Salmonella dry surface biofilm: morphology, single-cell landscape, and sanitization

ABSTRACT In this study, Salmonella Typhimurium dry surface biofilm (DSB) formation was investigated in comparison with wet surface biofilm (WSB) development. Confocal laser scanning microscopic analysis revealed a prominent green cell signal during WSB formation, whereas a red signal predominated during DSB formation. Electron microscopy was also used to compare the features of DSB and WSB. Overall, WSB was unevenly scattered over the surface, whereas DSB was evenly dispersed. In contrast to WSB cells, which have a distinct plasma membrane and outer membrane layer, DSB cells are contained in large capsules and compressed. Next, microbiome single-cell transcriptomics was used to investigate the functional heterogeneity of the Salmonella DSB microbiome, with nine clusters successfully identified. Although over 60% of the dried cells were metabolically inactive, the rest of the Salmonella cells still demonstrated specific antioxidative and virulence capabilities, suggesting a possible concern for low-moisture food (LMF) safety. Finally, because sanitization in LMF industries must be conducted without water, a list of 39 flavonoids was tested for their combined effect with 70% isopropyl alcohol (IPA) against DSB, and morin induced the greatest reduction in the green:red ratio from 3.67 to 0.43. Significantly higher reductions of Salmonella viability in DSB were achieved by 10-, 100-, 1,000-, and 10,000-µg/mL morin (1.69 ± 0.25, 3.21 ± 0.23, 4.32 ± 0.24, and 5.18 ± 0.16 log CFU/sample reductions) than 70% IPA alone (1.55 ± 0.20 log CFU/sample reduction) ( P < 0.05), indicating the potential to be formulated as a dry sanitizer for the LMF industry. IMPORTANCE DSB growth of foodborne pathogens in LMF processing environments is associated with food safety, financial loss, and compromised consumer trust. This work is the first comprehensive examination of the characteristics of Salmonella DSB while exploring its underlying survival mechanisms. Furthermore, morin dissolved in 70% IPA was proposed as an efficient dry sanitizer against DSB to provide insights into biofilm control during LMF processing.