**2. Antimicrobial properties of the antimicrobial peptide nisin**

A report published in 2016 projects that resistance to antibiotics could potentially lead to 10 million deaths per year by 2050 [17]. Moreover, the estimated economic impact of microbial resistance will be massive, costing nearly 100 trillion US dollars while leading to sharp decreases in the gross domestic product. Microbial resistance against conventional antibiotic agents is a serious hazard to the effective treatment of numerous diseases. This upsurge in antibiotic resistance has stimulated research into the development of alternative antimicrobial agents. Antimicrobial peptides are considered promising alternatives to current antibiotics and have the potential to replace certain antibiotics or to be used synergistically in combination with existing antimicrobial agents [2, 18].

### **2.1. Anti-bacterial effects of Nisin**

Nisin was discovered in the same year as penicillin, but was quickly overshadowed by this antibiotic due to penicillin's ease of mass production and low manufacturing costs [19]. Nisin is a 3.5 kDa polycyclic peptide consisting of 34 amino acids and is produced by the non-pathogenic bacteria *Lactococcus lactis* [20]. Two naturally occurring variants of this peptide are nisin A and nisin Z. These two variants are structurally identical with the exception a single amino acid at position 27, where histidine occurs in nisin A while asparagine is found in nisin Z [20]. Both variants display similar antimicrobial activity but nisin Z is more soluble at neutral pH [21, 22].

In Gram-positive bacteria, nisin exhibits a dual mode of action by binding to lipid II on the bacterial membrane resulting in the inhibition of cell wall synthesis and the formation of pores in the bacterial cell membrane [23]. The antimicrobial effects of nisin Z against Gramnegative bacteria are largely inadequate. However, the activity towards Gram-negative bacteria can be improved by using ethylenediaminetetraacetic acid (EDTA) and the non-ionic surfactant Tween®80 [24, 25] (**Figure 1**).

or serves as additives to current antibiotics [2–4]. Many of the more than 2000 known AMPs have been demonstrated to exhibit broad-spectrum antibacterial activity [5], and bacteria are less

The lantibiotic nisin, produced by *Lactococcus lactis*, has promising potential for clinical application with its *Generally Regarded as Safe* (GRAS) status. This AMP was approved by the World Health Organisation (WHO) in 1969 and the US Federal Food and Drug Administration (FDA) in 1988 for the use as a food preservative [8]. Despite being extensively utilised for food preservation for nearly 50 years, there is very little indication of resistant mutants arising in food

Nisin is primarily used for its antibacterial activity. However, AMPs, and especially bacteriocins, display selectivity towards cancer cells [10]. Due to the toxicity associated with many conventional chemotherapeutic agents, as well as the development of chemotherapy resistance [11–13], there is a need for the development of novel anti-cancer therapies. Furthermore, to overcome chemotherapy resistance, the efficacy of chemotherapeutic agents can be enhanced by the co-administration of multi-functional agents to achieve synergistic interactions [14, 15]. The ability of nisin to increase the activity of the chemotherapeutic drug doxorubicin was investigated *in vivo* by Preet and co-workers. Nisin, when used in combination with doxorubicin, enhanced the anti-cancer activities of doxorubicin. Apoptosis could be detected upon treatment of mice with induced skin carcinogenesis. However, the exact mechanism by which

likely to develop resistance to these peptides compared to conventional antibiotics [6, 7].

products treated with this AMP [8, 9].

22 Cytotoxicity

nisin exerts its anti-cancer activities was not known [16].

tion with existing antimicrobial agents [2, 18].

