The lantibiotics are the most extensively studied group of bacteriocins (Cotter et al., 2013). These bacteriocins are post-translationally modified antimicrobial peptides, characterized by the presence of unusual amino acids, and their structure involves lanthionine and β-methyl lanthionine intramolecular bridges. During the formation of these intramolecular bridges, serine and threonine residues are dehydrated to dehydroalanine and dehydrobutyrine respectively (Cotter et al., 2013). In one study, the most thoroughly investigated lantibiotic, nisin (at concentrations ranging from 1.5 to 16 μg/ml), was combined with the glycolipodepsipeptide ramoplanin (at concentrations ranging from 0.38 to 1.5 μg/ml), both of which target lipid II, and the combination resulted in synergistic interactions against 14 out of 20 MRSA strains assessed (Brumfitt et al., 2002). Resistance to methicillin in S. aureus is a significant problem and is mediated by the expression of PBP2a (penicillin binding protein 2a) instead of, or in addition to, the regular PBP (Hackbarth et al., 1995; Stapleton and Taylor, 2002; Chambers and Deleo, 2009). Furthermore, β-lactamase expression is also known to contribute to resistance to methicillin amongst S. aureus strains (Montanari et al., 1996), rendering such MRSA strains amongst the most challenging pathogens to target in clinical settings. In contrast to the effective nisin-rampolanin interactions however, the study by Brumfitt et al., showed that a combination of nisin with 3–12 μg/ml chloramphenicol (which inhibits protein synthesis in bacteria) yielded antagonistic effects against these MRSA strains (Brumfitt et al., 2002).

Enterococcus faecalis is another nosocomial pathogen and is an etiological agent of endocarditis, urinary tract infections and other systemic infections, and has exhibited resistance to several groups of antibiotics including aminoglycosides, daptomycin, quinolones, macrolides, rifampicin and β-lactams, amongst others (Murray, 1990; Johnston and Jaykus, 2004; Flores-Mireles et al., 2015). A study by Tong et al., demonstrated that the presence of 200 U/ml nisin led to a reduction in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 18 different antibiotics against E. faecalis. The combinations of 200 U/ml nisin with 1–16 μg/ml chloramphenicol or with 2 μg/ml penicillin were especially effective, with statistically significant improvements in MIC values in the presence of nisin. Synergistic interactions between nisin in combination with either penicillin or chloramphenicol against three E. faecalis strains were also apparent with checkerboard assays. In addition, transmission electron microscopy (TEM) demonstrated that these combinations were highly effective at destroying E. faecalis cells (Tong et al., 2014a). In another study, the lipid II-binding lantibiotic, actagardine, was combined with the antibiotics metronidazole, vancomycin and ramoplanin against several Clostridium difficile isolates (Mathur et al., 2013). C. difficile is primarily a nosocomial pathogen and is the causative agent of C. difficile-associated diarrhea (CDAD), which often occurs due to perturbations of the gut microbiota resulting from broad-spectrum antibiotics (Rea et al., 2010; Leffler and Lamont, 2015). Interestingly, it was revealed that actagardine in combination with ramoplanin behaved in a partial synergistic/additive fashion against 61.5% of the target C. difficile strains assessed in the relatively recent study (Mathur et al., 2013). Actagardine-metronidazole and actagardine-vancomycin combinations were also effective with partial synergistic/additive effects obtained against 54% and 38% of C. difficile strains respectively (Mathur et al., 2013).

With respect to veterinary pathogens, LeBel et al., reported that nisin behaved in a synergistic manner when combined with the β-lactams amoxicillin, penicillin or ceftiofur, and also when combined with streptomycin or tetracycline against Streptococcus suis (LeBel et al., 2013). Although S. suis is primarily a porcine pathogen, it has also been described as a zoonotic pathogen with transmission to humans possible (Hughes et al., 2009; Goyette-Desjardins et al., 2014; Huong et al., 2014). The novel lantibiotic, suicin 3908, was also shown to interact in an additive manner with amoxicillin or penicillin against S. suis in a recent study (Vaillancourt et al., 2015).

