Outbreaks of Typhlocolitis Caused by Hypervirulent Group ST1 Clostridioides difficile in Highly Immunocompromised Strains of Mice

Abstract

Clostridioides difficile is an enteric pathogen that can cause significant clinical disease in both humans and animals. However, clinical disease arises most commonly after treatment with broad-spectrum antibiotics. The organism's ability to cause naturally occurring disease in mice is rare, and little is known about its clinical significance in highly immunocompromised mice. We report on 2 outbreaks of diarrhea associated with C. difficile in mice. In outbreak 1, 182 of approximately 2, 400 NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) and related strains of mice became clinically ill after cessation of a 14-d course of 0.12% amoxicillin feed to control an increase in clinical signs associated with Corynebacterium bovis infection. Most mice had been engrafted with human tumors; the remainder were experimentally naïve. Affected animals exhibited 1 of 3 clinical syndromes: 1) peracute death; 2) severe diarrhea leading to euthanasia or death; or 3) mild to moderate diarrhea followed by recovery. A given cage could contain both affected and unaffected mice. Outbreak 2 involved a small breeding colony (approximately 50 mice) of NOD. CB17-Prkdc^scid/NCrCrl (NOD-scid) mice that had not received antibiotics or experimental manipulations. In both outbreaks, C. difficile was isolated, and toxins A and B were detected in intestinal content or feces. Histopathologic lesions highly suggestive of C. difficile enterotoxemia included fibrinonecrotizing and neutrophilic typhlocolitis with characteristic 'volcano' erosions or pseudomembrane formation. Genomic analysis of 4 isolates (3 from outbreak 1 and 1 from outbreak 2) revealed that these isolates were closely related to a pathogenic human isolate, CD 196. To our knowledge, this report is the first to describe naturally occurring outbreaks of C. difficile-associated typhlocolitis with significant morbidity and mortality in highly immunocompromised strains of mice.

Figure 1.

Number of mice affected by C. difficile in outbreak 1, including the proportions found dead or that required euthanasia, recovered, or were submitted for diagnostic evaluation in 2 multiple-investigator rooms after gradual cessation of amoxicillin administration. The total estimated population in both rooms at the time of the outbreak was approximately 1677 mice.

Figure 2.

Number of mice affected by C. difficile in outbreak 1, including the proportions found dead or required euthanasia, recovered, or submitted for diagnostic evaluation in a single-investigator room after abrupt cessation of amoxicillin administration. The total estimated population at the time of the outbreak was 724 mice.

Figure 3.

Cecum. Loss of continuity of the intestinal mucosa, with efflux of neutrophils and luminal accumulation of necrotic cellular debris. Numerous rod-shaped bacteria are present within the exudate. Prominent submucosal edema (asterisk) is shown. (A) Hematoxylin and eosin stain. (B) Gram stain. High-power magnification of luminal content containing gram-positive, rod-shaped bacteria (arrowhead; inset) with endospores (arrows). (C) Colon. Multifocal ‘volcano lesions’ (star) characterized by vertical neutrophilic exudation and sloughed off necrotic enterocytes through the damaged mucosa. Submucosal edema (asterisk) was consistently present. Hematoxylin and eosin stain. (D) Small intestine. Congestion of the villi and degeneration of the enterocyte lining (arrow). Hematoxylin and eosin stain.

Figure 4.

Analysis of percent presence or absence of proteome match for C. difficile strains CD 196, VPI 10463, Lem1, vendor-supplied sample, and C. difficile isolates from outbreaks 1 and 2 (investigators A, C, D, and E).

Figure 5.

Analysis of clustering genomes by using UMAP according to the presence or absence of PATRIC Local Families for C. difficile strains CD196, VPI 10463, Lem1, Vendor sample, 33 clinical isolates from Lewis and colleagues (2017) and C. difficile isolates from outbreaks 1 and 2 (investigators A, C, D, and E). Isolates are characterized by shape (mouse compared with human clinical isolates) and color (MLST grouping). Isolates are characterized by shape (triangle: mouse compared with circle: human) and color (MLST grouping). Proximity of isolates indicates greater genome similarity.