**2.1. Anti-bacterial effects of Nisin**

**2. Antimicrobial properties of the antimicrobial peptide nisin**

A report published in 2016 projects that resistance to antibiotics could potentially lead to 10 million deaths per year by 2050 [17]. Moreover, the estimated economic impact of microbial resistance will be massive, costing nearly 100 trillion US dollars while leading to sharp decreases in the gross domestic product. Microbial resistance against conventional antibiotic agents is a serious hazard to the effective treatment of numerous diseases. This upsurge in antibiotic resistance has stimulated research into the development of alternative antimicrobial agents. Antimicrobial peptides are considered promising alternatives to current antibiotics and have the potential to replace certain antibiotics or to be used synergistically in combina-

Nisin was discovered in the same year as penicillin, but was quickly overshadowed by this antibiotic due to penicillin's ease of mass production and low manufacturing costs [19]. Nisin is a 3.5 kDa polycyclic peptide consisting of 34 amino acids and is produced by the non-pathogenic bacteria *Lactococcus lactis* [20]. Two naturally occurring variants of this peptide are nisin A and nisin Z. These two variants are structurally identical with the exception a single amino acid at position 27, where histidine occurs in nisin A while asparagine is found in nisin Z [20]. Both variants display similar antimicrobial activity but nisin Z is more soluble at neutral pH [21, 22].

The glycopeptide antibiotic, vancomycin, also binds to lipid II to inhibit cell wall synthesis, albeit at a different amino acid moiety. Vancomycin is one of the last line treatments against several Gram-positive antibiotic-resistant bacteria including methicillin-resistant *Staphylococcus aureus* (MRSA) [26, 27]. Disturbingly, clinical variants of MRSA have been isolated of which the lipid II pentapeptide have mutated to acquire resistant to vancomycin. These strains contain the *vanA*-type gene cluster where the terminal D-Ala has been changed to D-Lactate in the lipid II pentapeptide [28]. Due to its different binding motif, nisin remains active against the *vanA*-type resistant strains [29]. This shows the potential of nisin to bolster the antimicrobial defences against antibiotic-resistant bacterial strains. Nisin has a promising potential for clinical application with its GRAS status and approval by both the FDA and WHO, considering its low cytotoxicity and the fact that it is considered safe for human consumption. Currently, it is employed as a food preservative in nearly 50 countries to guard food against spoilage resulting from pathogens such as *Staphylococcus aureus, Listeria monocytogenes*, and *Clostridium botulinum* [30]. In addition, nisin has also been demonstrated to possess antibacterial activity against several clinically

**Figure 1.** Minimum inhibitory concentrations (MIC) of nisin Z for Gram-positive and Gram-negative bacterial strains. The effect of EDTA (200 μM) on the MIC of *E. coli* is also demonstrated.

relevant pathogens including vancomycin-resistant *Enterococci*, *Streptococcus pneumonia*, and methicillin-resistant *Staphylococcus aureus* [31, 32].

penicillin, chloramphenicol or ciprofloxacin, biofilm formation of *E. faecalis* was significantly

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In a previous study, we also evaluated the interaction of the nisin Z variant with conventional antibiotics [24]. Antibiotic-nisin Z combinations (1:1) were evaluated on *Staphylococcus epidermidis* (ATCC 12228) and *Staphylococcus aureus* (ATCC 12600) seeing as nisin is principally effective against Gram-positive bacterial species. Several conventional antibiotics with different mechanisms of action against Gram-positive bacteria were selected and included methicillin; vancomycin; ampicillin; tetracycline; gentamicin and novobiocin. The minimum inhibitory concentration (MIC) was used as a reflection of the bacterial cytotoxicity following exposure to the antibiotic-nisin Z combinations. The MIC was determined using a modified broth microdilution method [44], where the p-iodonitrophenyltetrazolium violet (INT) was replaced with the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The interactions between the antibiotics and nisin were determined using the fractional inhibitory concentrations (FIC) [45] and values were interpreted as ƩFIC ≤0.5 synergistic, ƩFIC >0.5–1.0 additive, ƩFIC >1.0 and <4.0 indifferent and ƩFIC ≥4.0

Bacterial treatment with nisin Z-antibiotic combinations resulted in the identification of three additive and two synergistic combinations. Nisin Z displayed an additive effect (ƩFIC >0.5 to 1.0)

> Vancomycin (2.50) 2.55 Ampicillin (16.67) **0.71** Tetracycline (80.00) 3.65 Gentamicin (1.04) 2.23 Novobiocin (1.46) **0.50**

> Vancomycin (2.50) 2.50 Ampicillin (1.04) **0.66** Tetracycline (0.47) 1.40 Gentamicin (6.67) **0.94** Novobiocin (2.29) **0.17**

Highlighted values represent ƩFIC values which indicate positive interactions between nisin Z and antibiotics where;

**ƩFIC**

**Bacterial strain MIC of nisin Z (μg/ml) MIC of antibiotic (μg/ml) Nisin Z:antibiotic (1:1)**

when combined with ampicillin and gentamicin in *S. aureus* (**Table 1**).