The sactibiotics are a relatively newly-designated group of bacteriocins, characterized by post-translational modifications involving radical S-adenosyl methionine (SAM) methylases and the presence of sulfur to α-carbon linkages in their structure (Arnison et al., 2013; Mathur et al., 2015). In general, the sactibiotics tend to be relatively hydrophobic in nature with characteristic hairpin-shaped structures. The efficacy of the sactibiotic, thuricin CD, was assessed in combination with metronidazole, ramoplanin and vancomycin, with a view to targeting C. difficile strains. It was revealed that thuricin CD interacted in a partial synergistic manner when combined with ramoplanin against 31% of the target strains tested (Mathur et al., 2013). In contrast, combinations of thuricin CD-vancomycin and thuricin CD-metronidazole resulted in indifferent effects (1.0 ≤ FIC ≤ 2.0) against the majority of C. difficile isolates tested in the study (Mathur et al., 2013). Antimicrobial combination studies have also been conducted with the sactibiotic subtilosin A against Gardnerella vaginalis. This opportunistic pathogen is one of the predominant causative agents of bacterial vaginosis (Catlin, 1992). It was determined that subtilosin A exhibited Bliss synergy when combined with clindamycin phosphate, metronidazole, ε-Poly-L-Lysine (polylysine) and lauramide arginine ethyl ester (LAE) against the pathogen (Cavera et al., 2015). Bliss synergy is based on the principle that drug effects are outcomes of probabilistic processes and assumes that drugs act independently in such a manner that neither of them interferes with the other (different sites of action), but each contributes to a common result (Tang et al., 2015). However, only subtilosin A-metronidazole and subtilosin A-clindamycin combinations yielded synergistic effects against G. vaginalis when FIC indices were evaluated (Cavera et al., 2015). Subtilosin produced by Bacillus amyloliquefaciens was also shown to exhibit synergistic activity with lauric arginate, ε-poly-L-lysine and glycerol monolaurate against G. vaginalis in an earlier study (Noll et al., 2012). Finally, a recent study demonstrated that combinations of the class II bacteriocin, durancin 61A and the broad-spectrum antimicrobial reuterin yielded FIC indices of 0.2 against C. difficile, indicating highly synergistic activity (Schaefer et al., 2010; Hanchi et al., 2017). Interestingly, durancin 61A combinations with vancomycin were also synergistic against MRSA (S. aureus ATCC 700699) with FIC values of 0.3 obtained (Hanchi et al., 2017).

With respect to the treatment of oral pathogens, one study investigated the combination of the bacteriocin PsVP-10, a non-lantibiotic displaying characteristics akin to class II bacteriocins, with the antimicrobials triclosan and chlorhexidine in an effort to target Streptococcus mutans and Streptococcus sobrinus (Lobos et al., 2009). S. mutans is the predominant causative agent of dental caries (Loesche, 1986; Metwalli et al., 2013), while S. sobrinus has also been implicated in its causation, albeit to a lesser extent than S. mutans (Conrads et al., 2014). Synergistic effects were obtained when PsVP-10 was combined with chlorhexidine, whereas partial synergistic/additive effects were apparent with PsVP-10-triclosan combinations in the study by Lobos et al. (2009). Candida albicans is the etiological agent of many opportunistic yeast infections in the oral cavity as well as other parts of the body and it is noteworthy that the class II bacteriocins plantaricin E, F, J, and K have displayed activity against C. albicans when used in combination with several different antibiotics (Sharma and Srivastava, 2014). This opportunistic pathogen typically infects immunosuppressed individuals. Indeed, it is frequently implicated in oro-pharyngeal thrush in AIDS patients and can also be a causative agent of systemic infections such as vaginitis (Kim and Sudbery, 2011). Thus, effective combinations of therapeutics to target this pathogen are warranted.

