Rodent Citrobacter

Citrobacter is a common member of the normal gut microbiota in humans and animals, and it also serves as an important opportunistic pathogen. In the 1960s, two outbreaks of high morbidity and mortality from mouse diarrhea occurred among mouse populations in different regions—first at the ANL (Argonne National Laboratory) in the United States, and later at Japan’s National Institute of Public Health. Both outbreaks were characterized by colonic hyperplasia. In the U.S. cases, culturing fecal and intestinal samples from infected animals revealed that 90% of the aerobic bacterial flora isolated belonged to an atypical strain of Citrobacter freundii (C. freundii Ediger). Meanwhile, in the Japanese cases, the causative agent was identified as an atypical Escherichia coli strain designated Ex30, later known as Mouse Pathogenic Escherichia coli (MPEC). In the mid-to-late 1970s, additional reports emerged from the United States regarding spontaneous disease outbreaks in mouse populations. Notably, these later outbreaks differed from the earlier ones, primarily manifesting as frequent cases of rectal prolapse and colitis—though without overt diarrhea in the affected animals. Once again, the isolates from these cases were identified as the same atypical strain of Citrobacter freundii (C. freundii Ediger 4280). Based on genetic classification, the Citrobacter strains and MPEC isolates initially identified were eventually reclassified into a single species. In 1995, Schauer and colleagues formally named this new species Rodentia citrobacter.

Citrobacter rodentium, a member of the genus Citrobacter within the family Enterobacteriaceae, is a natural pathogen in rodents that shares similar virulence traits with human-pathogenic strains such as enterohemorrhagic E. coli and enteropathogenic E. coli. It was also listed by China’s Standards for Microbiological Classification and Monitoring of Laboratory Animals as a mandatory testing item when necessary—specifically, E. coli O115 a,C:K(B). In 2022, these standards were officially updated to rename the pathogen as Citrobacter rodentium.

Gram-negative bacilli, observed under the microscope as short rod-shaped bacteria with motility. These organisms can grow on DHL or MacConkey agar plates, forming colonies that are pinkish-red at the center and translucent at the edges. Biochemically, their reactions closely resemble those of Salmonella, making them极易混淆.

Generally transmitted via the fecal-oral route, the disease has a short course, typically lasting around 4 weeks. When infected mice are housed together with uninfected ones, the infection can spread within 6 days. Mice and gerbils are highly susceptible, while guinea pigs may also become infected. In contrast, hamsters and rats are less prone to infection.

After infection, different clinical manifestations may appear. Adult mice typically show no clinical symptoms, while pups that have not yet been weaned or are just weaned are highly susceptible, often leading to weight loss, diarrhea, rectal prolapse, and even death in young mice. However, most infected mice exhibit colonic hyperplasia, a lesion that can be detected as early as 5 to 14 days post-infection. The progression of the disease is closely linked to host factors: genetic background, age, immune system deficiencies, and concurrent infections have all been shown to influence both the course and severity of the illness. Following an outbreak of Citrobacter rodentium, both morbidity and mortality rates tend to be very high. On the other hand, animals with robust immunity are able to mount an effective immune response against this bacterium, ultimately generating protective immunity.

Rodent Citrobacter enters the host orally, initially detected in the cecum. By 3 to 4 days post-infection, it migrates to the distal colon, where it rapidly proliferates. Similar to EPEC and EHEC, it exploits adherent and effacing (A/E) lesions in the intestinal epithelium to colonize the host’s gastrointestinal tract. In the early stages of infection, A/E lesions are characterized by bacteria tightly adhering to the host epithelial cell plasma membrane, accompanied by the localized disappearance of brush-border microvilli and the formation of pedestal-like structures beneath the adherent bacteria. These three features collectively trigger remodeling of the intestinal epithelium. Typical pathological changes include thickening of the colonic mucosa, marked hyperplasia of colonic crypts, and atrophy of the cecum. Typically, bacterial loads peak around 2 to 3 weeks post-infection. Notably, at the height of proliferation, the bacteria can no longer be isolated from the intestinal tract. Two months after infection, the lesions begin to resolve, and the colonic mucosa gradually returns to normal. Moreover, the infection often elicits a robust inflammatory response, involving the release of multiple cytokines as well as the recruitment of innate lymphoid cells, neutrophils, macrophages, B cells, and T cells. Neutrophils are among the first immune cells to accumulate in the colon, peaking around day 4 post-infection, while macrophages and dendritic cells infiltrate the colon more prominently by day 8. The numbers of these three immune cell populations reach their peak around day 14 before gradually declining and returning to baseline by day 21. Meanwhile, B cells show a significant increase by day 8, and both CD4+ and CD8+ T lymphocytes reach their peak levels by day 14, with levels of both T-cell subsets subsequently decreasing by day 21.

Rodent Citrobacter species exhibit remarkable colonizing ability in the colon, making them an ideal model for studying gut pathogen-host immune interactions. However, animals infected with rodent Citrobacter not only experience disruptions to intestinal homeostasis and compromised immune responses during infection, but they may also develop disease symptoms or suffer impaired growth and development, potentially leading to secondary infections. As a result, these animals are unsuitable for research related to intestinal diseases or for animal breeding programs. Moreover, most immunodeficient mice fail to clear the infection, often progressing to chronic infections that could serve as a source of contagion, inadvertently affecting other animals. Additionally, mice lacking mast cells frequently succumb rapidly, further complicating experimental procedures.

Once an infection occurs, it can easily contaminate the rearing environment. To prevent infection by this bacterium, it is essential to strengthen management practices, eliminate disease-inducing factors, and maintain strict hygiene of both feed and drinking water. Notably, Citrobacter rodentium is highly sensitive to commonly used disinfectants targeting enteric bacteria, so increasing environmental disinfection measures can be particularly effective. While antibiotics can treat infected animals, they rarely eradicate the bacterial carriage state in carriers and often disrupt the natural balance of gut microbiota, potentially leading to increased antibiotic resistance. Moreover, antibiotic use may even interfere with research outcomes. Therefore, treatment is generally not recommended; instead, purification can be achieved through cesarean delivery or embryo cleansing techniques.

References

1. Bouladoux N, et al. The mouse model of infection with Citrobacter rodentium [J]. Curr Protoc Immunol. 2017 Nov 1;119:19.15.1-19.15.25.

2. Eve G D Hopkins, et al. Intestinal Epithelial Cells and the Microbiome Undergo Swift Reprogramming at the Onset of Colonic Citrobacter rodentium Infection[J]. mBio. 2019 Apr 2;10(2):e00062-19.

3. Danielle Carson, et al. Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease. Cell Microbiology[J]. 2020 Jan;22(1):e13126.

4. Rosanna Mundy, et al. Citrobacter rodentium in mice and humans. Cellular Microbiology [J]. (2005) 7(12), 1697–1706

5. Charles River Technical Sheet: Helicobacter spp.

6. Tian Kegong et al., Experimental Animal Epidemiology [M], Chapter 35, Section 2 of Part III, 2014:571-575