Low-Toxin Clostridioides difficile RT027 Strains Exhibit Robust Virulence

Abstract

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is lack of consensus on a single optimal diagnostic method for C. difficile infection (CDI). Nucleic acid amplification tests (NAAT) that detect toxin genes are highly sensitive, but their specificity limitations could inflate CDI rates. Alternate multi-step diagnostic algorithms emphasize the detection of C. difficile toxins TcdA/TcdB, and are premised on the rationale that stool toxin-negative (Tox-) CDI patients have less severe disease, shorter diarrhea duration, and fewer disease complications. There have been no systematic assessments, however, of the virulence of C. difficile strains from Tox-/NAAT+ (discrepant) specimens. In our prospective analysis of 1243 C. difficile-positive patient stool specimens from three Southern Arizona hospitals, 31% were discrepant. Ribotype 027 (RT027) strains were recovered from 221 specimens; of these, 23% were discrepant. Post-culture, RT027 strains produced a range of toxin amounts including levels lower than that of the non-epidemic strain CD630. These low-toxin RT027 (LT-027) strains harbored both tcdA and tcdB genes, and their culture supernatants were cytotoxic to cultured fibroblasts. We confirmed robust colonization and virulence of a subset of LT-027 strains using multiple rodent models; lethality in animals infected with LT-027 strains was comparable to that potentiated by a high-toxin RT027 strain. Comparative genomics and proteomics analyses of several LT-027 strains identified unique genes and altered protein abundances relative to closely-related high-toxin strains. Collectively our data highlight the robust virulence and clinical relevance of low-toxin-producing, RT027 C. difficile isolates.

Prevalence of diagnostically-discrepant Clostridioides difficile clinical specimens: insights from longitudinal surveillance

“…GDH+ stool specimens may be negative for the toxin EIA test either because they harbor non-toxigenic C. difficile strains, or because the amount of toxin in the stool sample is below the threshold of detection in the clinical test. We have demonstrated that C. difficile isolated from a subset of the latter group also produce lower levels of toxin during in vitro culture and also in the rodent intestine, relative to ribotype-matched isolates from GDH + Toxin+ specimens (16). Nevertheless, such low-toxin C. difficile strains exhibited comparable virulence to high-toxin strains in a hamster model of acute CDI (16).…”

“…We have demonstrated that C. difficile isolated from a subset of the latter group also produce lower levels of toxin during in vitro culture and also in the rodent intestine, relative to ribotype-matched isolates from GDH + Toxin+ specimens (16). Nevertheless, such low-toxin C. difficile strains exhibited comparable virulence to high-toxin strains in a hamster model of acute CDI (16).…”

“…Except for RT027, the most prevalent USA ribotypes are more likely to be isolated from GDH+/Toxin-specimens than GDH+/Toxin+ specimens. This is especially notable for rapidly-expanding ribotypes such as RT106, since they can be extremely virulent despite producing low amounts of toxin (15,16).…”

“…A previously published method was used to quantify toxin production from C. difficile isolates (16). Briefly, C. difficile strains were grown in 5 mL BHI broth for 72 h. The cultures were then centrifuged, and cell-free supernatants clarified using 0.22 μm filters (Argos Technologies, Elgin, IL, United States).…”