*S. epidermidis* Nisin Z (9.17) Methicillin (1.88) 2.68

*S. aureus* Nisin Z (10.00) Methicillin (1.88) 1.06

≤0.5 synergistic, >0.5–1.0 additive, 1.1–3.9 indifferent and ≥4.0 antagonistic.

**Table 1.** MIC values and ƩFIC values for antibiotic-nisin Z combinations.

reduced [43].

antagonistic.

Mastitis-causing *Staphylococcus* strains have a tendency to develop resistance to antibiotics [33, 34]. Nisin has been successfully applied as a sanitizer against mastitis causing *Staphylococcus* and *Streptococcus* species in lactating cows even when these species are antibiotic resistant [35, 36]. Three nisin-based products were developed for the treatment of bovine mastitis, namely Ambicin N® (Applied Microbiology, Inc., New York) and Mast Out® as well as Wipe Out® Dairy wipes (ImmuCel Corporation, Maine, USA) [30]. *In vivo* nisin has also been shown to be an effective and safe alternative to antibiotics in the treatment of staphylococcal mastitis during lactation in pregnant women [37].

Antibacterial agents possessing various modes of action are particularly of interest in the fight against antimicrobial resistance as it is considered to be more challenging for bacteria to develop resistance against multiple mechanisms concurrently. This has proven true in the case of nisin, as there is very little evidence of transmissible and stable resistance occurring after nearly 50 years of treating food products with this AMP [37–39].

## **2.2. AMPs as antibiotic adjuvant therapy**

The discovery and subsequent development of a wide range of antibiotics have revolutionised modern health care. Over the last century, the introduction of antibiotics drastically reduced morbidity and mortality. Today, antibiotics are readily available to the global population, and effective antibiotic agents have been developed against the majority of illness-causing bacteria. Ironically, the success of antibiotics has resulted in these drugs being misused, leading to the accelerated development of antimicrobial resistance amongst many bacterial species. Antibiotic resistance is making the effective treatment of numerous infections no longer achievable and there is a pressing need for alternative therapeutic approaches.

Antibiotic adjuvant therapy (to achieve synergistic interactions, although additive interactions are also favoured) can be considered as a promising strategy to combat antibiotic resistance. Combination of antibiotics and AMPs that possess different modes of action are valuable in the fight against antimicrobial resistance as it is unlikely for bacteria to develop resistance against multiple mechanisms simultaneously. Several studies have demonstrated synergism between nisin and conventional antibiotics. Nisin displayed synergism with the antibiotics, colistin and clarithromycin, against the common Gram-negative bacteria, *Pseudomonas aeruginosa* [40]. Synergistic effects were also observed with streptomycin, penicillin, rifampicin and lincomycin against *P. fluorescens* as well as the antibiotic-resistant variants of this strain [41]. Daptomycin, teicoplanin and ciprofloxacin displayed synergism against MRSA biofilms [4]. In a study by Dosler and Gerceker, nisin-antibiotic combinations were shown to have synergistic interactions against clinical isolates of methicillin-susceptible *S. aureus* (MSSA), MRSA and *Enterococcus faecalis*. A major finding from their study was that a high incidence of synergistic interactions occurred with a nisin-ampicillin combination against MSSA and nisin-daptomycin combination against *E. faecalis* strains [42]. When nisin is combined with penicillin, chloramphenicol or ciprofloxacin, biofilm formation of *E. faecalis* was significantly reduced [43].

