Diversity and Prevalence of Clostridium innocuum in the Human Gut Microbiota

Abstract

<i>Clostridia</i> are a polyphyletic group of Gram-positive, spore-forming anaerobes in the <i>Firmicutes</i> phylum that significantly impact metabolism and functioning of the human gastrointestinal tract. Recently, <i>Clostridia</i> were divided into two separate classes, <i>Clostridia</i> and <i>Erysipelotrichia</i>, based on phenotypic and 16S rRNA gene-based differences. While <i>Clostridia</i> include many well-known pathogenic bacteria, <i>Erysipelotrichia</i> remain relatively uncharacterized, particularly regarding their role as a pathogen versus commensal. Despite wide recognition as a commensal, the erysipelotrichial species Clostridium innocuum has recently been associated with various disease states. To further understand the ecological and potential virulent role of C. innocuum, we conducted a genomic comparison across 38 C. innocuum isolates and 194 publicly available genomes. Based on colony morphology, we isolated multiple C. innocuum cultivars from the feces of healthy human volunteers (<i>n</i> = 5). Comparison of the 16S rRNA gene of our isolates against publicly available microbiota data sets in healthy individuals suggests a high prevalence of C. innocuum across the human population (>80%). Analysis of single nucleotide polymorphisms (SNPs) across core genes and average nucleotide identify (ANI) revealed the presence of four clades among all available genomes (<i>n</i> = 232 total). Investigation of carbohydrate and protein utilization pathways, including comparison against the carbohydrate-activating enzyme (CAZyme) database, demonstrated inter- and intraclade differences that were further substantiated <i>in vitro</i>. Collectively, these data indicate genetic variance within the C. innocuum species that may help clarify its role in human disease and health. <b>IMPORTANCE</b> <i>Clostridia</i> are a group of medically important anaerobes as both commensals and pathogens. Recently, a new class of <i>Erysipelotrichia</i> containing a number of reassigned clostridial species has emerged, including Clostridium innocuum. Recent studies have implicated C. innocuum as a potential causative agent of diarrhea in patients from whom Clostridioides difficile could not be isolated. Using genomic and <i>in vitro</i> comparison, this study sought to characterize C. innocuum in the healthy human gut. Our analyses suggest that C. innocuum is a highly prevalent and diverse species, demonstrating clade-specific differences in metabolism and potential virulence. Collectively, this study is the first investigation into a broader description of C. innocuum as a human gut inhabitant.