Food Decontaminating Activity of Acyclovir Against Waterborne Salmonella enterica Persisters Using Drug Repurposing Strategy

ABSTRACT

This study aimed to repurpose the marketed antiviral drug, Acyclovir, by screening for food decontaminating activity against a strain (HCB1) isolated from contaminated water, Salmonella enterica persister cells. Salmonella isolation agar was used to isolate HCB1. The identification was based on colony morphology and 16S rRNA sequence analysis. A persister cell assay was carried out using an extended growth curve assay in the presence of Azithromycin, a prescription drug. Antibacterial and antipersister activity of Azithromycin and Acyclovir was assessed using a disc diffusion assay with determination of minimum inhibitory concentration (MIC), growth curve profiling, and optical microscopy. Surface morphology was also investigated using electron microscopy. Molecular docking was performed to elucidate interactions between Acyclovir and D‐Ala‐D‐Ala Ligase A, a cell wall enzyme of Salmonella enterica . Statistical analysis was carried out using one‐way ANOVA on the dependent variables, namely the incubation period and corresponding absorbance values. The chicken breast tissue Salmonella contamination assay was performed using a novel approach not reported previously. The bacterium HCB1 was identified as a strain of Salmonella enterica as evidenced by culture plates, microscopic features, and 16S rRNA sequencing. The presence of persisters was confirmed by a double‐zone inhibition pattern as well as a long stationary phase executed by the test bacteria in the presence of Azithromycin. In contrast, no double zone of inhibition, short stationary phase, and disrupted cellular integrity were observed in the presence of Acyclovir, at a concentration of 65 μg/ml. Both optical and electron microscopy inferred cellular disruption in Acyclovir‐exposed bacterial cells. The molecular docking simulations between Acyclovir and bacterial receptor proteins predicted the establishment of seven conventional H‐bonds, having binding affinities between Acyclovir and Ligase A (−6.9 kcal/mol), and hydrophobic interactions suggesting interference in cell wall synthesis. The one‐way ANOVA inferred statistical significance at p  < 0.05. There was a remarkable decontaminating activity of Acyclovir against the Salmonella‐contaminated chicken breast tissue, at a concentration of 26 μg/gm against 10 ³  CFU/ml. The findings demonstrate that Acyclovir possesses potent antibacterial activity against Salmonella enterica persisters, with evidence of cell wall targeting and bactericidal effects. This positions Acyclovir as a promising candidate for drug repurposing against drug‐tolerant Gram‐negative foodborne pathogens, Salmonella enterica . The Chicken breast tissue assay substantiated the decontaminating activity of Acyclovir.