Molecular clock complexities of Clostridioides difficile.

ObjectivesReconstruct the phylogenetic status of a collection of historical Clostridioides difficile isolates and evaluate the congruence of their evolutionary trajectories with established molecular clock models.MethodsPhylogenetic 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.ResultsMoxifloxacin-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 only a weak linear correlation.ConclusionsC. difficile molecular clock estimations may be more complex than previously considered, with periods of spore quiescence potentially complicating analyses.