César Rodríguez

In a recent One Health genomic survey of community-associated Clostridioides difficile in Japan, S. Yoshizawa, K. Komori, K. Aoki, T. Sawa, et al. (Appl Environ Microbiol 92:e00397-26, 2026, https://doi.org/10.1128/aem.00397-26 ) deposited the genome of strain TUM20227, a human tcdA ⁻ / tcdB ⁺ isolate assigned to the novel sequence type ST919. Reanalysis of its publicly available genome shows that TUM20227 belongs to cryptic clade C-III and carries a chromosomally encoded, monotoxin PaLoc-like locus encoding a 2,367-amino acid TcdB-family protein. This TcdB-like protein shares less than 83% amino acid identity with all recognized TcdB subtypes (B1–B12) and forms a deeply divergent branch in both full-length and domain-resolved phylogenies, with heterogeneous nearest neighbors across domains and an atypical predicted receptor-binding profile. TcdB _TUM20227 should therefore be regarded as representative of a previously unrecognized, deeply divergent TcdB lineage. IMPORTANCE: Clostridioides difficile is a leading cause of diarrhea, and its principal virulence factor, the toxin TcdB, is both a diagnostic target and a template for therapeutics. The recognized diversity of TcdB is captured in a scheme composed of 12 subtypes largely built from strains of the five C. difficile clades, whereas the Clostridioides cryptic clades and their toxins remain comparatively underexplored, even though they can encode divergent toxins with altered receptor use and antigenicity. Here, reanalysis of a single, previously unremarked genome from a One Health survey uncovers a deeply divergent and apparently complete and intact TcdB-family toxin gene whose product falls outside the established subtype framework. This finding shows that systematic data mining of animal, human, and environmental strains can unearth novel toxin genes and provide critical insight into the evolution, receptor specificity, and subtype boundaries of the toxin genes of this important human pathogen.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that coordinates lifestyle transitions, virulence, and developmental processes in bacteria. In Clostridioides difficile , elevated c-di-GMP levels inhibit sporulation, but the underlying mechanism remained unclear. Here, we identified a conserved family of small, membrane-associated proteins encoded by c-di-GMP riboswitch-regulated genes. Transcriptomic analyses revealed that c-di-GMP represses these genes, and reporter assays demonstrated riboswitch-dependent transcriptional regulation via premature termination mechanism. Overexpression of a single gene, CD1980.2 , was sufficient to trigger the transcriptional activation of sporulation genes, including sigma factors and their regulons, and to increase spore formation. Conversely, sporulation efficiency decreased proportionally with the number of deleted small-protein genes, and the strain lacking all seven genes displayed a severe sporulation defect, underscoring their cumulative and functionally redundant roles. Elevating c-di-GMP levels in the deletion mutant did not further reduce sporulation, supporting a model in which c-di-GMP inhibits spore formation by repressing the expression of this small-protein family. Thus, our work establishes these small proteins, encoded by c-di-GMP riboswitch-regulated genes, as important regulators of spore formation in C. difficile , a process essential for pathogen persistence and transmission.

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.

Clostridioides difficile infection (CDI), primarily mediated by toxin B (TcdB), is a leading cause of healthcare-associated and community-acquired diarrhea worldwide. Although this genus exhibits substantial genomic diversity, functional studies have focused mainly on a limited set of isolates, leaving other genomospecies understudied in this regard. Here, we characterized five isolates derived from five distinct patients with suspected CDI cases that tested negative for the tcdC PaLoc marker. Integrated genomic, proteomic, and phenotypic analyses confirm that these strains constitute three novel toxin-producing species and their distinct carbon utilization profiles, exoproteomes, and spore protein repertoires suggest niche specialization within the human gut ecosystem. These species harbor monotoxin PaLoc architectures encoding TcdB7 or TcdB11, located on either chromosomal or extrachromosomal elements, which frequently co-occur with binary toxin loci. Functional assays confirmed secretion of active TcdB, cytotoxicity in mammalian cells, and moderate enterotoxicity in a murine ileal loop model, although virulence was attenuated relative to C. difficile R20291 from Clade 2. Collectively, these findings expand the taxonomy of toxigenic Clostridioides, highlight the dynamic evolution of TcdB-mediated pathogenicity, and emphasize the importance of refining diagnostic workflows and surveillance strategies to address emerging diarrheal diseases. The three novel species were designated as Clostridioides cryptodifficilis sp. nov., Clostridioides divergens sp. nov., and Clostridioides subdifficilis sp. nov.

