Assessment of Genetic Stability During Serial In Vitro Passage and In Vivo Carriage

In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.