VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Abstract Underlying mutation ratesand other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestisisolates from a rodent laboratory challenge with strain CO92 using whole genome sequencingand multi-locus variable-number tandem repeat(VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitroduring laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation ratesand the calculated number of Y. pestis generations that occurred during the in vitroversus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitromutations should be considered, particularly when highly variable genetic markerssuch as VNTRs are used.