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Control of Listeria monocytogenes on Food-Contact and Noncontact Surfaces by Antimicrobial Coatings

foodsafetymagazine.com by Keith Warriner, Ph.D., and Kayla Murray, M.Sc.,, February March 2018

The number of product recalls attributed to suspected Listeria monocytogenes contamination has increased exponentially since 2013. In 2017, there were at least 100 separate recalls of a diverse range of products, from the traditional soft cheese to the more unusual energy bars. The reasons for the increased recalls are a combination of testing with more sensitive diagnostics along with the power of next-generation sequencing-based typing techniques.[1–3] Consequently, whereas previously Listeria in foods would have been missed or attributed to an unknown source, today, the probability of detection is much higher.

From a food safety prospective, product recalls are an essential step to prevent foodborne illness. On the counter side, recalls lead to multimillion-dollar losses, in addition to eroding consumer confidence, or consumers becoming fatigued and inclined not to notice recall announcements.[4] More importantly, recalls represent the failure of industry to control L. monocytogenes despite the implementation of food safety management systems.

Getting to the Root of Listeria Contamination
In the course of foodborne listeriosis outbreak investigations, a breakdown in plant sanitation is frequently cited as the root cause.[5] It is well established that L. monocytogenes can become endemic within processing environments by building, or becoming incorporated into, biofilms that can persist for years.[6] Individual studies have correlated the persistence of L. monocytogenes with phenotypic traits such as attachment strength, biofilm formation, high growth rate, sanitizer resistance, phage resistance, expression of stress genes and low virulence.[7] Yet, an equal body of evidence has suggested that the aforementioned phenotypes do not differ between persistent and transient L. monocytogenes strains. Moreover, no genetic differences have been identified to differentiate persistent versus transient strains.[8] It has been suggested that rather than genotypes and phenotypes of L. monocytogenes, it is the endogenous population that aids the persistence of the pathogen by the formation of biofilms and/or the modulation of the activity of Listeria.[9]

Sanitation Is Key to L. monocytogenes Control
The benefits of even the deepest sanitation are lost early into a processing activity, as both organics and contamination start accumulating on lines along with the continuation of biofilm formation.[10] Therefore, even a processing facility operating a diligent sanitation routine can be open to the risk of establishing an endemic L. monocytogenes population.[11] It follows that prevention of biofilm formation is key to controlling Listeria given that once formed, the films can be difficult, if not impossible, to remove.

Antimicrobial Coatings That Prevent Biofilm Formation
Antimicrobial coatings
As a supplement to routine sanitation, antimicrobial coatings as a means of continuous protection against biofilm formation have attracted interest. Antimicrobial coatings are based on several principles, the simplest (first generation) being as a protective coating to cover or prevent scratches that would provide nucleation sites for biofilm formation.[12] Other coatings are hydrophobic and act to prevent attachment of microbes to surfaces (Figure 1). For example, polyethylene glycol and diamond-like carbon are effective at preventing microbial adhesion on plastic surfaces. A further approach is to alter the surface topography to impart high hydrophobicity that resists microbial attachment.[13] For example, coatings that mimic sharkskin or leaf surfaces support weak microbial attachment. Although effective, such coatings only displace bacteria rather than inactivate them, which ultimately limits their utility.[14] 

The next generation of coatings developed impregnated antimicrobial agents within a polymer base (Figure 1). The most commercially successful were coatings based on polymers impregnated with triclosan to inactivate microbes on contact. Yet, the triclosan leaches out over time, thereby diminishing the protective effect. The decreased antimicrobial efficacy coupled with the environmental impact of triclosan has led to phasing out of this antimicrobial coating. Moreover, exposure of Listeria to sublethal concentrations of triclosan has been linked to development of drug resistance.[15]

The antimicrobial properties of silver are well established.[12] The function of the coatings is based on the leaching of silver ions over time that are assimilated into the microbial cells where the ions disrupt proteins (Figure 1). L. monocytogenes exposed to silver has been shown to initially decrease in number, but regrowth occurs due to emergence of resistant mutants.[16] In addition, silver gradually leaches out of films, thereby providing only short-term benefits. The same disadvantages have been observed for copper coatings, with additional cytotoxic concerns being voiced.[17] Antimicrobial coatings have been prepared using films impregnated with quaternary ammonium salts, chitosan, bacteriophages and bacteriocins. However, the majority of coatings have been tested only under laboratory conditions, with very few being commercialized due to economic feasibility and technical challenges.

