Bentonite-Based Functional Nanoclay Enhances Bacteriophage Therapy against Enteric Infections via Toxin Adsorption...

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.