Genomic Delineation of Zoonotic Origins of Clostridium difficile

Abstract

Clostridium difficile is toxin-producing antimicrobial resistant (AMR) enteropathogen historically associated with diarrhea and pseudomembranous colitis in hospitalized patients. In recent years, there have been dramatic increases in the incidence and severity of C. difficile infection (CDI), and associated morbidity and mortality, in both healthcare and community settings. C. difficile is an ancient and diverse species that displays a sympatric lifestyle, establishing itself in a range of ecological niches external to the healthcare system. These sources/reservoirs include food, water, soil, and over a dozen animal species, in particular, livestock such as pigs and cattle. In a manner analogous to human infection, excessive antimicrobial exposure, particularly to cephalosporins, is driving the expansion of C. difficile in livestock populations worldwide. Subsequent spore contamination of meat, vegetables grown in soil containing animal feces, agricultural by-products such as compost and manure, and the environment in general (households, lawns, and public spaces) is contributing to a persistent community source/reservoir of C. difficile and the insidious rise of CDI in the community. The whole-genome sequencing era continues to redefine our view of this complex pathogen. The application of high-resolution microbial genomics in a One Health framework (encompassing clinical, veterinary, and environment derived datasets) is the optimal paradigm for advancing our understanding of CDI in humans and animals. This approach has begun to yield critical insights into the genetic diversity, evolution, AMR, and zoonotic potential of C. difficile. In Europe, North America, and Australia, microevolutionary analysis of the C. difficile core genome shows strains common to humans and animals (livestock or companion animals) do not form distinct populations but share a recent evolutionary history. Moreover, for C. difficile sequence type 11 and PCR ribotypes 078 and 014, major lineages of One Health importance, this approach has substantiated inter-species clonal transmission between animals and humans. These findings indicate either a zoonosis or anthroponosis. Moreover, they challenge the existing paradigm and the long-held misconception that CDI is primarily a healthcare-associated infection. In this article, evolutionary, and zoonotic aspects of CDI are discussed, including the anthropomorphic factors that contribute to the spread of C. difficile from the farm to the community.

Keywords: Clostridium difficile; evolution; livestock; one health; transmission; zoonosis.

Figure 1

Global prevalence of C. difficile in farm animals, food and the environment. Data were taken from 86 studies in 23 countries worldwide (, –122). Categories: Poultry (hens and broilers, Seafood (salmon, perch, clams, shrimp, and mussels), Meat (veal, beef, pork, lamb, chicken, goat, buffalo, and turkey), Vegetables (salads, lettuce, pea sprouts, ginger, carrots, beetroot, potatoes, onions, and spinach), Household (sandbox, shoes, toilet, vacuum, sink, floor), and Natural Environment (compost, lawn, soil, sediment, lake, and river). Two-letter country codes (International Organization for Standardization, ISO): AU, Australia; BE, Belgium; CA, Canada; CH, Switzerland; CI, Ivory Coast; CR, Costa Rica; CZ, Czech Republic; DE, Germany; ES, Spain; FR, France; GB, Great Britain and Northern Ireland; IR, Iran; IT, Italy; JP, Japan; KR, Korea; NE, Nigeria; NL, The Netherlands; SA, Saudi Arabia; SE, Sweden; SI, Slovenia; TW, Taiwan; US, United States of America; ZW, Zimbabwe. NAP, North American Pulse Type. RT027 and all ST11 RTs listed are binary toxin-positive.

Figure 2

Evolutionary clade 5. Maximum-likelihood phylogeny based on concatenated MLST allele sequences (seven loci, 3,501 bp) for 32 known clade 5 STs. For global phylogenetic context, well-characterized representatives of major MLST clades C1 (ST54, RT012), C2 (ST1, RT027), C3 (ST22, RT023), and C4 (ST37, RT017) are also shown. Branches for clade 5 are shown in red. The inset box highlights major ST11 RT sublineages of clinical and agricultural/livestock importance with associated toxin gene profiles and graphical representation of 16s−23s rRNA PCR ribotyping banding patterns.

Figure 3

Transmission networks and community reservoirs of C. difficile RT014 in Australia. (A) summary of ST13 (n = 8) and ST2 (n = 10) RT014 clonal groups found in pigs and humans with CDI in Australia, adapted from Knight et al. (128). A clonal group is defined as two or more strains differing by <2 SNVs in their core genome. HCF, healthcare facility; NSW, New South Wales; SA, South Australia; VIC, Victoria; QLD, Queensland; INDET, indeterminate. (B) summary of RT014 ecological niches in Australia. ¹Knight et al. (186); ²Collins et al. (188); ³Knight et al. (52); ⁴Moono et al. (151); ⁵Lim et al. (84); ⁶Lim et al. (122).