Feasibility of using nitric oxide donors to disperse biofilms of industrial significance to strengthen the efficacy of current industrial disinfectants.

Biofilms formed on industrial surfaces in post-harvest production facilities are recalcitrant reservoirs of pathogens, which are difficult to control. Pathogens in biofilms are resistant to common disinfectants and can contaminate produce during post-harvest handling. Even though mechanical removal of biofilms is one of the most common methods for controlling them in the production facilities, this approach is only partially effective and only removes biofilms that are easily accessible. Therefore, novel approaches for controlling biofilms are needed. We will test how effectively hydrogels containing Nitric Oxide Donors disperse biofilms under the conditions that mimic production environment and increase sensitivity of the dislodged biofilms to quaternary ammonium salts or chlorine dioxide. Four compounds will be tested at nano- to picomolar concentrations to determine how effectively they dislodge biofilms (individually or in combination with disinfectants or detergents) formed by mixtures of Salmonella, Listeria or E.coli and Leuconostoc. Upon completion of this feasibility study, we expect to have identified nitric oxide donors that are capable of dislodging existing biofilms. We envision that further studies will focus on designing foaming agents containing these molecules for dislodging biofilms from surfaces in post-harvest facilities prior to their cleaning with common disinfectants.

Biofilms formed on industrial surfaces in post-harvest production facilities are particularly problematic. Biofilms serve as recalcitrant reservoirs of pathogens and are difficult to control. While there are several innovative strategies for reducing microbial attachment to the surfaces of industrial significance, the tool-set for disrupting existing biofilms is significantly less versatile. Recent discoveries of the function of nitric oxide in dispersing existing biofilms offer an opportunity to test the feasibility of using this gas in industrial applications. The rationale for this approach is offered by the studies that demonstrate that nitric oxide “donor” molecules (NODs) significantly decrease surface areas of biofilms under the laboratory conditions. More than 105 NODs are currently available, but only few of them were tested on enteric pathogens; fewer yet were explored for the potential industrial applications. If proven effective in dispersing biofilms formed on industrial surfaces in post-harvest facilities, we see several potential cost-effective applications for disrupting biofilms. The dual action of the NOD more the disinfectant can be significant: while NO proceed to the dispersion of the biofilm, disinfectant can easily kill planktonic cells. Therefore, with this one-year pilot study, four non-toxic NODs and their mixtures with disinfectants and detergents will be tested to determine efficacy of these compounds in dispersing biofilms and disrupting attachment of mixtures of Salmonella enterica, pathogenic E. coli, Listeria and Leuconostoc to polypropylene, polystyrene and stainless steel when associated with quaternary ammonium salts and chlorine dioxide.

Objective 1. To establish which of the NODs is most effective at low concentration in disrupting existing biofilms formed on polypropylene, polystyrene and stainless steel by mixtures of Salmonella enterica pathogenic E. coli and Listeria monocytogenes isolated from recent outbreaks 

Objective 2. To test whether hydrogels enriched with NODs associated with quaternary ammonium salts or chlorine dioxide possesses a synergistic effect in promoting biofilm dispersion from plastics and stainless steel

The scientific findings are summarized in five key-points and they are supported by recommendation for their future development. 

1. Dispersal potential. All the nitric oxide donors tested were effective as biofilm dispersal. All five nitric oxide donors induced significant (15-80%) dispersal of 24-hour old biofilms, however, the degree of dispersal and the optimal conditions varied when dissolved in phosphate saline buffer. Some donors are more efficient than others, for example moldisomine and MAHMA NONOate have shown the strongest activity, possibly due to their chemical structure. In addition, the donors showed different dispersion potentials when used in different conditions (for example different temperatures, contact times, or materials affected the biofilm dispersal). Further research should be focused in characterizing the more effective chemical structures and the best conditions for the treatments. 

2. Association of nitric oxide donors with other molecules. Our research has demonstrated the synergistic effect of the nitric oxide donors moldisomine and MAHMA NONOate in association with disinfectant and cellulose nanocrystals hydrogel (CNC). This aspect is extremely important in industry: for example the association with CNC has proved to reduce the effective contact time up to 2 hours on well established biofilms (1 week-old Salmonella biofilms). The association with molsidomime and MAHMA NONOate with the disinfectant Sanidate12.0 has also shown that the association was ~15% more effective in removing the preformed biofilm when compared with the Sanidate 12.0 treatment only. Further research should focus on the study of the association of donors with other disinfectants, hydrogels and foaming solutions. 

3. Nitric oxide diffusion (in collaboration with the Co-PI Dr. Eric McLamore). Diffusion is the predominant transport process within hydrogels and cell aggregates. To that end, we measured the nitric oxide diffusion of nitric oxide in MAHMA NONOate-CNC. We characterized the flux, diffusion, and the releasing time; all these data are important in order to define the chemical characteristic of this association and to predict the nitric oxide release profile in different conditions. However, for a better understanding of nitric oxide diffusion we advise that future research should be focused on the measurement of nitric oxide penetration into the biofilm under various donor exposures.

 4. Genetics. In this project we established that the recA-hydN genomic region is involved in Salmonella biofilm dispersal. Further genetic studies need to well characterize this region and clarify the metabolic cascade upon nitric oxide perception. Further research should be addressed to determine how physico-chemical properties of surfaces, temperatures and nitric oxide donors contribute to the regulation of Salmonella regulatory and structural determinants during nitric oxide-mediated biofilm dispersal. 

5. Clinical trials. Nitric oxide donors are not considered as “Generally Recognized As Safe” (GRAS). Even though some donors are used as therapeutic agents for treatment of cardiovascular diseases, the concentrations we are showing as effective in biofilm dispersal are about 10-7 less than the therapeutic dose. It is important to recognize that it is almost certain that no GRAS compound or an herbal extract would ever be costeffective and efficient for disrupting industrial biofilms. Clearly, addressing potential toxicity of nitric oxide donors will be an important consideration before proceeding to scale up.