Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

Abstract

Upon colonization of the intestinal epithelium, the attaching and effacing (AE) pathogen Enterohemorrhagic Escherichia coli (EHEC) effaces microvilli and forms pedestal-like structures beneath the adherent bacterium. The production of one of its virulence factors, the phage-encoded Shiga toxin (Stx) results in systemic disease, including the development of renal failure. Although EHEC does not productively infect conventional mice, EHEC infection can be modeled in mice utilizing a derivative of the natural murine AE pathogen Citrobacter rodentium (CR). Gavage of mice with CR(ΦStx2dact), a C. rodentium lysogenized by a phage encoding an Stx variant with high potency in mice, features AE lesion formation on intestinal epithelium and Stx-mediated systemic disease, including renal damage. This model is somewhat limited by mouse-to-mouse variation in the course of disease, with the time to severe morbidity (and required euthanasia) varying by as many as 5 days, a feature that limits pathological analysis at defined stages of disease. In the current study, we altered and optimized the preparation, dose, and mode of delivery of CR(ΦStx2dact), using food-borne route of infection to generate highly synchronous disease model. We found that food-borne inoculation of as few as 3 × 104 CR(ΦStx2dact) resulted in productive colonization and severe systemic disease. Upon inoculation of 1 × 108 bacteria, the majority of infected animals suffered weight loss beginning 5 days post-infection and all required euthanasia on day 6 or 7. This enhanced murine model for EHEC infection should facilitate characterization of the pathology associated with specific phases of Stx-mediated disease.

Efficacy of EHEC gold nanoparticle vaccines evaluated with the Shiga toxin-producing Citrobacter rodentium mouse model

“…Although Stx2d-producing C. rodentium was previously established to induce full lethality in young C57BL/6 mice, we sought to confirm the disease kinetics in the conditions of our animal facility, as well as verify that the model would elicit similar outcomes in older mice, considering the length of our vaccine regimens ( 24 , 25 ). Therefore, we orally challenged 10- to 12-week-old female C57BL/6 mice with 1 × 10 9 CFU of either Stx2d-producing C. rodentium strain (DBS770) or the Stx2d-negative (DBS771) strain ( n = 6 for each group).…”

“…Mice treated with phosphate-buffered saline (PBS) were used as a control (mock, n = 6). Furthermore, we utilized a feeding method of infection, which involves inoculating a small piece of rodent chow with the bacterial suspension and presenting it to fasted mice until consumption, because it was previously demonstrated that this method exhibits more consistent disease kinetics at even lower inoculum ( 25 ). Also, this technique is safer to administer, does not require anesthesia, and more closely resembles natural human infection with EHEC.…”

“…Here, we demonstrated that the Stx2d-producing C. rodentium , which more closely recapitulates human intestinal disease as seen with EHEC, is a viable model to assess the effectiveness of novel vaccine candidates. Former studies with this pathogen, specifically using the feeding model of infection, utilized mice corresponding to 6 to 8 weeks of age ( 24 , 25 ). To mitigate concerns regarding the age of mice used in our vaccine regimen, which equals around 8 full weeks starting from arrival at our facilities to infection, we initially evaluated this model in 10- to 12-week-old C57BL/6 female mice.…”

Bacteriophage Transcription Factor Cro Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli

“…EHEC is a human pathogen that does not cause GI disease in rodents (Mallick et al, 2012). The natural murine pathogen C. rodentium , has been extensively used as a surrogate murine infection model for EHEC infection (Flowers et al, 2016). It harbors the same virulence genes as EHEC (i.e.…”

Infection and antibiotic-associated changes in the fecal microbiota ofC. rodentiumϕstx2dact-infected C57BL/6 mice

“…Animal infections. Six-week-old female C57BL/6Rj mice purchased from Janvier Labs (Le Genest-Saint-Isle, France) barrier A02 were infected with 5 x 10 8 CFU C. rodentium DBS770 (C. rodentium stx2dact) following the feeding protocol described in Flowers et al (55) or left uninfected. From 4 days post-infection, drinking water was supplemented with 2% glucose and either of the following antibiotics: enrofloxacin (0.25 mg/ml), kanamycin (2.6 mg/ml), tetracycline (1 mg/ml), rifampicin (1 mg/ml), or trimethoprim/sulfamethoxazole (Trimetotat oral suspension 48% (Livisto)).…”