The Cytopathic Effect of Different Toxin Concentrations From Different Clostridioides difficile Sequence Types Strains in Vero Cells.

Zohar Hamo,Maya Azrad,Boris Fichtman,Avi Peretz

doi:10.3389/fmicb.2021.763129

Zohar Hamo, Maya Azrad + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2021.763129

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Abstract

Clostridioides difficile is one of the leading causes of healthcare-associated diarrhea, with severity ranging from mild, self-limiting disease, to life-threatening toxic megacolon. C. difficile infection (CDI) pathogenesis is mediated by the TcdA and TcdB toxins. This work aimed to draw correlations between toxin levels, bacterial strains, and disease severity in 63 CDI patients. C. difficile typing was performed by multi-locus sequence types (MLST). Toxin concentrations were measured using the TOX A/B test. In addition, cell cytotoxicity assay was performed following Vero cell exposure to stool extracts (24 h). The most prevalent sequence types (ST) were ST2, ST4, ST6, ST13, ST37, ST42, and ST104, with highest toxin levels produced by ST42 and ST104 (302.5 and 297.1 ng/ml, respectively). These strains had a stronger cytopathic effect (CPE) on Vero cells as compared to strains with lower toxin concentrations (p < 0.001), as manifested by lower cell counts and higher percentages of cell rounding and adhesion loss. Although no association was found between ST, toxin concentrations, and disease severity, a diverse in vitro effect of different STs on the viability and activity of Vero cells was observed. These findings suggest that disease severity is affected by both host immune responses and by bacterial characteristics.

Highlights

Clostridioides difficile (C. difficile) is one of the most prevalent causes of nosocomial diarrhea. It is responsible for approximately 25% of antibiotic-associated diarrhea (AAD) infections and most events of pseudomembranous colitis associated with antibiotic use (Nakao et al, 2020)
The sample was mixed with a test solution that contains mouse monoclonal antibodies anti-Glutamate Dehydrogenase (GDH)/Toxin A/Toxin B conjugated to red polystyrene latex
It is intriguing to observe that high-magnification images show differences between supernatants ST6, ST13, and ST37 on cells morphology

Summary

Introduction

Clostridioides difficile (C. difficile) is one of the most prevalent causes of nosocomial diarrhea. It is responsible for approximately 25% of antibiotic-associated diarrhea (AAD) infections and most events of pseudomembranous colitis associated with antibiotic use (Nakao et al, 2020). The epidemiology of C. difficile infection (CDI) has changed, with increasing rates of incidence and recurrences. The main virulence factors contributing to CDI pathogenesis are toxin A (TcdA) and toxin B (TcdB). Following their release, these toxins catalyze the inactivation of small GTPases (such as Rho, Rac, and Ras), which results in cytoskeletal disorganization, hemorrhage, and the release

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