Isolation, Characterization, and Epizootiology of Clostridioides cuniculi from Immunodeficient Mice with Enteric Disease
Mouse strains deficient in adaptive and innate immune functions, such as NSG, NSG-SGM3, and NBSGW, are highly susceptible to opportunistic infections. Over a period of 7 mo, 1,193 mice from the above 3 strains in an SPF barrier were observed with mild loose stool (LS). Affected mice had minimal weight loss and mortality. Histopathology revealed erosion of the jejunal villi with neutrophilic inflammation and Gram-positive bacterial rods adhering to the cecal mucosa with varying degrees of mucosal hyperplasia, epithelial vacuolation, and apoptosis. Anaerobic culture revealed a clostridial species that could not be speciated using standard biochemical phenotyping. Further, Clostridioides difficle and Clostridioides perfringens ELISA on intestinal contents were negative for toxins. We performed a challenge study by exposing naïve NSG mice to dirty bedding from affected cages; metagenomics on pre- and postchallenge feces identified and associated the etiopathogenesis to Clostridioides cuniculi. Whole genome sequencing and phylogenetic analysis confirmed the identity of C. cuniculi. The isolate was sensitive to trimethoprim-sulfamethoxazole (TMS). TMS was effective in abrogating signs of LS and clearing infection in mice in studies. A probe-based real-time PCR specific for C. cuniculi was established. This assay was used to screen environmental and fomite contamination and potential use in rack-level screening. We traced the source of the outbreak to a NBSGW breeding colony. However, in our observation, spontaneous C. cuniculi-induced disease was only seen in the presence of an irradiated diet in the breeding NBSGW strain and not in the breeding colonies of NSG or NSG-SGM3 strains. Interestingly, we observed that exposure to infected feces from NBSGW-induced LS in both NSG and NSG-SGM3 mice. This investigation provides insights into the etiopathogenesis and probable source of sporadic clostridial infections in immunodeficient mice and lays the groundwork for its prevention and surveillance in immunodeficient mouse colonies.
Separate and dedicated breeding rooms for (A) NSG and NSG-SGM3 with 8- to 10-wk old mice transferred to study rooms for almost 5 years with no history of LS or enteric pathology until (B; red asterisk depicts infected mice) the introduction of NBSGW for breeding to the room housing breeding NSG-SGM3 colony. All breeding colonies were on the same irradiated rodent diet before and during the outbreak of LS and enteric disease.
Histopathology of intestinal tract from index cases and challenge study. (A) Jejunum from an index case depicting erosion of the apical mucosa (arrows) with associated clusters of degenerate neutrophils (*) in the lumen. (B) Cecum from the same mouse shows mucosal hyperplasia and apoptosis (circles) with rodlike bacteria adhering to the epithelial surface (inset: Gram stain, arrows). (C) Gram strain of bacteria cultured from an index case, revealing they are Gram-positive, rod-shaped bacteria arranged in chains. (D) Romanowsky stain of the same bacteria, in which the formation of central spores (arrows) is present. Challenge study showing no pathology of (E) the cecum from an unexposed NSG mouse and (F) the cecum from an exposed mouse with loose stool exhibiting mucosal hyperplasia (note increased mucosal thickness) and apoptosis of the epithelium (circled).
Shotgun metagenomics from the challenge study, in which naïve, culture-negative NSG mice were exposed to infected bedding from LS cases. (A) PCoA of beta diversity using UniFrac distances. Green ellipses represent 95% CI for samples and exclude exposed mice from week 3. (B) Relative abundance for Clostridiaceae family. C. cuniculi is only present in the exposed group at week 3 (dotted outline and purple). (C) Heatmap visualizing the relative abundances of core bacterial species, defined as those with a mean relative abundance greater than 1%. Hierarchical clustering analysis revealed 2 distinct clusters: one comprising the week 3 samples from the exposed group, and the other comprising the remaining 6 samples. (D) LEfSE on Cluster 1 or Cluster 2 from C. C. cuniculi, the only pathogenic species identified, was restricted to Cluster 2 (i.e. exposed group at week 3).
Comparative genomics of C. cuniculi isolates relative to other Clostridioides species using average nucleotide to perform comparisons identifying homologous regions. Pairwise comparisons are used to identify (A) the proportion of identical aligned regions, (B) the amount of each genome that is aligned, (C) the number of unaligned or nonidentical bases, and (D) the total number of aligned bases in each genome.
Concatenated sequences from 49 clusters of orthologous genes for each genome were aligned to determine relatedness similarity. C. cuniculi genomes are bold and include information about host species from which the isolate was obtained.
Agar disc diffusion for assessing antibiotic effectiveness against C. cuniculi.
Real-time PCR values from environmental testing (black) in comparison to fecal samples from LS mice (red) or asymptomatic animals (blue).
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