Molecular clock complexities of Clostridioides difficile.

OBJECTIVES: Reconstruct the phylogenetic status of a collection of historical Clostridioides difficile isolates and evaluate the congruence of their evolutionary trajectories with established molecular clock models. METHODS: Phylogenetic analysis was performed on Illumina sequence reads from previously analysed historic C. difficile isolates (1980-86; n = 75) demonstrating multiple antimicrobial resistances. Data was grouped by ribotype (RT), including comparators from European surveillance (2012-13) and phylogenetic studies (1985-2010). Reads were mapped to CD630/CD196 reference genomes and compared using recombination-adjusted maximum likelihood trees. Prediction intervals for expected SNP differences by age were calculated using a Poisson distribution and molecular clock estimates (0.74 SNPs per genome/per year). Root-to-tip analysis was performed to determine the date of most common recent ancestor of genomes sharing a ribotype. RESULTS: Moxifloxacin-resistant (>16 mg/L) RT027 isolate JV67 (1986) was two SNPs distinct from a 2006 genome, fewer than the expected lower estimate (4.4 SNPs) under current molecular clock calculations; (p = 3.93x10-5). For isolate JV02 (1981), the 13 SNP divergence from a 2008 isolate was consistent with expectations (5.9 SNPs; p = 0.07). JV73 (1983) demonstrated an 8 SNP difference, which although above the expected lower limit (5.5 SNPs), was outside the 95 % prediction interval; (p = 4.51x10-3). Only sixty-nine percent of historical genomes fit within the prediction interval for the number of SNPs expected compared to recent isolates, with fewer SNPs observed more frequently than expected. Root-to-tip analysis demonstrated a weak linear correlation. CONCLUSIONS: C. difficile molecular clock estimations may be more complex than previously considered, with periods of spore quiescence potentially complicating analyses.