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Background    Clostridium difficile  whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals.    Methods  Serial isolates from two scenarios were whole genome sequenced. Firstly, 62 isolates from 29 timepoints from three  in vitro  gut models, inoculated with a NAP1/027 strain. Secondly, 122 isolates from 44 patients (2–8 samples/patient) with mostly recurrent/on-going symptomatic NAP-1/027  C. difficile  infection. Reference-based mapping was used to identify single nucleotide variants (SNVs).    Results  Across three gut model inductions, two with antibiotic treatment, total 137 days, only two new SNVs became established. Pre-existing minority SNVs became dominant in two models. Several SNVs were detected, only present in the minority of colonies at one/two timepoints. The median (inter-quartile range) [range] time between patients’ first and last samples was 60 (29.5–118.5) [0–561] days. Within-patient  C. difficile  evolution was 0.45 SNVs/called genome/year (95%CI 0.00–1.28) and within-host diversity was 0.28 SNVs/called genome (0.05–0.53). 26/28 gut model and patient SNVs were non-synonymous, affecting a range of gene targets.    Conclusions  The consistency of whole genome sequencing data from gut model  C. difficile  isolates, and the high stability of genomic sequences in isolates from patients, supports the use of whole genome sequencing in detailed transmission investigations.

Short-Term Genome Stability of Serial Clostridium difficile Ribotype 027 Isolates in an Experimental Gut Model and Recurrent Human Disease