Bacteria present in a biofilm are inherently more resistant to antimicrobials than those present in a planktonic state predominantly due to the complex polymeric matrix which biofilms are composed of, and this matrix often impedes penetration of the antimicrobial to the deepest strata of the biofilm (Mah and O'Toole, 2001; Davies, 2003; Flemming and Wingender, 2010). Thus, there is an increased emphasis on trying to seek alternative therapeutic options and/or effective antimicrobial combinations to target biofilms. A recent study showed that nisin interacts synergistically with several antibiotics and such combinations were effective against Staphylococcus biofilms (Field et al., 2016a). In particular, the bioengineered variant of nisin, M21V, in combination with penicillin was effective at inhibiting biofilms of S. aureus SA113. In contrast to SA113 biofilms however, M21V was most effective in combination with chloramphenicol against SA113 planktonic cells (Figure 1C). Furthermore, another variant, nisin I4V, was reported to be highly effective in conjunction with chloramphenicol against Staphylococcus pseudintermedius DSM21284 biofilms in the same study, while combinations of I4V with penicillin were particularly potent against DSM21284 planktonic cells (Field et al., 2016a; Figures 1B, 2). S. pseudintermedius is an opportunistic pathogen, primarily affecting dogs (van Duijkeren et al., 2011) and an outbreak of methicillin-resistant S. pseudintermedius in Finland has been documented in recent years (Grönthal et al., 2014). Significantly, a case of S. pseudintermedius in a human was also described for the first time in 2006 (Van Hoovels et al., 2006). While the use of the colorimetric XTT assay revealed that nisin I4V-chloramphenicol combinations were effective at diminishing the viability of the biofilms of a S. pseudintermedius strain in the study by Field and co-workers, the authors found that there was no synergy with nisin-vancomycin combinations against Staphylococcus biofilms (Field et al., 2016a). Indeed, variations with respect to the nature of interactions between nisin and vancomycin when targeting MRSA or methicillin-sensitive S. aureus (MSSA) have also been reported in previous studies (Dosler and Gerceker, 2011, 2012). A separate study by Mataraci and Dosler also investigated the potency of several antibiotics combined with nisin against MRSA ATCC43300 biofilms and it was determined that antibiotic-nisin combinations were effective at preventing biofilm formation at 1x MIC. However, biofilm-associated bacteria were highly resistant to antibiotics or antibiotic combinations (Mataraci and Dosler, 2012). In another study, it emerged that nisin in combination with ciprofloxacin or daptomycin was effective against 24 h-old MRSA biofilms. The combination elicited a reduction in MRSA CFU counts by 3 logs, whereas the individual antimicrobials were unable to decrease the CFU to such an extent (Dosler and Mataraci, 2013). A recent interesting study evaluated the activity of nisin and lysostaphin against pre-formed biofilms of S. aureus involved in bovine mastitis (Ceotto-Vigoder et al., 2016). After treatment for 24 h with a combination of nisin and lysostaphin, the biofilm pre-formed by all eight strains tested in the study was reduced by >50%, as suggested by biofilm detachment from the microtiter plates. Although no remarkable detachment could be noticed by confocal laser scanning microscopy (CLSM) analysis after a 4 h treatment, when the biofilm matrix of S. aureus 4181 was assayed for cell viability, most cells were shown to be dead (Ceotto-Vigoder et al., 2016). In contrast to the synergistic activity between nisin and lysostaphin, significantly higher concentrations of lysostaphin used on its own, up to 50-fold higher, were required to cause the same level of biofilm detachment achieved by nisin-lysostaphin combinations. A separate study evaluated the antimicrobial effects of the class IIc bacteriocin enterocin AS-48 both independently and in combination with several biocides against three MRSA and three MSSA strains (Caballero Gómez et al., 2013). It was determined that the anti-biofilm activity of the biocides triclosan, benzalkonium chloride and polyhexamethylene guanidinium chloride (PHMG) were highly effective in combination with 50 μg/ml of AS-48 (Caballero Gómez et al., 2013). Thus, these combinations may prove to be useful therapeutic options for MRSA. Finally, Tong and co-workers also reported the efficacy of nisin in combination with chloramphenicol, ciprofloxacin or penicillin at targeting biofilms of the nosocomial pathogen, E. faecalis (Tong et al., 2014a).

