Ecology of Clostridioides (Clostridium) difficile in Western Australia

Karla E Cautivo Reyes

Clostridioides (Clostridium) difficile is a common cause of infectious diarrhoea in humans in high-income countries. The high mortality associated with infections caused by toxigenic strains of C. difficile, combined with the considerable clinical and economic burden on healthcare systems worldwide, demands a better understanding of C. difficile ecology. C difficile can be found in the gastrointestinal tract of human and non-human animals, and in a wide variety of environmental sources. Based on the prevalence and high diversity of strains of C. difficile found in Australian production animals and the similarity with strains found in humans, it is imperative to examine other environmental sources/hosts like soils and native animals to find potential links. Thus, the overarching aim was to investigate the ecology of C. difficle in Western Australia. First, a state-wide epidemiological study was conducted examining soils of the Australian state of Western Australia (WA), the largest state of Australia comprising the western third of the continent, which covers 2.6 million km2. This study described the prevalence, molecular epidemiology and biogeographic distribution of C. difficile across the eight health regions in WA. A total of 321 soil samples from residential and remote locations of WA were collected by “citizen scientists” through the collaboration with the MicroBlitz project and analysed following enrichment culture and ribotyping. A high diversity and heterogenicity of C. difficile strains were identified; a total of 52 different strains were isolated including 14 new strains with similar ribotyping banding patterns and toxin profiles to strains belonging to C. difficile clade 5. Alfa and beta diversity indices were calculated to determine the ecological richness in each health region and to determine how they were related. The high diversity and heterogenicity of C. difficile found suggest that soils may act as a substantial vehicle for transmission among reservoirs of infection and accidental hosts. The second epidemiological study investigated C. difficile carriage in native animals using 324 faecal samples from wild and captive native reptiles, mammals and birds. . C difficile prevalence was 55.4% in reptiles, and 42.7% in mammals, both significantly higher than in birds (8%, p-value < 0.0001). Ecological indices, alpha and beta diversity were determined and the relationship between the presence of C. difficile with animal class, habitat and feeding behaviour were assessed using logistic regression. There were significant variations in C. difficile presence across animal classes and environmental factors. Mammals in captivity were linked to reduced odds of detection (OR = 0.77, 95% CI: 0.68–0.86), while wild status decreased the odds (OR = 0.63, 95% CI: 0.45–0.82). Among dietary groups, insectivorous mammals showed the highest odds of carrying C. difficile, with an odds ratio (OR) of 1.66 (95% CI: 1.52–1.81), indicating a greater likelihood of detection. In contrast, omnivores exhibited the lowest odds (OR = 0.35, 95% CI: 0.23–0.46), followed by carnivores (OR = 0.77, 95% CI: 0.55–0.98), both suggesting reduced presence relative to herbivores. Arboreal-terrestrial mammals and reptiles exhibited increased odds compared to fully arboreal species (OR = 2.33 and OR = 3.29, respectively), and semiaquatic adaptation showed the highest association in both groups (mammals OR = 4.96, 95% CI: 4.66–5.26; reptiles OR = 7.00, 95% CI: 6.77–7.23). These results highlight behavioural and habitat-related traits as important predictors of C. difficile occurrence in wildlife. This study describes for the first time the ecology of C. difficile in native Australian animals providing insights into the prevalence, molecular epidemiology, and its ecology. In addition, a molecular evolutionary analysis of C. difficile strains of One Health importance recovered from two native animals was performed. Both isolates share the same sequence type (ST167) despite coming from different species and locations, indicating possible similarities in their genomic makeup or environmental exposures. Finally, a third epidemiological study was performed on Rottnest Island, a small island of approximately 19 km2 about 20 km off the coast near Perth the capital of WA, to evaluate the prevalence and population dynamics of C. difficile among the local quokka population and in soil samples. In 1696, the Dutch sea captain Willem de Vlamingh landed on the island, giving it the famous name of Rottnest Island. This means ‘rat’s nest’ in Dutch, as he mistook the island’s abundant quokka (Setonix brachyurus) population for giant rats. This seasonal study involved collecting faecal samples from quokkas and soil samples during winter (August 2020) and summer (January 2021 months) in two batches of 174 and 160 samples, respectively. The overall prevalence of C. difficile was 53.9% (n=180/334); 64.4% of soil samples and 45.7% of faecal samples from quokkas were positive. Ecological relationships between seasonal sampling and sample type and C. difficile prevalence were determined and diversity ecological tests were performed. Logistic regression showed that both seasonal sampling and sample type significantly influenced the presence of C. difficile. Samples collected in summer had lower odds of being positive compared to winter (OR = 0.35, 95% CI: 0.23–0.54, p < 0.0001), while soil samples were more likely to yield positive results than faecal samples (OR = 3.14, 95% CI: 2.03–4.90, p < 0.0001). The ribotypes diversity, measured by Shannon index, showed no significant differences across batches or sample types. Faecal samples had slightly higher diversity (2.92 vs 2.83), and samples from summer showed marginally greater diversity than those collected in winter (2.94 vs 2.83), with all p-values > 0.05. Toxin-profile diversity also varied modestly, with higher diversity in faecal (1.44) and winter samples (1.38), though these differences were not statistically significant (p = 0.72). This chapter compiled data not previously generated in Australia. In conclusion, there was a high diversity of C. difficile strains found in the environment, inferring that soil is an important source of transmission. The high prevalence of C. difficile in native animals suggests that the local population might act as a reservoir of infection to humans and animals of One Health importance.

Ecology of Clostridioides (Clostridium) difficile in Western Australia

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