Clostridioides difficile in Australian horses

Natasza M Hain-Saunders

Clostridioides difficile is an ongoing public health threat as a cause of severe gastrointestinal disease in both human and animal populations. Over the last two decades, community-associated infection has increased dramatically, with evidence of a zoonotic link. While C. difficile is regularly isolated from equine populations, its role in disease is poorly understood as is the role that equines play in further disease dispersal. In Australia, horses represent a unique mix of production, companion and feral animals. This study aimed to investigate the epidemiology of C. difficile infection (CDI) in Australian horse populations, providing insight into the extent of dissemination and characteristics of the strains isolated. Faecal samples were collected from free-roaming feral horses across five Australian jurisdictions (n=380), as well as from Western Australian domestic horses (n=752 from 387 individuals) between 2017 and 2022. Samples were cultured for C. difficile by enrichment methods and isolates characterised by PCR ribotyping and toxin gene profiling. Antimicrobial susceptibility testing was performed by an agar incorporation method. A subset of strains was further interrogated through a combination of whole genome sequencing, single nucleotide polymorphism (SNP) analysis and single linkage clustering with a comparator dataset of strains of human, environmental and animal origin. In domestic populations, C. difficile was isolated from 19 (37.8%) horses with gastrointestinal (GI) symptoms and 104 (30.9%) without GI symptoms - a prevalence significantly higher than previous studies within Australia. Of these 123 horses, 68 (55.3%) harboured toxigenic strains of C. difficile. Thirteen (6.9%) horses sampled at an equine hospital shed multiple strains, suggesting both true colonisation and transient shedding exists simultaneously within this population. Both novel and known C. difficile ribotypes (RTs) were identified including prominent toxigenic human strains, RTs 012 (n=14), 014/020 (n=10), 087 (n=7), 002 (n=5) and 103 (n=3). Whole-genome analysis of 45 RT 012 strains and 160 RT 014/020 strains of equine, human and environmental origin identified clusters indicating recent shared ancestry. Evidence of possible clonal inter-species transmission or common source exposure was identified for three subgroups. Isolates in these subgroups spanned 7 years (2014 to 2021) and extended across six reservoirs/sources (horses, humans, water, compost, lawn and vegetables), indicative of an underlying and persistent community reservoir or source of RT 014/020 and RT 012. In feral horses, C. difficile was isolated from 45 of the 380 samples (11.8%), a significantly lower proportion than domestic horses (P<0.001) (Risk Ratio: 0.37, 95%CI: 0.27-0.51, P<0.001), however, like wild animal species worldwide. This may reflect the impact that antimicrobial use in domesticated populations and closer and more frequent contact with humans has on disease aetiology. Eighteen toxigenic C. difficile strains were isolated, eight containing binary toxin genes. Forty RTs were identified, 28 of which (70%) were novel; other RTs had been seen previously in humans, livestock and soils. All strains were susceptible to metronidazole, the antimicrobial of choice for CDI in animals, with minimal resistance to human CDI treatments, vancomycin (domestic, n=2; feral, n=2) and fidaxomicin (domestic, n=1). Resistance was seen in clindamycin, with potential for inter-bacterial transfer adding to the current global antimicrobial resistance threat. Multi-drug resistance was seen in four domestic horse strains, all of which were toxigenic RT 012 from equine hospital samples. This study provided comprehensive epidemiological data for C. difficile and CDI in domestic horses in Western Australia and developed a better understanding of the role animal manures may play in community-acquired CDI. For the first time, C. difficile in feral horses was also described providing an opportunity for comparison with their domestic counterparts. The findings highlight the potential for horses as a reservoir of C. difficile within the community and solidifies the need for a One Health approach and enhanced surveillance for effective CDI monitoring, prevention and control.

Clostridioides difficile in Australian horses

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