# Bentonite-Based Functional Nanoclay Enhances Bacteriophage Therapy against Enteric Infections via Toxin Adsorption and Microbiome Recovery

## TL;DR

This study shows that combining bacteriophage therapy with bentonite clay improves treatment of E. coli-induced diarrhea by removing toxins and restoring gut health.

## Contribution

A novel dual-action strategy using phage and bentonite for toxin adsorption and microbiome recovery in treating enteric infections.

## Key findings

- Combination therapy with phage EC.W2-6 and bentonite achieved 100% survival in a murine diarrheal model.
- Bentonite reduced outer membrane vesicles from ETEC by up to 3.56-fold and neutralized phage surface charge.
- Dual therapy restored gut microbial diversity and suppressed Proteobacteria expansion.

## Abstract

Diarrheal infections caused by antibiotic-resistant Escherichia coli pose a serious threat to human and animal health, driving the need for innovative therapeutic strategies. This study introduces a dual-action strategy that integrates bacteriophage EC.W2-6 with bentonite to enhance bacterial clearance and macromolecular toxin removal. Phage EC.W2-6 demonstrated high specificity against enterotoxigenic E. coli (ETEC) H10407, achieving nearly 100% adsorption to host cells within 15 min and a moderate burst size of approximately 80 plaque-forming units per infected cell. Bentonite exhibited substantial dose-dependent binding of ETEC-secreted proteins and outer membrane vesicles (OMVs), with the 30-g treatment showing the highest efficiency. Nanoparticle tracking analysis confirmed a 3.56-fold reduction in OMVs at 5 g bentonite and near-complete removal at 30 g. Physicochemical analysis indicated a stabilizing effect of bentonite, showing that bentonite–phage association partially neutralized the phage surface charge (from −34.2 to −13.4 mV), forming a more stable colloidal complex with an approximately 2-fold decrease in colloidal size. In a murine diarrheal model, single therapy with either EC.W2-6 (multiplicity of infection = 0.1) or 8% bentonite conferred 60% survival, whereas combination treatment provided 100% protection with a synergistic effect. Microbiome analysis revealed that dual therapy restored gut microbial diversity and suppressed Proteobacteria expansion, closely resembling healthy controls. These findings highlight the therapeutic potential of combining bentonite with phage therapy as an integrated macromolecular intervention against ETEC-induced diarrhea and intestinal dysbiosis.