Comparison of Anaerobic Culture Methods for Detecting Clostridioides difficile in Bovine Faeces

ABSTRACT Background The study of the epidemiology of Clostridioides difficile in populations is greatly facilitated by the ability to isolate and further characterize individual organisms, which requires effective culture protocols. In cattle, where little is known about the epidemiology of C. difficile, no studies have assessed or compared the performance of different assays for detecting C. difficile. Objectives This study compared two culture protocols for detecting C. difficile in bovine faeces from 121 gestating cows and 70 of their neonatal calves, while situating results obtained with each protocol relative to those obtained with shotgun metagenomic sequencing. Methods Protocol 1 involved direct plating enrichment onto taurocholine‐cycloserine‐cefoxitin‐fructose agar (TCCFA), while Protocol 2 included an ethanol shock step before plating on CCFA/ChromID agar. For both protocols, one aliquot underwent broth enrichment prior to plating, while the other aliquot did not. Results Clostridioides difficile was detected following broth enrichment in two of the same calf samples using both protocols, and an additional cow sample was found to be positive with Protocol 2, though the difference in detection rates was not statistically significant (p = 1.0). Conclusions The detection of C. difficile in a much high number of these samples by shotgun metagenomics, albeit at low levels of relative abundance, suggests that neither of these culture protocols is sensitive when levels of abundance are low. This study compared two culture protocols for detecting C. difficile in bovine faeces from 121 gestating cows and 70 of their neonatal calves. No statistically significant difference in detection rates was observed with either protocol, but results from shotgun metagenomic sequencing from a subset of samples indicated C. difficile was present at very low levels in most samples, suggesting that anaerobic culture is a relatively insensitive method of detecting this organism in bovine faeces.