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).

Production and Antimicrobial Activity of Nisin Under Enological Conditions

“…These results indicate that nisin was able to reduce the capacity of both strains to generate their biofilms, and that in combination with ethanol (4–6%) a nisin concentration of 6 μg/mL was able to prevent the formation of biofilms. These results are in agreement with recent studies that indicate that antimicrobial peptides, such as nisin, can act as biofilm disruptors due to their ability to permeabilize bacterial membranes ( Smith et al, 2016 ; Grassi et al, 2017 ). Nevertheless, when we performed the experiments of addition of the culture broth containing binary combinations of 6 μg/mL nisin and ethanol (0, 2, 4, 6, 8% v/v) on a previously formed biofilm, instead of on planktonic cells of O. oeni IS151 and L. mesenteroides J32, the results were completely different: in all cases the pre-formed biofilms enlarged after 24 h incubation with the culture broth containing the subinhibitory concentrations of both nisin and ethanol ( Figure 3 ).…”

“…With respect to nisin, to our knowledge this is the first report on the activation of biofilm synthesis by a subinhibitory concentration of this bacteriocin. Previous studies reported the inhibitory effect of nisin on the growth and biofilm formation of pathogenic bacteria, such as Listeria ( Smith et al, 2016 ), Staphylococcus ( Dosler and Mataraci, 2013 ; Okuda et al, 2013 ; Field et al, 2016a ), Streptococcus ( Tong et al, 2014 ), Enterococcus ( Taneja et al, 2015 ), Bacillus and Salmonella ( Bag and Chattopadhyay, 2017 ), Bacillus and Clostridium spores ( Egan et al, 2016 ), Pseudomonas ( Field et al, 2016b ), and Escherichia coli ( Al Atya et al, 2016 ), but none of them reported any biofilm activating effect by subinhibitory concentrations. Regarding O. oeni , a biocide effect of nisin on O. oeni cells in biofilms formed on stainless steel surfaces was already reported ( Nel et al, 2002 ).…”

“…Bacteriocins are antimicrobial peptides secreted by certain bacterial strains, which are immune to them, to compete by inhibiting the growth of other bacteria present in their environment. Nisin is a 35-mer bacteriocin of the lantibiotics group, whose use as a food preservative was approved by the European Union (food additive number E234, EEC, 1983) and the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO), and it is currently the most studied and characterized of all the bacteriocins produced by LAB ( Smith et al, 2016 ; EFSA, 2017 ).…”

Co-expression of Nisin Z and Leucocin C as a Basis for Effective Protection Against Listeria monocytogenes in Pasteurized Milk

“…Organic acids are widely used for food preservation ( Young and Foegeding, 1993 ). The antimicrobial activity of nisin or other bacteriocins against L. monocytogenes F6854 can be enhanced in the presence of organic acids ( Smith et al, 2016 ). Overall, the antibacterial activity of L. lactis N8-r-lecCI supernatant can be further enhanced in the presence of the low concentration of EDTA.…”

Biofilm formation and control strategies of foodborne pathogens: food safety perspectives

“…Nisin A bioengineered derivative in combination with available food additives, such as cinnamaldehyde (35 mg ml À1 ) or citric acid (175 mg ml À1 ), can eradicate the L. monocytogenes biolm. 73 It can be concluded that food additives could enhance the antimicrobial treatment of biolms in the food industry. 4.1.2.…”