Sublethal chlorine stress promotes the biofilm-forming ability of Salmonella enterica serovars enteritidis and expression of the related genes

Chlorine treatment is the most common disinfection method in food-related environments. In addition to being simple and inexpensive, this method is very effective if used properly. However, insufficient chlorine concentrations only cause a sublethal oxidative stress in the bacterial population and may alter the growth behavior of stressed cells. In the present study, the effect of sublethal chlorine stress on the biofilm formation characteristics of Salmonella Enteritidis was evaluated. Our results demonstrated that, sublethal chlorine stress (350 ppm total chlorine) activates the biofilm (csgD, agfA, adrA and bapA) and quorum-sensing (sdiA and luxS) related genes in planktonic cells of S. Enteritidis. The higher expression of these genes illustrated that the chlorine stress induced the initiation of the biofilm formation process in S. Enteritidis. Results of the initial attachment assay confirmed this finding. In addition, the number of chlorine-stressed biofilm cells was significantly higher than non-stressed biofilm cells after 48 h incubation at 37 °C. In S. Enteritidis ATCC 13076 and S. Enteritidis KL19, the number of chlorine-stressed biofilm cells were 6.93 ± 0.48 and 7.49 ± 0.57 log CFU/cm2, while the number of non-stressed biofilm cells were 5.12 ± 0.39 and 5.63 ± 0.51 log CFU/cm2, respectively. These findings were confirmed by measurements of the major components of biofilm, i.e., eDNA, protein and carbohydrate. The amount of these components in 48-h biofilms was higher when the cells were initially subjected to sublethal chlorine stress. However, the up-regulation of the biofilm and quorum sensing genes was not observed in 48-h biofilm cells, indicating that the effect of chlorine stress had vanished in the subsequent generations of Salmonella. In total, these results revealed that sublethal chlorine concentrations can promote the biofilm-forming ability of S Enteritidis.