Andam, C. P. | Search bioRxiv

bioRxiv2026

Contact dependent suppression of Clostridioides difficile sporulation by enterococci requires the endocarditis and biofilm pilus

Clostridioides difficile is a healthcare-associated infection that arises when broad-spectrum antibiotic treatment disrupts the gut microbiota and is transmitted by highly resistant spores. Vancomycin-resistant Enterococcus faecium (VRE) is an opportunistic pathogen frequently co-isolated from C. difficile patients. We found that C. difficile sporulation is significantly reduced in VRE-C. difficile co-culture. Physical separation of C. difficile and VRE in transwell co-culture restored sporulation. Mixed macrocolony culture assays on solid agar confirmed physical contact is necessary for sporulation inhibition. We screened a panel of enterococci and found that most strains reduce sporulation, except Enterococcus saccharolyticus, which lacks predicted surface displayed virulence factors in its genome. We performed a candidate gene screen using an Enterococcus faecalis OG1RF transposon library and found that an insertion in the major pilin ebpC partially restored C. difficile sporulation in co-culture. These data were confirmed with in-frame deletions in the ebpABC pilus operon and a clinical isolate of E. feacalis lacking ebpABC. These findings suggest enterococci modulate C. difficile sporulation through a contact-dependent mechanism involving the Ebp pilus. ImportanceA characteristic of C. difficile infection is multiple episodes of acute disease. Spores are the transmission vector of C. difficile and are necessary for recurrence in models of disease. Our research demonstrates that C. difficile spore production is significantly reduced in the presence of enterococci, a common group of beneficial and pathogenic bacteria present in the gut microbiota. Physical contact with enterococci reduces C. difficile spore production. We attribute this effect to a protein structure on the surface of enterococci. This finding suggests a potential role for enterococci and the gut microbiota in general to uncover regulators of C. difficile spore formation. This may provide an avenue for innovative treatment strategies that reduce spore formation.

microbiology↗

bioRxiv2025

Biogeographical and phylogenetic constraints on horizontal gene transfer and genome evolution in Streptomyces

The role of horizontal gene transfer (HGT) in shaping bacterial genomes is well recognized, but constraints on gene exchange and the degree to which these constraints shape genome evolution remain poorly described. In this study, we sought to determine whether geographic and phylogenetic distance constrains HGT within and between bacterial species. To address this question, we isolated strains (n = 17) of two closely related bacterial species, Streptomyces griseus and Streptomyces pratensis from two ecologically similar sites. We identified homologous recombination events within the core genomes of these species (557 recent and 457 ancient) and determined that patterns of recombination were constrained primarily by phylogeny rather than geography. Notably, shell accessory genes were over three times more likely to be shared between the same species than with non-related geographical neighbors. The richness of secondary metabolite gene clusters is highly variable with an average of 35 - 55 clusters per genome, depending on clade membership. The majority of secondary metabolite gene clusters (60%) are found in all strains, indicating that they were present in the most recent common ancestor of S. griseus and pratensis. We conclude that most HGT in the core and accessory genome is phylogenetically constrained, while HGT of shell genes is more likely constrained by geography. This outcome indicates that the predominant mechanisms of HGT favor high phylogenetic relatedness, and that rapid gene acquisition and loss in the accessory genome could aid with adaptation to contemporary environmental conditions. ImportanceHorizontal gene transfer (HGT) is a vital ecological and evolutionary force in microbiology, but we still lack a precise understanding of how precisely HGT acts on the gene pool of a species or genus. While HGT can complicate phylogenetic analyses and assumptions of homology, its role in adaptation and acquiring secondary metabolites should not be overlooked. Microbial ecologists agree that the pangenome is a shifting collection of genes that can be influenced by both vertical inheritance and ecological factors. This study examines how the Streptomyces pangenome is shaped by these two forces and offers an important quantitative insight into how HGT shapes bacterial genome dynamics.

microbiology↗