Culture-dependent screening of endospore-forming clostridia in infant feces

Background Only a few studies dealt with the occurrence of endospore-forming clostridia in the microbiota of infants without obvious health complications. Methods A methodology pipeline was developed to determine the occurrence of endospore formers in infant feces. Twenty-four fecal samples (FS) were collected from one infant in monthly intervals and were subjected to variable chemical and heat treatment in combination with culture-dependent analysis. Isolates were identified by MALDI-TOF mass spectrometry, 16S rRNA gene sequencing, and characterized with biochemical assays. Results More than 800 isolates were obtained, and a total of 21 Eubacteriales taxa belonging to the Clostridiaceae, Lachnospiraceae, Oscillospiraceae , and Peptostreptococcaceae families were detected. Clostridium perfringens, C. paraputrificum, C. tertium, C. symbiosum, C. butyricum , and C. ramosum were the most frequently identified species compared to the rarely detected Enterocloster bolteae, C. baratii , and C. jeddahense. Furthermore, the methodology enabled the subsequent cultivation of less frequently detectable gut taxa such as Flavonifractor plautii, Intestinibacter bartlettii, Eisenbergiella tayi , and Eubacterium tenue . The isolates showed phenotypic variability regarding enzymatic activity, fermentation profiles, and butyrate production. Conclusions Taken together, this approach suggests and challenges a cultivation-based pipeline that allows the investigation of the population of endospore formers in complex ecosystems such as the human gastrointestinal tract.