In this research, mono- and dual-species biofilms of Escherichia coli (O45:H2 and O121:H19) and Salmonella enterica serovar Typhimurium formed on stainless-steel coupons were treated with 100 mg/L NaClO for 1 min. Confocal laser scanning microscopy (CLSM) was applied to investigate the spatial structural dynamics of mono- and dual-species biofilms, and nuclear magnetic resonance (NMR) spectroscopy was employed to further investigate their metabolic responses toward chlorine. CLSM results indicated that mixed-species biofilms (total biovolume, 148,000 to 167,000 μm³) stimulated the growth of biomass 2 to 6 times that of single-species biofilms. Upon chlorine treatment, E. coli O45 and S. Typhimurium achieved less reduction (P < 0.05) when coexisting in mixed biofilms (0.70 and 1.17 log CFU/coupon reductions, respectively) compared with their corresponding single-species biofilms (1.97 and 2.01 log CFU/coupon reductions, respectively), while for E. coli O121, more reduction (P < 0.05) was achieved in a mixed biofilm (1.37 log CFU/coupon reductions) compared with its single-species biofilm (0.59 log CFU/coupon reductions). Moreover, NMR results suggested that the increase of putrescine (antioxidation regulator) and the decrease of glucose (enhanced glycolysis for energy replenishment) might contribute to the improved chlorine tolerance in mixed biofilms. Overall, dual-species biofilms promoted biofilm growth and their chlorine tolerance. This study improved our knowledge of the metabolic difference of single- and mixed-species biofilms of E. coli and Salmonella to chlorine sanitization and raised an urgency to investigate the effectiveness of common disinfectants against multispecies consortia. IMPORTANCE Outbreaks of Escherichia coli and Salmonella in food might be associated with the cross-contamination of biofilms on food-contact surfaces. The knowledge of the sanitization of mono-species biofilm on the food-contact surface is well established, while mixed-species biofilm occurs more naturally, which could profoundly affect the efficacy of sanitizer. Therefore, this research aims to evaluate the efficacy of using chlorine against single- and dual-species biofilms of E. coli and Salmonella along with the underlying bacterial metabolic responses. The responses of a mixed biofilm of E. coli and Salmonella to chlorine sanitization were clarified, providing insights to develop a targeted and green sanitization strategy against specific pathogens by perturbing their most susceptible metabolism pathway without sanitizer residue.

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