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

Diarrheal infections caused by antibiotic-resistant <i>Escherichia coli</i> 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 <i>E. coli</i> (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 <i>Proteobacteria</i> 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.