Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model

Abstract

Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI.

Advances in Therapeutic Strategies for the Management of Clostridioides difficile Infection

“…Moreover, direct competition with a non-toxic C. difficile strain (NTCD) can be used as preventive strategy of CDI. In vitro models of the human gut have shown that inoculation with an NTCD successfully prevents the development of CDI with the hypervirulent strain RT027 after the administration of a number of different antibiotics [ 54 ].…”

Clostridioides difficile infections; new treatments and future perspectives

“…Another method in the prevention of CDI is through direct competition with a nontoxigenic C. difficile strain (NTCD). In vitro models of the human gut have shown that prior inoculation with a NTCD strain successfully prevented the development of simulated CDI with the hypervirulent RT027 strain after administering a range of different antibiotics [ 54 ]. Recently, oral immunization of mice with spores from a genetically modified NTCD strain (NTCD_Tcd169) to target both C. difficile toxins and adhesion/colonization factors resulted in effective protection against hypervirulent strain RT027 R20291 and reduced excretion of R20291 spores when compared to treatment with NTCD strains [ 55 ].…”