With a specific goal of finding therapeutics against oral biofilms, Tong and co-workers also examined the anti-biofilm activities of the lantibiotic nisin, independently and in combination with free amino acids against S. mutans biofilms. The results of crystal violet biofilm assays indicated that mixtures of either the L or D-enantiomers of Glu, Asp or Cys in combination with nisin could ameliorate the potency of the lantibiotic against biofilms of S. mutans (Tong et al., 2014b). In an earlier study, the same group found that nisin interacted synergistically with sodium fluoride against S. mutans biofilms, in that the combination was more effective than sodium fluoride used independently at inhibiting the formation of biofilms at 4 and 16 h (Tong et al., 2011). The same investigators also assessed the effect of adding nisin to the dental irrigant, MTAD, and its anti-biofilm activity against oral E. faecalis isolates. Importantly, it was found that nisin in combination with doxycycline successfully inhibited E. faecalis biofilms whereas MTAD on its own was ineffective against such biofilms (Tong et al., 2013). E. faecalis, best known as a notorious nosocomial pathogen causing systemic infections, is also implicated in root canal infections. These successful combinations mentioned above could be harnessed as effective antimicrobial combinations against E. faecalis and S. mutans biofilms or as part of an endodontic irrigant following root canal treatments to prevent post-operative infections.

With respect to Gram negative organisms, a recent study found that nisin was effective when used together with polymyxins against Pseudomonas aeruginosa biofilms (Field et al., 2016b). P. aeruginosa is an important opportunistic pathogen and its biofilm-forming abilities contribute to its pathogenicity (Gellatly and Hancock, 2013). It is particularly common in lungs of patients with cystic fibrosis and thus warrants further extensive research to target its biofilm-forming and consequent pathogenic properties (Reen et al., 2016). The study by Field and co-workers demonstrated that decreased concentrations of polymyxins (either 0.5x MIC or even 0.2x MIC) are required to prevent P. aeruginosa PA-01 biofilm formation, when combined with 0.25x MIC nisin. Combinations of 0.33x MIC of nisin with 0.5x MIC of polymyxin were also effective against PA-01 planktonic cells (Field et al., 2016b; Figure 1A). Nisin was also used in combination with other antimicrobial agents used in the clinic against planktonic cells of P. aeruginosa ATCC 27853, as well as 40 other strains of P. aeruginosa in a separate study (Giacometti et al., 1999). A synergistic interaction of this nature can be hugely beneficial as it has the potential to attenuate the undesired nephrotoxicity associated with polymyxins.

A recent study using the sactibiotic, subtilosin, in combination with LAE was undertaken with a view to targeting G. vaginalis biofilms (Algburi et al., 2015). A combination of two methods; ATP viability assays and resazurin assays, were conducted to show the efficacy of these combinations against biofilms of the pathogen. Encouragingly, this combination of subtilosin-LAE was ineffective against healthy vaginal Lactobacillus biofilms. This highlights the specific nature of antimicrobial combinations and such a combination could be harnessed as a targeted therapeutic option against bacterial vaginosis. Furthermore, bacteriocins can potentially be combined with agents that target biofilms through non-microbiocidal means such as by altering molecular pathways which are responsible for regulating biofilm formation. Examples of such agents include thiazolidinone derivatives and diterpenoids (Buommino et al., 2014).

A summary of studies involving bacteriocins in combination with other stressors with a view to targeting clinical and veterinary pathogens, as well as biofilms, is found in Tables 1, 2.

