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Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of  Salmonella  outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used  Salmonella  typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the  Salmonella  genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either  Salmonella  Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of  Salmonella  Mbandaka.  Salmonella  shedding in both single and co-infected birds was variable over the course of the 16-week experiment.  Salmonella  Typhimurium and  Salmonella  Mbandaka were identified in feces of co-infected birds.  Salmonella  colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in  Salmonella  Typhimurium strains isolated from co-infected birds. Isolates of  Salmonella  Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness