Research Article: Gut microbiota features associated with Clostridioides difficile colonization in puppies

Date Published: August 30, 2019

Publisher: Public Library of Science

Author(s): Alexander S. F. Berry, Brendan J. Kelly, Denise Barnhart, Donna J. Kelly, Daniel P. Beiting, Robert N. Baldassano, Laurel E. Redding, Christopher Staley.


In people, colonization with Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, has been shown to be associated with distinct gut microbial features, including reduced bacterial community diversity and depletion of key taxa. In dogs, the gut microbiota features that define C. difficile colonization are less well understood. We sought to define the gut microbiota features associated with C. difficile colonization in puppies, a population where the prevalence of C. difficile has been shown to be elevated, and to define the effect of puppy age and litter upon these features and C. difficile risk. We collected fecal samples from weaned (n = 27) and unweaned (n = 74) puppies from 13 litters and analyzed the effects of colonization status, age and litter on microbial diversity using linear mixed effects models.

Partial Text

Clostridioides difficile is a spore-forming anaerobic, gram-positive bacillus that is the leading cause of antibiotic-associated and nosocomial diarrhea in humans [1, 2] and a significant enteric pathogen in many species of animals [3–5]. Administration of antibiotics is the primary risk factor for the development of C. difficile infection (CDI) [1, 6, 7]. However, patients can develop CDI outside of a healthcare facility without the prior use of antibiotics, and community-acquired CDIs are now thought to account for one quarter of infections [8, 9].

Asymptomatic carriage of C. difficile is common in the young of many species, including humans [43], dogs [20], pigs [12, 44, 45], and cattle [46]. In people, colonization with C. difficile has been shown to be associated with altered gut microbial diversity [24, 26, 47–49], but no studies have examined this association in young dogs. In adult dogs, C. difficile colonization was associated with reduced gut bacterial species and diversity [50]. In puppies, we found that the association between lower bacterial community diversity and C. difficile colonization was statistically significant even when accounting for age, and certain bacterial taxa were preferentially associated with C. difficile colonization.

We found that puppies with C. difficile-positive fecal samples had reduced gut microbiota diversity, even when adjusting for the puppy’s age, and that there were differentially-abundant taxa in C. difficile-positive and C. difficile-negative fecal samples. These differences in microbial features may be permissive in promoting the colonization and establishment of C. difficile, though longitudinal studies are needed to confirm this hypothesis. Though this effect was not observed at the level of the litter, and even though the litter explained a large proportion of the gut microbiota diversity, heterogeneity in the gut microbiota and in C. difficile colonization within litters was observed in more than half of the litters, suggesting that the gut microbiota and potentially other unmeasured factors contribute to colonization resistance against C. difficile in puppies.




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