Short-term genomic stability of Clostridium difficile within patients and a gut model

Background: Clostridium difficile (CD) whole genome sequencing (WGS) has potential to identify related isolates for transmission studies, even among otherwise indistinguishable strains, but depends on understanding the genomic variation within isolates and individuals. Methods: Serial isolates (n=11) were taken over 31 days from a triple stage chemostat human gut model inoculated with a CD NAP1/027 strain for WGS, including baseline, clindamycin exposure, toxin induction, and antibiotic treatment phases. Multiple isolates were also obtained for WGS from 10 patients (range 2-8 isolates per patient, n=29) with recurrent symptomatic NAP1/027 CD infection during 2007-09. Extracted DNA from each isolate was sequenced using the Illumina GA2 and HighSeq platforms and mapped to the CD196 reference genome using STAMPY. Base and variant calls were made using SAMTOOLS, PICARD and bespoke Python scripts. Results: The first 9 isolates from the gut model were identical by WGS. During antibiotic treatment, 1 single nucleotide variant (SNV) emerged and persisted in the subsequent isolate, introducing a non-synonymous change in the coding sequence for a glucose transport protein. WGS of 3 duplicate DNA extract pairs produced fully concordant data. In serially sampled patients, the median (IQR) time between baseline and subsequent tests was 76 (1-95) days. In 8 of the 10 patients no SNVs were observed up to a maximum of 192 days later. A single SNV occurred transiently in another patient after 82 days. Among 8 isolates from the final patient, 1 SNV occurred after 75 days and another the next day, and both persisted over the next 32 days. A further SNV was identified in a single sequence and not found in subsequent isolates. Conclusions: The consistency of WGS data for gut model CD isolates demonstrates the reliability of our sequencing and analysis techniques. The high stability of mapped genomic sequences in CD isolates from patients supports their use in detailed transmission studiesNon peer reviewe