Schubert, C.; Kim, J.; Näpflin, N.; Hoos, M.; Huuskonen, J.; von Mering, C.; Hardt, W.-D.

Background: Barcoding of isogenic strains is a powerful approach to assess pathogen population dynamics during infection. Here, we adapted WISH-barcoding to Salmonella Typhimurium ATCC14028s to evaluate its suitability for pooled infection experiments in streptomycin-pretreated mouse models. Results: WISH-barcoded wild-type pools showed pronounced population instability, characterized by stochastic strain loss and segregation into high- and low-fitness subpopulations. Whole-genome sequencing identified recurrent mutations in the methyltransferase rsmG and loss of the P3 plasmid carrying streptomycin resistance in low-fitness strains; neither was observed in {Delta}invG or {Delta}ssaV pools. We propose that rsmG mutations were enriched during strain construction carried out under streptomycin selection, following loss of the P3 plasmid. Control experiments demonstrated that rsmG mutations and P3 loss are counter-selected in vivo and attenuate gut-luminal colonization in streptomycin-pretreated mice. Conclusion: While population dynamics experiments with ATCC14028s are feasible in principle, wild-type strains are prone to acquiring fitness-altering mutations during in vitro construction when using the P3 plasmid and streptomycin, highlighting the need for careful pool validation prior to use.