A Bioengineered Nisin Derivative, M21A, in Combination with Food Grade Additives Eradicates Biofilms of Listeria monocytogenes

Abstract

The burden of foodborne disease has large economic and social consequences worldwide. Despite strict regulations, a number of pathogens persist within the food environment, which is greatly contributed to by a build-up of resistance mechanisms and also through the formation of biofilms. Biofilms have been shown to be highly resistant to a number of antimicrobials and can be extremely difficult to remove once they are established. In parallel, the growing concern of consumers regarding the use of chemically derived antimicrobials within food has led to a drive toward more natural products. As a consequence, the use of naturally derived antimicrobials has become of particular interest. In this study we investigated the efficacy of nisin A and its bioengineered derivative M21A in combination with food grade additives to treat biofilms of a representative foodborne disease isolate of Listeria monocytogenes. Investigations revealed the enhanced antimicrobial effects, in liquid culture, of M21A in combination with citric acid or cinnamaldehyde over its wild type nisin A counterpart. Subsequently, an investigation was conducted into the effects of these combinations on an established biofilm of the same strain. Nisin M21A (0.1 μg/ml) alone or in combination with cinnamaldehyde (35 μg/ml) or citric acid (175 μg/ml) performed significantly better than combinations involving nisin A. All combinations of M21A with either citric acid or cinnamaldehyde eradicated the L. monocytogenes biofilm (in relation to a non-biofilm control). We conclude that M21A in combination with available food additives could further enhance the antimicrobial treatment of biofilms within the food industry, simply by substituting nisin A with M21A in current commercial products such as Nisaplin^® (Danisco, DuPont).

Keywords: Listeria monocytogenes; bioengineered; biofilm; cinnamaldehyde; citric acid; combinations; nisin.

FIGURE 1

Agar diffusion assays depicting the antimicrobial effects of Lactococcus lactis NZ9800 strains producing the mutants M21V, M21A, AAA and nisin A (wild type) against Listeria monocytogenes F6854. Diameter zone sizes (mm) are the average of triplicate experiments.

FIGURE 2

Growth curve depicting the effect of both M21A and Nisin A (Nis A) alone and in combination with citric acid (CA) against L. monocytogenes F6854. Growth was measured via triplicate readings at OD_{600 nm} over a period of 22 h.

FIGURE 3

Growth curve depicting the effect of both M21A and Nisin A (Nis A) alone and in combination with cinnamaldehyde (CN) against L. monocytogenes F6854. Growth was measured at OD_{600 nm} over a period of 24 h. Results were recorded in triplicate.

FIGURE 4

Treatment of an established F6854 biofilm – NisA and M21A in combination with cinnamaldehyde. Treatment of established (48 h) biofilms of L. monocytogenes F6854 with M21A, nisin A (Nis A) and cinnamaldehyde (CIN) alone and in combination. Treatment was performed in triplicate over 24 h. Graph indicates the level of metabolically viable cells remaining following treatment as determined through XTT assay. ^∗∗P-value ≤ 0.01; ^∗∗∗P-value ≤ 0.001.

FIGURE 5

Treatment of an established F6854 biofilm – NisA and M21A in combination with citric acid. Treatment of established (48 h) biofilms of L. monocytogenes F6854 with M21A, nisin A (Nis A) and citric acid (CA) alone and in combination. Treatment was performed in triplicate over 24 h. Graph indicates the level of metabolically viable cells remaining following treatment as determined via XTT assay. ^∗∗P-value ≤ 0.01; ^∗∗∗P-value ≤ 0.001.

FIGURE 6

Treatment of an established F6854 biofilm – Nisaplin^® and cinnamaldehyde. Treatment of established (48 h) biofilms of L. monocytogenes F6854 with Nisaplin^® and cinnamaldehyde (CN) alone and in combination. Treatment was over 24 h and performed in triplicate. Graph indicates the level of metabolically viable cells remaining following treatment as determined through XTT assay. ^∗∗∗P-value ≤ 0.001.

FIGURE 7

Treatment of an established F6854 biofilm – Nisaplin^® and citric acid. Treatment of established (48 h) biofilms of L. monocytogenes F6854 with Nisaplin^® and citric acid alone and in combination. Treatment was over 24 h and performed in triplicate. Graph indicates the level of metabolically viable cells remaining following treatment as determined via XTT assay. ^∗∗∗P-value ≤ 0.001.