Foodborne infection of mice with Salmonella Typhimurium

Abstract

The bacterial pathogen Salmonella enterica serovar Typhimurium is one of the most common causes of foodborne disease in humans and is also an important model system for bacterial pathogenesis. Oral inoculation of C57Bl/6 mice, which are genetically susceptible to Salmonella, results in systemic infection but the murine intestine is not efficiently colonized unless the intestinal microbiota is disrupted. Pretreatment of C57Bl/6 mice with streptomycin, followed by oral inoculation with Salmonella Typhimurium results in colitis resembling human intestinal Salmonellosis. The predominant method of delivery of bacteria is oral gavage, during which organisms are deposited directly into the stomach via a feeding needle. Although convenient, this method can be stressful for mice, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we developed a method for oral infection of mice by voluntary consumption of regular mouse chow inoculated with bacteria. Mice readily ate chow fragments containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in reproducible infections with doses as low as 103 CFU. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, voluntary consumption of chow inoculated with Salmonella represents a natural route of infection for foodborne salmonellosis and a viable alternative to oral gavage.

Fig 1. Salmonella survival on mouse chow.

(A) Representative image of a regular mouse chow pellet and prepared chow fragments. Ruler scale in centimeters. (B, C) Survival of Salmonella on chow and in SPGS, 1 and 3 h after inoculation. Original inoculum added as a comparison. Data represent the mean ± SD of three independent experiments (n = 1 per experiment).

Fig 2. Comparison of tissue loads in mice infected by VC and OG.

(A) Schematic representation of the OG (top) and VC (bottom) infections. (B) Bacterial numbers in tissues from mice infected either by OG or VC 3 days p.i. n = 10 mice. Symbols represent individual mice. Error bars represent the mean ± SD. Tissues where bacterial load was below the level of detection are given a value of “1” for visualization purposes.

Fig 3. Colonization of B6 and B6N mice following VC inoculation.

(A) Bacterial loads in tissues 3 days p.i. in strep+ B6 mice infected with indicated CFUs. (B) Bacterial loads in tissues 3 days p.i. in strep+ B6N mice infected with 10⁴ or 10⁵ CFUs. n = 5 mice and symbols represent individual mice. Tissues where bacterial load was below the level of detection are given a value of “1” for visualization purposes.

Fig 4. Infection of strep- mice.

Bacterial numbers in tissues 3 days p.i. in strep- B6 and B6N mice infected with 10⁸ CFU Salmonella. n = 5 mice. Filled symbols represent individual B6 mice and open symbols represent individual B6N mice. Tissues where bacterial load was below the level of detection are given a value of “1” for visualization purposes. Asterix indicates statistical significance; * p < 0.05, ** p < 0.01, ns, not statistically different, two-tailed Mann-Whitney U test.

Fig 5. Kinetics of disease progression in mice inoculated by VC.

Survival and bacterial loads of B6 (A, C, E) and B6N (B, D, F) mice. Strep- (not pretreated with antibiotic) and strep+ (pretreated with antibiotic in drinking water) mice were infected with 10⁸ or 10⁴ CFU respectively. n = 5 mice in each experiment and symbols represent individual mice. Open symbols in Fig 5D indicate the B6N mouse that was euthanized 15 days p.i. Tissues where bacterial load was below the level of detection are given a value of “1” for visualization purposes.

Fig 6. Fasting duration affects consumption time and bacterial loads.

(A) Time required for mice to completely consume fragments of chow with the indicated inoculum and fasting time. Symbols represent individual mice and error bars represent the mean ± SD. 5 mice per experiment. (B) Consumption time (left panel) and bacterial loads (right panel) in tissues 3 days p.i. following 4 h of fasting and an inoculum of 10⁸ CFU Salmonella. n = 5 mice, symbols represent individual mice, error bars represent the mean ± SD.