Inhibition of L. monocytogenes Biofilms by Plasma-Deposited Antibacterial Layers

Bacteria attach to surfaces of food processing equipment forming biofilms that can contaminate food and cause foodborne diseases. We will use plasma technologies to modify these surfaces to inhibit bacterial attachment and biofilm formation, thereby enhancing food safety.
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Since bacterial attachment is a first step in biofilm formation, any surface that can inhibit attachment should also decrease the potential for biofilm formation. It has been shown that poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) deposited onto the surfaces of various materials reduces the adsorption of proteins, cells such as platelets, and bacteria. We will use cold plasma to deposit PEG-like structures onto a variety of substrate (e.g., stainless steel, rubber, plastic) surfaces. The chemical structures and characteristics of the resulting macromolecular structures obtained under different plasma parameters will be evaluated, and the relative potential of the modified surfaces to inhibit bacterial attachment and biofilm formation will be determined. In addition, we will evaluate the structures and the stability of the biofilms formed. Survival of biofilm cells stored under different conditions encountered in a food-processing environment will be determined. We will also determine the relative ability of different detergents and sanitizers to remove and inactivate biofilm cells formed on the various modified and unmodified surfaces. The stability of the plasma-deposited macromolecules and their ability to inhibit bacterial attachment on storage will be evaluated. The findings will enable us to determine which types of macromolecular structures on a given substrate are effective in inhibiting biofilm formation, and the specific plasma conditions for generating these structures. Silver and its compounds have been used as antimicrobial agents for many years. Currently, silver is used in many medical applications and water treatment. It was recently approved as a preservative in polymeric coatings for polyelefin films intended for use in contact with food. We have developed a plasma-mediated method for deposition of silver onto polymer surfaces. In the proposed research, anti-bacterial silver coatings will be developed on plasma-functionalized polymer surfaces in a two stage reaction. The presence of silver layers and surface morphologies will be analyzed. We will determine the ability of these silver coated polymer surfaces to inhibit attachment and biofilm formation by L. monocytogenes. The stability of the modified surfaces on storage will also be monitored. Modified and unmodified surfaces with and without biofilms will be analyzed by survey and high resolution electron spectroscopy for chemical analyses, attenuated total reflectance Fourier transformed infrared spectroscopy, auger electron microscopy, energy dispersive X-ray spectroscopy, and contact angle measurements. Surface topographies will be evaluated by scanning electron microscopy and atomic force microscopy. The nature and relative ratios of plasma generated gas phase species will be monitored by mass spectrometry and optical emission spectroscopy. The degree of injury and survival of biofilms will be assessed by plate counts on nonselective and selective agar media with and without prior enrichment.
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The overall goal of this research was to use plasma-aided technologies to develop antibacterial surfaces that will inhibit biofilm formation by L. monocytogenes. One strategy is to deposit or incorporate bactericidal compounds on surfaces. A second strategy is to coat surfaces with structures, such as poly(ethylene) glycol (PEG), that will inhibit bacterial attachment and biofilm formation. Silver has been used as a disinfectant due to its strong bactericidal activity and low toxicity to mammalian cells and tissues. Silver nano-particle thin layers were deposited onto formaldehyde-radio frequency (RF)-plasma-functionalized food- and medical-grade silicone rubber, stainless steel, and paper surfaces. The silver deposition was carried out under ex-situ plasma conditions using the Tollens reaction. Results from survey and high-resolution electron spectroscopy for chemical analysis (ESCA), scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy dispersive X-ray spectroscopy investigations confirmed the presence of thin silver layers on the plasma-exposed and subsequently modified substrate surfaces. In addition, SEM and AFM demonstrated the nano-particle-based morphology of the deposited layers. Our results showed that thin macromolecular layers bearing aldehyde functionalities could be deposited onto silicone rubber, stainless steel, and paper surfaces. The bactericidal properties of the silver-coated surfaces were demonstrated by exposing them to Listeria monocytogenes. Decreases of over 60 to 90% in bacteria were observed after 6 h on silver-coated silicone rubber surfaces, and no viable bacteria were detected after 12 to 18 h. To generate PEG structures on silicone rubber (both food and medical grade) surfaces, we used several approaches: grafting primary amine-terminated PEG onto argon plasma modified surfaces; grafting PEG onto dichlorosilane functionalized surfaces; generation of polyglycidol branched structures; and generation of linear polyglycidol brush structures. ESCA results showed that PEG-like structures were present on all surfaces plasma modified by the different approaches. The most efficient method was grafting PEG 400 onto a dichlorosilane-functionalized surface. We showed that argon-plasma generated surface free radicals could react efficiently with dichlorosilane in the absence of plasma, generating halo-silane groups, which enabled covalent bonding of PEG molecules to the modified rubber surface. The presence of a PEG coating layer was confirmed by chemical derivatization with trifluoroacetic anhydride, and analysis by AFM and SEM. About 90% reduction in L. monocytogenes biofilm formation was observed with the plasma-treated and PEG-grafted medical grade rubber compared to the untreated surfaces, while less reduction (about 50%) was observed with the PEG-grated food grade rubber, which could be due to the lower density of PEG on this surface. PEG-like structures generated by the other approaches did not inhibit biofilm formation by L. monocytogenes. The mechanisms by which different PEG structures exhibit anti-fouling characteristics require further study.
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Results generated from this study will be useful in developing surfaces used in food processing that can minimize the potential for contamination by foodborne pathogens such as L. monocytogenes.