Background:Gut microbiota plays a key role in health, immunity, digestion, and production in layers. Factors such as environment, diet, diseases, stress, and flock management significantly affect gut microbiota;however, it is not known how potential stressors such as intramuscular injections or feed withdrawal alter the composition of gut microbiota that result in increased the shedding level of foodborne pathogens. In the current study, the effects of intramuscular corticosterone injection and feed withdrawal were evaluated to understand their role in Salmonella Typhimurium shedding and changes in the composition of gut microbiota in layers. Results:Salmonella shedding was observed for 8 weeks post-infection. There was a significant increase in Salmonella Typhimurium count after intramuscular injection and feed withdrawal. The Salmonella infected and the negative control groups showed significant differences in the abundance of different genera in gut microbiota at week 1 and up to week 7 post infection. The infected group showed a significant reduction in alpha diversity of gut microbiota. Firmicutes reduced significantly (P<0.05) after intramuscular injection, while the feed withdrawal groups did not cause any significant changes in Proteobacteria-Firmicutes ratio. Furthermore, intramuscular injection resulted in a significant change in alpha diversity of gut microbiota. Conclusions:Exposure of chicks to relatively low dose of Salmonella Typhimurium can lead to persistent shedding in pullets. The Salmonella Typhimurium infection disrupted the gut microbiota composition immediately after infection. The potential stress of intramuscular injection and feed withdrawal significantly increased the Salmonella Typhimurium count in faeces. The intramuscular injection also resulted in a significant alteration of the Proteobacteria-Firmicutes ratio, which could increase the risk of dysbiosis.