Minimally Disturbed Tropical Soils Harbour Diverse Clostridioides Genomospecies With Mosaic Population Structures, Functional TcdB homologues, and Clini

ABSTRACT

Community‐acquired Clostridioides difficile infection is increasing, yet the ecology of toxin B (TcdB)‐encoding lineages in natural environments remains poorly understood. We investigated whether minimally disturbed tropical soils function as active reservoirs for toxigenic Clostridioides rather than passive sinks for clinical spillover. From 108 soil samples across 11 protected sites, we recovered 48 isolates: seven C. difficile strains (Clades 1 and 4) and 41 from cryptic clades C‐I, C‐III, and C‐V. Genomic analyses revealed extensive microdiversity with allelic mosaicism and site restriction, indicating structured populations shaped by local persistence and potential lateral gene exchange. Four C. difficile isolates carried chromosomal tcdB1 , whereas four C‐V isolates encoded tcdB7 on extrachromosomal elements. Two C‐V isolates with identical tcdB7 subtypes showed neutralizable in vitro cytopathic effects but divergent in vivo virulence. Resistome profiling identified diverse gyrB substitutions and, in one C. difficile strain, ermB and tetA(P)/tetB(P) . Phenotypic testing confirmed non‐susceptibility to fluoroquinolones, clindamycin, or tetracycline. These findings demonstrate that protected tropical soils host genetically structured Clostridioides populations with functional toxin loci and clinically relevant resistance genes, redefining our understanding of environmental origins and diversification of TcdB‐mediated disease.