Direct food-contact antimicrobial coatings
The key limitation of current food-contact surface coatings is that many have to be applied at the point of manufacture of the equipment. Consequently, when the antimicrobial agent (e.g., silver) is depleted, the units have to be disassembled, then sent for recoating. An alternative approach is to apply antimicrobial paint. Although more convenient to apply compared with other coatings, it cannot be used on food-contact surfaces due to the risk of toxic residues from the coating migrating into the product.[18] In this regard, relatively few coatings that include binders to adhere to substrates such as stainless steel, plastic or rubber can be directly applied on food-contact surfaces.[19] The U.S. Food and Drug Administration regulatory approval for food-contact coatings is described in 21 C.F.R. 177.168, which essentially provides a list of permitted polymer and resin materials.[20]

Considering the permitted polymers/materials for food-contact surfaces, several antimicrobial coatings have been developed, with those modified with titanium dioxide being of particular interest.[21] Anatase titanium dioxide is widely used as a photocatalyst and antimicrobial surface coating, among other applications (Figure 1).[22] The underlying mechanism relies on the formation of free radicals from the breakdown of water and oxygen following illumination with UV light in the range of 254–395 nm (UV-C to UV-A).

Titanium dioxide has been introduced within a range of polymer bases, which retain the antimicrobial agent and also provide mechanical robustness to the coating. For example, polyurethane modified with titanium dioxide was assessed for inactivating a surface inoculated with L. monocytogenes, Salmonella, Pseudomonas aeruginosa and Escherichia coli.[23] Although the surface had good durability, the log reductions of the aforementioned bacteria were limited to 0.5–1.0 log CFU despite illumination of the modified film with UV-A.

Polylactide as a Food-Contact Antimicrobial Coating
Polylactide (PLA) is a biodegradable polymer produced from lactate and lactide monomers derived from the fermentation of raw materials such as corn, sugar cane or other sources of sugar. PLA has a generally regarded as safe status and has been applied in food-contact applications such as tea bags, single-serve coffee containers, jugs, cups, linings and coatings (Figure 2).[24]

PLA can be coated on a range of materials, including paper, ceramic, glass, rubber and stainless steel to produce high-adherent coatings with mechanical robustness.[25–27] 

As a coating, PLA has a weak negative charge that prevents strong attachment of microbial cells and exhibits a degree of antimicrobial activity.[25,28] Antimicrobial agents such as silver and titanium dioxide can be incorporated into PLA by inclusion during film formation or via surface modification.[29–31] PLA modified with silver ions has been reported to support a 5-log CFU reduction of L. monocytogenes, Staphylococcus aureus, E. coli and Salmonella, although the leaching rate of silver ions was high.[32] PLA modified with titanium dioxide has been shown to inhibit the growth of Klebsiella pneumoniae and S. aureus using an agar plate assay.[33]

In a further report, Fonseca et al.[34] fabricated polylactide modified with titanium dioxide nanoparticles and assessed antimicrobial activity by submerging coupons in brain-heart infusion broth inoculated with E. coli, then illuminated with UV-A. The authors reported a 94 percent reduction on E. coli numbers compared with control cultures, although it was unclear if this could be attributed to bacteriostatic or bactericidal activity. A more detailed study examined the antilisterial properties of biodegradable PLA coatings modified with titanium dioxide.[35] It was demonstrated that PLA alone could support a 2.84 ± 0.10-log CFU reduction of L. monocytogenes when incubated at 23 °C for 2 hours, although the log reduction was increased to greater than 4-log CFU when titanium dioxide was incorporated into the PLA film during casting and illuminated with UV-A. The coating was stable in repeated (up to five) sanitation cycles consisting of detergent and sodium hypochlorite rinses.[35]

N-halamine is a further food-contact antimicrobial coating gaining interest with the added advantage of being regenerated by activation with hypochlorite.[36] However, how halamine films function under commercial processing conditions remains to be assessed.

Conclusions
The number of product recalls due to suspected L. monocytogenes contamination is forecast to increase, while our ability to detect the pathogen outperforms our ability to control it. The first protective coatings were introduced in the 1990s and have undergone several generations of development, especially given innovations in packaging and the clinical sector. The generation of food-contact-compatible polymers modified with antimicrobial agents has strong potential for Listeria control. The most successful coatings would probably be applied as a film following a sanitation activity as opposed to surfaces being manufactured with impregnated antimicrobials. This is based on cost aspects in addition to the short-term stability of impregnated coatings. In this respect, the biggest knowledge gap that exists relates to how antimicrobial coatings perform under commercial conditions. This and other aspects of antimicrobial coatings will be explored in the near future.