# Whole-genome sequencing-based typing methods for Clostridium butyricum strains from clinical, animal, plant, and environmental sources

## TL;DR

This study develops high-resolution genotyping methods for Clostridium butyricum to improve tracking of this bacteria in clinical and environmental settings.

## Contribution

The paper introduces novel cgMLST and cgSNP schemes for high-resolution typing of C. butyricum.

## Key findings

- Most C. butyricum strains cluster independently of their origin or spatiotemporal context.
- cgSNP analysis provides finer resolution for NEC-related strains compared to cgMLST.
- Botulinum neurotoxin type E-producing strains are grouped within a single clade.

## Abstract

Clostridium butyricum exhibits a dual role, acting not only as a probiotic but also as an opportunistic pathogen associated with neonatal necrotizing enterocolitis (NEC) and infant botulism. We aimed to establish high-resolution genotyping frameworks to improve molecular surveillance and outbreak investigations. We analyzed 297 C. butyricum genomes, including 200 isolates from preterm neonates across 13 French neonatal intensive care units over a 20-year period and 97 publicly available genomes. A core-genome multilocus sequence typing (cgMLST) scheme was developed using chewBBACA, defining 2,621 loci, and applied to genomes with ≥95% locus presence. Core-genome single-nucleotide polymorphism (cgSNP) analysis was performed for complementary resolution. Phylogenetic cgMLST classified isolates into nine major clades. Some clinical strains displayed clonal relationships, whereas others were geographically and temporally unrelated. All botulinum neurotoxin type E-producing strains were grouped within a single clade. NEC-associated isolates showed geographic and temporal clustering, but no clade was uniquely linked to NEC. cgSNP analysis identified 11 clusters with overall discriminatory power similar to cgMLST while providing finer resolution for NEC-related strains. We propose robust cgMLST and cgSNP schemes for C. butyricum, enabling high-resolution genotyping and supporting epidemiological surveillance and outbreak investigation of this emerging opportunistic pathogen in neonatal settings.

Clostridium butyricum has been identified in fecal samples from both asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Using a large collection of strains from different origins and spatiotemporal contexts, we developed and established a cgMLST scheme for the molecular typing of C. butyricum. Our results show that most C. butyricum strains cluster independently of origin and spatiotemporal context factors. However, specific cgMLST clades of C. butyricum were found for plant and botulinum neurotoxin type E strains. Clonal strains were also identified. No specific cgMLST clade was found to be genetically associated with NEC. cgSNP showed higher discriminatory power compared to cgMLST. Importantly, cgSNP provided better discriminatory power for strain relatedness with respect to strains isolated from NEC patients.