Abstract
Salmonella enterica serovar Typhimurium (S. Tm) mouse infection models commonly rely on antibiotic pretreatment to disrupt microbiota-mediated colonization resistance and enable reproducible infection. Streptomycin is the primary antibiotic used, but alternative pretreatment regimens like ampicillin are increasingly employed, particularly for infections with attenuated S. Tm mutants. However, the impact of different antibiotic pretreatments on Salmonella infection kinetics and host responses remains poorly characterized. Here, we compared S. Tm infection outcomes in streptomycin- versus ampicillin-pretreated mice. While both antibiotics supported comparable luminal colonization, ampicillin pretreatment induced earlier, stronger, and more sustained intestinal inflammation and histopathology during S. Tm infection. In germ-free mice, these differences were absent, indicating a microbiota-dependent mechanism, further supported by microbiota transfer experiments. The distinct infection outcomes were associated with antibiotic-specific alterations in the microbiota composition, while targeted analysis of luminal metabolites by mass spectrometry revealed no pronounced differences. Strikingly, ampicillin pretreatment coincided with higher early TTSS-1 expression in S. Tm, consistent with increased epithelial invasion. Together, our findings demonstrate that antibiotic pretreatment profoundly shapes the intestinal environment and is associated with differences in Salmonella virulence expression, host responses, and infection kinetics. The choice of antibiotic pretreatment is therefore a crucial variable in murine Salmonella infection models.