Binding and neutralization of C. difficile toxins A and B by purified clinoptilolite-tuff.

Carmen Ranftler,Dietmar Nagl,Andreas Sparer,Andreas Röhrich,Florian Koban,Cornelius Tschegg,Ali El-Kasaby,Shahrooz Nasrollahi Shirazi,Stephane Nizet,Michael Freissmuth,Pradeep Dudeja

doi:10.1371/journal.pone.0252211

Abstract

Clostridioides difficile (C. difficile) infection is a major public health problem worldwide. The current treatment of C. difficile-associated diarrhea relies on the use of antibacterial agents. However, recurrences are frequent. The main virulence factors of C. difficile are two secreted cytotoxic proteins toxin A and toxin B. Alternative research exploring toxin binding by resins found a reduced rate of recurrence by administration of tolevamer. Hence, binding of exotoxins may be useful in preventing a relapse provided that the adsorbent is innocuous. Here, we examined the toxin binding capacity of G-PUR®, a purified version of natural clinoptilolite-tuff. Our observations showed that the purified clinoptilolite-tuff adsorbed clinically relevant amounts of C. difficile toxins A and B in vitro and neutralized their action in a Caco-2 intestinal model. This conclusion is based on four independent sets of findings: G-PUR® abrogated toxin-induced (i) RAC1 glucosylation, (ii) redistribution of occludin, (iii) rarefaction of the brush border as visualized by scanning electron microscopy and (iv) breakdown of the epithelial barrier recorded by transepithelial electrical resistance monitoring. Finally, we confirmed that the epithelial monolayer tolerated G-PUR® over a wide range of particle densities. Our findings justify the further exploration of purified clinoptilolite-tuff as a safe agent in the treatment and/or prevention of C. difficile-associated diarrhea.

Highlights

Over the past three decades, Clostridioides difficile (C. difficile), previously known as Clostridium difficile, a gram positive, anaerobic, spore-forming bacillus has emerged as a major nosocomial pathogen [1, 2]
We assessed the proportion of dead cells by flow cytometry after propidium iodide staining: it is evident from Fig 1B that–regardless of the presence or absence of G-PUR1 –over 95% of Caco-2 cells excluded propidium iodide
Our experiments showed that the purified clinoptilolite-tuff preparation G-PUR1 bound C. difficile toxins A and B with high affinity

Summary

Introduction

Over the past three decades, Clostridioides difficile (C. difficile), previously known as Clostridium difficile, a gram positive, anaerobic, spore-forming bacillus has emerged as a major nosocomial pathogen [1, 2]. A substantial fraction of C. difficile-associated diseases (CDAD) are community-acquired [3]. The Center for Disease Control and Prevention (CDC) estimated that, annually, there were about 500,000 C. difficile infections (CDI) and close to 30,000 C. difficile-related deaths in the USA [4]. A Europe-wide coordinated surveillance program, which was initiated by the European Centre for Disease. Binding of clostridial toxins to purified clinoptilolite-tuff additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

Methods

Results

Conclusion