antibiotic-resistant Enterococcus spp. from farm-to-fork in intensive pig production in KwaZulu-Natal, South Africa.

Abstract

Background: Substantial antibiotic use and high population densities in intensive farming systems results in the emergence and spread of antibiotic-resistant commensals and pathogens. This study investigated the molecular epidemiology of antibiotic resistance (ABR) and virulence in Enterococcus spp. from pigs in an intensive food production continuum from farm-to-fork in the uMgungundlovu district, Kwa-Zulu Natal. Methods: A total of 174 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) were subjected to the quantification and putative identification of Enterococcus spp. using the IDEXX Enterolert® method and selective media, respectively. Up to three presumptive enterococcal colonies were picked per sampling point for molecular confirmation by real-time PCR, targeting the genus- and species-specific (tuf and sodA) genes, respectively. Antibiotic resistance profiles were determined by the Kirby-Bauer disk diffusion method against a panel of antibiotics for Enterococcus spp. recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. Results: A total of 284 isolates constituted the final sample. Real-time PCR confirmed 79.2% of the isolates as E. faecalis, 6.7% as E. faecium, 2.5% as E. casseliflavus, 0.4% as E. gallinarum, and 11.2% as other Enterococcus spp. Antibiotic susceptibility testing revealed resistance to sulfamethoxazole-trimethoprim (78.8%), tetracycline (76.9%), erythromycin (68.1%), streptomycin (62.6%), chloramphenicol (27.0%), ciprofloxacin (8.5%), gentamicin (8.1%), and levofloxacin (5.6%) but no vancomycin, teicoplanin, tigecycline or linezolid resistance was detected. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. A total of 78% of enterococcal isolates were MDR. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3’)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most commonly detected virulence genes were: gelE, efaAfs, and cpd in 89.1%, 78.5%, and 77.1% of isolates conferring autolysin and biofilm formation capabilities, cell adhesion, and conjugative plasmid accumulation, respectively. Clonality evaluated by REP-PCR revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, largely consisting of isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. There was minimal evidence of clonal transmission across the continuum. Conclusion: Multi-drug resistant Enterococcus spp. were isolated along the farm-to-fork continuum. Isolates harboured a diversity of antibiotic resistance and virulence genes in different combinations forming reservoirs for the potential transfer of these genes from pigs to occupationally exposed workers and consumers via direct contact with animals and animal products/food, respectively. The results highlight the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.