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium and a major nosocomial pathogen, notable for its high genetic diversity. C. difficile has been classified into five classical phylogenetic clades (C1–C5) and five cryptic clades (C-I to C-V), reflecting its extensive genome plasticity. In addition, C. difficile spores are considered essential for the onset, persistence, and transmission of the disease, and their exosporium layer features hair-like projections formed by the BclA family of proteins (BclA1, BclA2, and BclA3). Previous work in C1 and C2 strains has demonstrated that the absence of these proteins affects spore germination, pathogenesis, persistence, and recurrence of CDI. Nevertheless, the conservation of BclA proteins across different C. difficile clades remains unclear. In this work, genomic analysis of more than 25,000 C. difficile genomes revealed that the prevalence and variability of BclA are not consistent across classical and cryptic clades. The most represented clade in the data set was C1, where roughly 50% of the genomes possessed all three bclA genes. Pseudogenization of bclA1 and all bclA was observed in C2 and C3, respectively. Additionally, the absence of bclA1 in C4 and both bclA1 and bclA3 in C5 further demonstrated the divergence of BclA among C. difficile clades. Subsequent analysis revealed high variability in the central collagen-like region (CLR) of all three BclA and a highly conserved bclA1 pseudogenization event in most members of C2. Restoration of a full-length bclA1 from strain 630 (C1) into C. difficile R20291 (C2) slightly decreased the length of the hair-like projections. Overall, the extensive variability of bclA , primarily focused on their CLR, prevalence of a bclA1 pseudogenization, and absence of bclA in specific clades likely impact spore morphogenesis and pathogenesis across the species. IMPORTANCE: Clostridioides difficile is a major cause of healthcare-associated infections, with spores playing a central role in disease transmission, persistence, and recurrence. The outermost spore layer, the exosporium, is critical for interactions with the host and environment, and its structure is shaped in part by the BclA family of proteins. While previous studies have linked BclA proteins to virulence traits, their distribution and conservation across the species’ genetically diverse clades have remained unexplored. This study provides the first comprehensive analysis of bclA gene prevalence and variability across more than 25,000 genomes, revealing substantial divergence among classical and cryptic clades. The frequent pseudogenization or absence of bclA genes, particularly bclA1 , and high variability in the collagen-like regions suggest that BclA-mediated exosporium architecture is not universally conserved and may have clade-specific implications for spore morphology and pathogenicity. These findings highlight the need to account for phylogenetic context when studying C. difficile spore biology and open new avenues for understanding how structural variation in spores contributes to strain-specific virulence and transmission dynamics.

Among the five MLST clades that define the global population structure of the bacterial pathogen Clostridioides difficile, Clade 2 has received special attention due to the global spread, clinical severity and hospital prevalence of ST01 strains. To identify features potentially contributing to the historically attributed higher virulence and epidemic potential of ST01 strains, we compared a range of phenotypic traits across the infection cycle between clinical Clade 2 ST01 and non-ST01 strains from ST41, ST47, ST67, ST154 and ST638. We found no significant differences in canonical virulence-associated characteristics such as spore adherence, motility, biofilm formation and resistance to a disinfectant. However, ST01 strains exhibited distinct profiles in surface layer protein A (SlpA)-mediated immune activation and toxin B (TcdB)-induced cytotoxicity that were consistent with allelic divergence. These findings highlight the need to reconsider current paradigms of Clade 2 hypervirulence and underscore the importance of allele-specific phenotypic variation in developing targeted public health strategies.

Among the five MLST clades that define the global population structure of the bacterial pathogen Clostridioides difficile, Clade 2 has received special attention due to the global spread, clinical severity, and hospital prevalence of ST01 strains. To identify features potentially contributing to the historically attributed higher virulence and epidemic potential of ST01 strains, we compared a range of phenotypic traits across the infection cycle between clinical Clade 2 ST01 and non-ST01 strains from ST41, ST47, ST67, ST154, and ST638. We found no significant differences in canonical virulence-associated characteristics such as spore adherence, motility, biofilm formation, and resistance to a disinfectant. However, ST01 strains exhibited distinct profiles in surface layer protein A (SlpA)-mediated immune activation and toxin B (TcdB)-induced cytotoxicity that were consistent with allelic divergence. These findings highlight the need to reconsider current paradigms of Clade 2 hypervirulence and underscore the importance of allele-specific phenotypic variation in developing targeted Public Health strategies.

Clostridioides difficile infections (CDI) cause almost 300,000 hospitalizations per year, of which ~15%–30% are the result of recurring infections. The prevalence and persistence of CDI in hospital settings have resulted in an extensive collection of C. difficile clinical isolates and their classification, typically by ribotype. While much of the current literature focuses on one or two prominent epidemic ribotypes (e.g., RT027), recent years have seen several other ribotypes dominate the clinical landscape (e.g., RT106 and RT078). Some ribotypes are associated with severe disease and/or increased recurrence rates, but why certain ribotypes are more prominent or harmful than others remains unknown. Because C. difficile has a large, open pan-genome, this observed relationship between ribotype and clinical outcome could be a result of the genetic diversity of C. difficile . Thus, we hypothesize that the core biological processes of C. difficile are conserved across ribotypes/clades. We tested this hypothesis by observing the growth kinetics, sporulation, germination, production of toxin A and toxin B, bile acid sensitivity, bile salt hydrolase activity, and surface motility of 15 strains belonging to various ribotypes spanning each known C. difficile clade. In viewing these phenotypes across each strain, we see that core phenotypes (growth, germination, sporulation, and resistance to bile salt toxicity) are remarkably consistent across clades/ribotypes. This suggests that variations observed in the clinical setting may be due to unidentified factors in the accessory genome or due to unknown host factors. IMPORTANCE: Clostridioides difficile infections impact thousands of individuals every year, many of whom experience recurring infections. Clinical studies have reported an unexplained correlation between some clades/ribotypes of C. difficile and disease severity/recurrence. Here, we demonstrate that C. difficile strains across major clades/ribotypes are consistent in their core phenotypes. This suggests that such phenotypes are not responsible for variations in disease severity/recurrence and are ideal targets for the development of therapeutics meant to treat C. difficile -related infections.