In vivo animal models confirm an increased virulence potential and pathogenicity of the NAP1/RT027/ST01 genotype within the Clostridium difficile MLST Clade 2

Abstract

Background Based on MLST analyses the global population of C. difficile is distributed in eight clades, of which Clade 2 includes the “hypervirulent” NAP1/RT027/ST01 strain along with various unexplored sequence types (STs). Methods To clarify whether this clinically relevant phenotype is a widespread feature of C. difficile Clade 2, we used the murine ileal loop model to compare the in vivo pro-inflammatory (TNF-α, IL-1β, IL-6) and oxidative stress activities (MPO) of five Clade 2 clinical C. difficile isolates from sequence types (STs) 01, 41, 67, and 252. Besides, we infected Golden Syrian hamsters with spores from these strains to determine their lethality, and obtain a histological evaluation of tissue damage, WBC counts, and serum injury biomarkers (LDH, ALT, AST, albumin, BUN, creatinine, Na+, and Cl−). Genomic distances were calculated using Mash and FastANI to explore whether the responses were dictated by phylogeny. Results The ST01 isolate tested ranked first in all assays, as it induced the highest overall levels of pro-inflammatory cytokines, MPO activity, epithelial damage, biochemical markers, and mortality measured in both animal models. Statistically indistinguishable or rather similar outputs were obtained for a ST67 isolate in tests such as tissue damage, neutrophils count, and lethal activity. The results recorded for the two ST41 isolates tested were of intermediate magnitude and the ST252 isolate displayed the lowest pathogenic potential in all animal experiments. This ordering matched the genomic distance of the ST01 isolate to the non-ST01 isolates. Conclusions Despite their close phylogenic relatedness, our results demonstrate differences in pathogenicity and virulence levels in Clade 2 C. difficile strains, confirm the high severity of infections caused by the NAP1/RT027/ST01 strain, and highlight the importance of C. difficile typing.

Characterization of the virulence of three novel clade 2 Clostridioides (Clostridium) difficile strains and a two-year screening in animals and humans in Brazil

“…After 48 h, animals from groups I to IV received 100 μL (containing 10 4 colony forming unit (CFU) spores) of the control reference strain (RT027/CD196), RT883, RT884, and RT885 strains by esophageal gavage, respectively. Hamsters from group V were subjected to gavage with 0.85% saline for 48 h. All animals were monitored, by trained veterinarians, every 4 h for 15 days for signs of infection, such as weight loss and wet tail (diarrhea), and to record their mortality [27]. Animals considered moribund were immediately submitted to euthanasia [28].…”

Low-toxin Clostridioides difficile RT027 strains exhibit robust virulence

“…The past 20 years have seen the emergence of outbreak-associated strains, particularly those belonging to the ribotype 027 [RT027; also called (REA) type BI or North American Pulsed-Field (NAP) type NAP1]. RT027 strains have been associated with increased prevalence and persistence in clinical settings as well as severe disease [ 5 , 6 ]. The emergence and dominance of this clade have been attributed to various properties such as fluoroquinolone resistance, expression of a variant TcdB toxin [ 7 ], and the ability to metabolize the sugar trehalose [ 8 ].…”