Identification of Novel Cryptic and Classical Clades in Clostridioides difficile

Cid-Rojas, F.; Guerrero-Araya, E.; Rodriguez, C.; Paredes-Sabja, D.

doi:10.1101/2025.08.01.668003

Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.08.01.668003

Identification of Novel Cryptic and Classical Clades in Clostridioides difficile

Cid-Rojas, F.·Guerrero-Araya, E.·Rodriguez, C.·Paredes-Sabja, D.

View original source ↗Export metadata ↓

Abstract

Clostridioides difficile is a major cause of nosocomial infections and antimicrobial-associated diarrhea, with significant global health implications. While five classical clades (C1 to C5) have traditionally encompassed most pathogenic strains, recent genomic studies have uncovered several highly divergent cryptic clades (C-I to C-V), suggesting greater taxonomic and evolutionary complexity. In this study, we performed a comprehensive whole-genome analysis of 25,144 publicly available C. difficile genomes, supplemented with 21 novel isolates from Costa Rica and Brazil. Using average nucleotide identity (ANI), recombination-corrected core-genome phylogenies, we confirmed the presence of all known classical and cryptic clades and identified two novel classical clades (C6 and C7) and two previously undescribed cryptic clades (C-VI and C-VII). We further detected evidence for bifurcation within cryptic clade C-III, supporting its division into two lineages C-IIIa and C-IIIb. ANI comparisons revealed that cryptic clades share less than 95% identity with classical C. difficile strains but remain more similar to C. difficile than to any other Peptostreptococcaceae species, reinforcing their placement within the species boundary. Core-genome recombination analyses revealed limited gene flow between cryptic and classical clades, except for Clade C7, which exhibited intermediate recombination patterns and may represent a transitional lineage. These findings expand the known diversity of C. difficile, provide a revised genomic framework for clade classification, and underscore the evolutionary depth and ecological breadth of cryptic lineages with potential clinical relevance.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Cid-Rojas, F., Guerrero-Araya, E., Rodriguez, C., Paredes-Sabja, D.. 2025-08-01. Identification of Novel Cryptic and Classical Clades in Clostridioides difficile. https://doi.org/10.1101/2025.08.01.668003

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗

bioRxiv2026

Mapping virulence-associated protein interaction networks reveals regulators of thermotolerance in Cryptococcus neoformans

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

microbiology↗

bioRxiv2026

Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

microbiology↗