Synergistic Effect of Nitric Oxide Donors in Association with Sanitizers in Dispersing Biofilms of Industrial Interest

Introduction: Biofilms formed in post-harvest production facilities are recalcitrant reservoirs of pathogens, which are difficult to control. Pathogens in biofilms are resistant to common disinfectants and their mechanical removal is only partially effective. Therefore, novel approaches for controlling biofilms are needed. Recent discoveries of the function of nitric oxide in dispersing preformed biofilms offer an opportunity to test the feasibility of using this gas in industrial applications. 

Purpose: Determine the dispersal effect of several nitric oxide donors (molsidomine, MAHMA-NONOate and diethylamine NONOate sodium salt hydrate) in association with disinfectant on biofilm of Salmonella enterica, Escherichia coli and Listeria innocua preformed on surfaces of industrial interest.

Methods: Biofilms were pre-formed in appropriate media for 24 hours or 1 week in 96-well plastic plates (polypropylene, polystyrene). After incubation, medium was removed and nitric oxide donors were resuspended in Phosphate Saline Buffer (PBS) and added to the wells using concentrations of 10µM, 10nM and 10pM. Biofilms were exposed to the donors for 6 hours at 22°C and 4°C to mimic the post-harvest environments. Biofilms were further treated with selected disinfectants (DiQuat, H₂O₂, peracetic acid and Pheno-Tek II) according with the manufacturer's reccomendations. Dispersal was measured by staining the remaining biofilms using crystal violet or using a GFP labeled Salmonella strain. The donors were also tested in association with a cellulose nanocrystals hydrogel (CNC). 

Results: Molsidomine and MAHMA-NONOate alone were able to disperse at least 50% of the biofilms preformed by human pathogens Salmonella enterica 14028 and Escherichia coli O157 (EHEC). The association of the two nitric oxide donors with cellulose nanocrystals was also effective in dispersing Salmonella preformed biofilms on polypropylene up to 15% of the total biomass. In addition, the effectiveness of peracetic acid was enhanced up to 25% by pre-treating L. innocua biofilms with the donor diethylamine NONOate sodium salt hydrate.

Significance: Our results show that nitric oxide donors expand the toolset of proactive solutions for removing industrial biofilms.