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

Abstract

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.