Receptor-specific phage resistance induced phenotypic and gene expression shifts in Salmonella Typhimurium impacting biofilm formation on food contact surfaces

초록

Bacteriophages are gaining increasing attention as targeted biological control agents against multidrug-resistant (MDR) foodborne pathogens. However, repeated exposure to bacteriophages can induce the emergence of bacteriophage-resistant mutant (BIM) strains, which may exhibit altered physiological characteristics that impair therapeutic efficacy. This study investigated the phenotypic and molecular adaptive mechanisms of multidrug-resistant Salmonella Typhimurium BIMs that acquired resistance to receptor-specific bacteriophages (STP-1 and STP-2) targeting O-antigen and flagella. Two representative BIM strains—MO-4 (O-antigen mutant) and MF-6 (flagella mutant)—were analyzed using phenotypic microarray, motility and biofilm experiments, confocal microscopy, and qRT-PCR. MO-4 exhibited extensive metabolic changes, including 10 alterations in carbon source utilization and increased resistance to 11 antibiotics, while MF-6 showed changes in sensitivity to osmotic and pH stress and increased susceptibility to 5 antibiotics. Both BIM strains exhibited reduced motility but maintained similar or enhanced biofilm formation ability on food contact surfaces. Confocal microscopy analysis revealed structurally distinct biofilms with reduced thickness and increased density. qRT-PCR analysis showed receptor-specific transcriptional changes: MO-4 lost rfaL expression, supporting O-antigen deficiency, while MF-6 showed increased fljB expression and decreased fliK expression, indicating changes in flagellar regulation. Both BIMs showed differences in the expression of tolC, rpoS, and luxS, suggesting changes in efflux, stress response, and quorum sensing. These results highlight receptor-dependent adaptation of phage-resistant Salmonella variants and emphasise the need to evaluate both genetic and phenotypic outcomes when designing effective phage-based control strategies for food safety.

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