relevant pathogens including vancomycin-resistant *Enterococci*, *Streptococcus pneumonia*,

Mastitis-causing *Staphylococcus* strains have a tendency to develop resistance to antibiotics [33, 34]. Nisin has been successfully applied as a sanitizer against mastitis causing *Staphylococcus* and *Streptococcus* species in lactating cows even when these species are antibiotic resistant [35, 36]. Three nisin-based products were developed for the treatment of bovine mastitis, namely Ambicin N® (Applied Microbiology, Inc., New York) and Mast Out® as well as Wipe Out® Dairy wipes (ImmuCel Corporation, Maine, USA) [30]. *In vivo* nisin has also been shown to be an effective and safe alternative to antibiotics in the treatment of staphylococcal mastitis

Antibacterial agents possessing various modes of action are particularly of interest in the fight against antimicrobial resistance as it is considered to be more challenging for bacteria to develop resistance against multiple mechanisms concurrently. This has proven true in the case of nisin, as there is very little evidence of transmissible and stable resistance occurring

The discovery and subsequent development of a wide range of antibiotics have revolutionised modern health care. Over the last century, the introduction of antibiotics drastically reduced morbidity and mortality. Today, antibiotics are readily available to the global population, and effective antibiotic agents have been developed against the majority of illness-causing bacteria. Ironically, the success of antibiotics has resulted in these drugs being misused, leading to the accelerated development of antimicrobial resistance amongst many bacterial species. Antibiotic resistance is making the effective treatment of numerous infections no longer achievable and there is a pressing need for alternative therapeutic

Antibiotic adjuvant therapy (to achieve synergistic interactions, although additive interactions are also favoured) can be considered as a promising strategy to combat antibiotic resistance. Combination of antibiotics and AMPs that possess different modes of action are valuable in the fight against antimicrobial resistance as it is unlikely for bacteria to develop resistance against multiple mechanisms simultaneously. Several studies have demonstrated synergism between nisin and conventional antibiotics. Nisin displayed synergism with the antibiotics, colistin and clarithromycin, against the common Gram-negative bacteria, *Pseudomonas aeruginosa* [40]. Synergistic effects were also observed with streptomycin, penicillin, rifampicin and lincomycin against *P. fluorescens* as well as the antibiotic-resistant variants of this strain [41]. Daptomycin, teicoplanin and ciprofloxacin displayed synergism against MRSA biofilms [4]. In a study by Dosler and Gerceker, nisin-antibiotic combinations were shown to have synergistic interactions against clinical isolates of methicillin-susceptible *S. aureus* (MSSA), MRSA and *Enterococcus faecalis*. A major finding from their study was that a high incidence of synergistic interactions occurred with a nisin-ampicillin combination against MSSA and nisin-daptomycin combination against *E. faecalis* strains [42]. When nisin is combined with

after nearly 50 years of treating food products with this AMP [37–39].

and methicillin-resistant *Staphylococcus aureus* [31, 32].

during lactation in pregnant women [37].

**2.2. AMPs as antibiotic adjuvant therapy**

approaches.

24 Cytotoxicity

In a previous study, we also evaluated the interaction of the nisin Z variant with conventional antibiotics [24]. Antibiotic-nisin Z combinations (1:1) were evaluated on *Staphylococcus epidermidis* (ATCC 12228) and *Staphylococcus aureus* (ATCC 12600) seeing as nisin is principally effective against Gram-positive bacterial species. Several conventional antibiotics with different mechanisms of action against Gram-positive bacteria were selected and included methicillin; vancomycin; ampicillin; tetracycline; gentamicin and novobiocin. The minimum inhibitory concentration (MIC) was used as a reflection of the bacterial cytotoxicity following exposure to the antibiotic-nisin Z combinations. The MIC was determined using a modified broth microdilution method [44], where the p-iodonitrophenyltetrazolium violet (INT) was replaced with the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The interactions between the antibiotics and nisin were determined using the fractional inhibitory concentrations (FIC) [45] and values were interpreted as ƩFIC ≤0.5 synergistic, ƩFIC >0.5–1.0 additive, ƩFIC >1.0 and <4.0 indifferent and ƩFIC ≥4.0 antagonistic.

