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

Perezimor Etifa; César Rodríguez; Céline Harmanus; Ingrid M J G Sanders; Igor A Sidorov; Olufunmilayo A Mohammed; Emily Savage; Andrew R Timms; Jane Freeman; Wiep Klaas Smits; Mark H Wilcox; Simon D Baines

doi:10.3390/antibiotics12030435

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

Antibiotics (Basel). 2023 Feb 22;12(3):435. doi: 10.3390/antibiotics12030435.

Authors

Affiliations

¹ Department of Food and Nutritional Sciences, School of Chemistry, Food and Pharmacy, Reading RG6 6DZ, UK.
² Facultad de Microbiología & CIET, Universidad de Costa Rica, San Pedro 11501-2060, Costa Rica.
³ Leiden University Medical Center, Department of Medical Microbiology, Albinusdreef, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
⁴ Department of Clinical, Pharmaceutical and Biological Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK.
⁵ Healthcare Associated Infections Research Group, Leeds Institute of Medical Research, University of Leeds, Leeds LS2 9JT, UK.
⁶ Department of Microbiology, Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK.
⁷ Centre for Microbial Cell Biology, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

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.

Keywords: Clostridioides difficile; RT027; antibiotics; colonisation; gut model; infection; non-toxigenic; resistance.

Grants and funding

Work at the University of Hertfordshire in this study was supported by the Centre for Agriculture, Food and Environmental Management Research (CAFEM).