Tolerance development in Listeria monocytogenes-Escherichia coli dual-species biofilms after sublethal exposures to pronase-benzalkonium chloride combined treatments

Abstract

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 μg/ml PRN followed by 50 μg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli. Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design.

Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride

“…Biofilm formation can protect bacteria on environmental surfaces of food processing facilities from the action of the antimicrobials used during sanitizing operations. Previous studies on multi-species biofilms that included L. monocytogenes have been limited by the use of single strains of environmental microbiota [ [60] , [61] , [62] , [63] ], or the use of other foodborne pathogens that may not be relevant in a food production environment [ 62 , [64] , [65] , [66] ]. Our study aimed to investigate the biofilm formation ability of microbial assemblages composed of environmental microbiota that co-occur with L. monocytogenes to determine whether L. monocytogenes can benefit from the production of biofilms by other bacteria.…”

Impact of Environmental Sub-Inhibitory Concentrations of Antibiotics, Heavy Metals, and Biocides on the Emergence of Tolerance and Effects on the Mutant Selection Window in E. coli

“…fliL is one of the seven genes within the flagellar-associated flaA locus that works with specific proteins to increase bacteria motility [ 33 , 34 ]. Cell motility contributes to bacteria survival and virulence, and survival due to motility does not increase the MIC of the survivors [ 35 ]. This result agrees with previous studies indicating that the exposure of E. coli to sub-MIC concentrations of BAC resulted in the expression of genes associated with efflux, outer membrane porins and motility, increasing tolerance to BAC [ 36 , 37 , 38 , 39 ].…”

Zinc mediates resuscitation of lactic acid‐injuredEscherichia coliby relieving oxidative stress

“…Antibacterial agents and physical treatments are widely used in food industry to kill or inhibit the growth of bacteria. It has been reported that many treatments caused sublethal injury of bacteria, such as organic acids, antibiotics, chlorine, iodine, heating, freezing, radiation, neutral electrolysed oxidizing water and so on (Wesche et al ; Mathieu et al ; Rodríguez‐López and Cabo ; Han et al ). Sublethal injury is induced by stresses that are not severe enough to destroy cells, and includes reversible injury of envelop, DNA, RNA and proteins.…”