Bacterial treatment with nisin Z-antibiotic combinations resulted in the identification of three additive and two synergistic combinations. Nisin Z displayed an additive effect (ƩFIC >0.5 to 1.0) when combined with ampicillin and gentamicin in *S. aureus* (**Table 1**).


Highlighted values represent ƩFIC values which indicate positive interactions between nisin Z and antibiotics where; ≤0.5 synergistic, >0.5–1.0 additive, 1.1–3.9 indifferent and ≥4.0 antagonistic.

**Table 1.** MIC values and ƩFIC values for antibiotic-nisin Z combinations.

Furthermore, *S. epidermidis* treated with ampicillin-nisin Z combination also showed an additive interaction. Novobiocin-nisin Z combinations showed synergistic interactions when used against *S. epidermidis* and *S. aureus.* Novobiocin, as part of the aminocoumarins antibiotic group, is able to indirectly block DNA replication by effectively inhibiting bacterial DNA gyrase. Novobiocin-nisin Z combination was particularly effective in the treatment of *S. aureus* as a dramatic reduction in the ƩFIC was witnessed. This may be due to the different, but complementary, mechanisms of actions of nisin Z and novobiocin. As the lipid II-nisin Z complex forms pores in the bacterial membrane, hydrophobic novobiocin can pass through the cell membrane to interact with the DNA gyrase of *S. aureus*. This is only speculation and the exact synergistic mechanism of should be examined further. This *in vitro* study shows the potential of nisin Z for the use as an adjuvant with conventional antibiotics. AMPantibiotic combination therapy may aid in reinforcing the defences against resistant organisms by making it more challenging for a bacterial strain to adapt to multiple antimicrobial mechanisms. Furthermore, novobiocin is used for the treatment of mastitis in lactating cows [46]; and as previously mentioned, some nisin-based products have been developed for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination especially of interest for developing novel formulations for the treatment of mastitis.

We also investigated cytotoxicity of nisin Z towards mammalian cells using the MTT assay to measure metabolic activity and the lactate dehydrogenase (LDH) assay to indicate membrane integrity. The non-malignant human immortalised keratinocyte (HaCaT) cells were employed for cytotoxicity testing and cultured under normal conditions [24]. Briefly, HaCat cells were seeded in a 96-well plate and incubated until ~90% confluent. Synthetic melittin was used (≥97% HPLC from Sigma-Aldrich) as a positive AMP control for cytotoxicity. After 24 h of exposure to nisin Z or melittin (2.5–40 μg/ml), the MTT assay was performed as described previously [24]. The ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan is considered a reflection of the number of viable cells present. Cell viability is expressed as a percentage relative to the untreated control, which was set as being 100% viable. For an assay positive control, cells were exposed to 0.01% Triton-X 100 (Sigma-Aldrich,

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To investigate the effect of the two AMPs on cell membrane integrity, the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, Madison, WO, USA) was employed. This assay determines the release of lactate dehydrogenase (LDH) into the culture media from cells with impaired cell membranes. HaCat cells were exposed to melittin and nisin Z as described earlier. A lysis solution (Promega) was used as a maximum LDH release positive control. The LDH release assay was performed as described previously [24]. Results are conveyed relative to the untreated control (set to 0% LDH release) and the maximum release

Cytotoxicity data (**Figure 2**) shows that nisin Z did not negatively affect the cell viability of

The MTT assay indicates that the ability of NAD(P)H-dependent cellular oxidoreductase enzymes to reduce MTT to formazan was not affected by the exposure to the tested nisin

**Figure 2.** Cytotoxicity assay of HaCat cells exposed to the AMPs melittin and nisin Z. (A) MTT assay and (B) LDH release assay. Vehicle control groups are represented by 0 mg/ml. Values represent mean stdev n = 3. \*\*\*p < 0.001 compared to

St Louis, MO, USA).

HaCat cells.

the vehicle control group.

sample (set to 100% LDH release).
