Probiotic Nutrient Competition Drives Adaptive Evolution in Salmonella Typhimurium

Salmonella Typhimurium, a leading cause of food-borne gastro-enteritis, infects thousands of people globally each year. The rising antimicrobial resistance crisis has driven the exploration of microbiome-based therapeutics such as probiotics as an alternative to traditional antibiotics in combating pathogens. Escherichia coli Nissle (EcN), a widely studied probiotic, has shown promising results in reducing Salmonella infections both in vitro and in vivo. However, the underlying mechanisms of EcN's inhibitory effects on Salmonella remain incompletely understood. Previous studies have identified two microcins (H47 and M) encoded by EcN that can inhibit Salmonella growth. Nevertheless, these microcins alone do not fully explain EcN’s anti-Salmonella activity. Our experiments demonstrate that even microcin-deficient EcN strains can still outcompete Salmonella, implicating resource competition rather than direct interference competition as the primary inhibitory mechanism, at least in iron-rich conditions. By performing growth assays, supernatant analyses and competition experiments, we confirm that nutrient competition is central to EcN’s ability to suppress Salmonella. To further investigate whether Salmonella could adapt to EcN-mediated resource competition and develop resistance to this inhibitory mechanism, we co-evolved both strains over a four-week period. In two independent experiments, we observed a significant increase in Salmonella frequency in some replicate populations (Figure 1), suggesting that Salmonella can indeed adapt to resource competition with probiotics. In the evolved Salmonella lineages, we identified four notable mutations. These mutations include an early stop codon in the iraP gene, as well as a frameshift in the stationary phase sigma factor rpoS. Both mutations likely enhance nutrient uptake and metabolic activity, but as a trade-off may decrease stress tolerance. A third mutation appears to constitutively activate malT, a positive regulator of the maltose operon, thereby also enhancing nutrient uptake and metabolic activity. Finally, a mutation in yjcC, a c-di-GMP phosphodiesterase, was observed. The exact phenotypic effect of this mutations remains unclear, but our current hypothesis is that this mutation results in increased c-di-GMP levels, hence promoting biofilm formation and colonization of the air-liquid interface. To conclude, our work demonstrates that resource competition can be an important mechanism by which probiotics inhibit pathogens, and that pathogens can evolve and adapt to resource competition through various metabolic adaptations.