Clostridioides difficile in the oral microbiome: an in-silico analysis

Introduction. High rates of recurrent Clostridioides difficile infection (CDI) and environmental contamination are attributed to its ability to form spores. Periodontal diseases are characterized by gingival inflammation, caused by dental plaque accumulation.

Hypothesis. Periodontal plaque could harbour C. difficile spores, acting as a reservoir for reinfection.

Aim. Compare the prevalence and abundance of C. difficile in metagenomic sequences of saliva and dental plaque from healthy and periodontal disease patients.

Methodology. Publicly available metagenomic reads from oral samples of healthy (n=80) and periodontitis (n=204) patients were analysed for C. difficile presence through an in-house bioinformatic pipeline. Briefly, reads underwent quality control (cutadapt/fastQC) prior to subsampling of 3 million reads (seqtk). Reads and MEGAHIT-assembled contigs were aligned to a C. difficile reference genome (ASM1888508v1) or a full non-redundant protein DIAMOND database. Outputs were filtered, annotated (Entrez Direct) and top hits identified via National Center for Biotechnology Information blast. Abundance and prevalence were compared between cohorts.

Results. Low levels of C. difficile sequences were observed, with significantly higher prevalence in periodontitis (7.4%, n=15/204) vs. healthy cohorts (5.0%, n=4/80) (P=0.0087) with reference genome alignment. Using the full non-redundant database, prevalence was also higher in periodontitis (14.2% vs. 3.8%; P=0.012), along with significantly greater average C. difficile sequence counts (0.608 vs. 0.075; P=0.018) and relative abundance (0.00029% vs. 0.0000003%; P=0.009).

Conclusion. Sequences pertaining to C. difficile were detected in oral samples, with significantly more observed in periodontal disease compared to healthy cohorts. This highlights the possibility for dental plaque to act as a reservoir, potentially contributing to reinfection in CDI patients.