The lantibiotic nisin has been the subject of several antimicrobial combination studies with a view to targeting food-borne pathogens. Indeed, it has been utilized for decades as a food preservative in more than 50 countries (Delves-Broughton, 1990). L. monocytogenes, the causative agent of listeriosis, is a notorious food-borne pathogen, and can cause opportunistic infections which can lead to meningitis and sepsis in severe cases (Low and Donachie, 1997; Schuppler and Loessner, 2010). In one study, it was found that 6,400 IU of nisin in combination with a green tea extract (GTE) or in combination with a grape seed extract (GSE) resulted in effective cell damage in a target L. monocytogenes strain (Sivarooban et al., 2008). The purified phenolic compounds present in GTE (0.02% epicatechin and 0.02% caffeic acid), as well as the pure phenolic compounds present in GSE (0.02% catechin and 0.02% epicatechin) were also used in the study (Sivarooban et al., 2008). When nisin was combined with GTE or GSE compounds, a compromised cell membrane and a condensed cytoplasm were apparent with TEM. With a starting inoculum of 10⁶ CFU/ml, the combination of nisin with GSE resulted in Listeria levels which were undetectable after 24 h of incubation, whereas the combination of nisin and GTE led to a decrease in Listeria levels to a mere 3.76 CFU/ml (Sivarooban et al., 2008). In another study, it was shown that semi-purified preparations of nisin A and a bioengineered derivative nisin V, combined effectively with the essential oils carvacrol, thymol and trans-cinnamaldehyde to inhibit L. monocytogenes in a validated food model system, as well as in laboratory media. It was shown that a greatly delayed lag phase was apparent during growth curves when nisin V was combined with 0.02% of the above-mentioned essential oils, compared to the nisin A equivalent (Field et al., 2015). Additionally, in time-kill assays, a 2-log decrease in cell numbers over and above that achieved by the nisin A combination with carvacrol or cinnamaldehyde was observed against the target L. monocytogenes EGDe, which was also maintained in a number of food settings (Field et al., 2015). In a separate study, the antimicrobial activity of nisin Z was potentiated when combined with thymol at sub-inhibitory concentrations, against the target strain L. monocytogenes ATCC7644 (Ettayebi et al., 2000). Significantly, the study showed that 40 IU/ml nisin combined with 0.02% thymol was more effective at inhibiting ATCC7644 than either of the antimicrobials used on their own. A combination of this nature may also preclude the emergence of bacterial sub-populations displaying resistance to the lantibiotic (Ettayebi et al., 2000). Another study investigated the effects of combining nisin with carvacrol or mountain savory essential oils on the viability of target L. monocytogenes strains subsequent to γ-irradiation. There was an increase in the relative sensitivity of L. monocytogenes to γ-irradation by a factor of 6.31 when nisin and mountain savory essential oils were combined and a corresponding increase in sensitivity by a factor of 4.19 with nisin-carvacrol combinations (Ndoti-Nembe et al., 2013).

With regards to combinations of bacteriocins with preservatives, a study reported that combinations of nisin-potassium sorbate-ethylene diamine tetraacetic acid (EDTA), as well as combinations of nisin-sodium-diacetate-EDTA were effective at reducing the numbers of L. monocytogenes on shrimp inoculated with the pathogen (Wan Norhana et al., 2012). Synergistic interactions between nisin and low concentrations of EDTA in targeting L. monocytogenes were also described in a study by Branen and Davidson. In addition, the authors showed that the antimicrobial activity of nisin against L. monocytogenes was potentiated in the presence of lactoferrin in the same study (Branen and Davidson, 2004). Combinations of 10 IU/ml nisin with 250 μg/ml lactoferrin were also shown to perform synergistically to inhibit L. monocytogenes in a separate study, similar to Branen and Davidson's findings (Murdock et al., 2007).

With respect to other classes of bacteriocins, Grande and co-workers reported synergistic effects when the class IIc bacteriocin enterocin AS-48 was combined with the phenolic compound carvacrol (Grande et al., 2007). The authors conducted a food trial with vegetable sauces whereby they investigated the efficacy of AS-48 both independently and when combined with phenolic substances with a view to inhibiting S. aureus. The activity of AS-48 was significantly enhanced against S. aureus, when combined with eugenol, geraniol, terpineol, carvacrol, hydrocinnamic acid, caffeic acid, citral and p-coumaric acid. The levels of the pathogen were decreased to less than detectable levels when 80 μg/ml of AS-48 was mixed with 126 nM carvacrol or 20 mM hydrocinnamic acid, in carbonara sauce kept at 22°C. Overall, the degree of synergy obtained depended on the concentration of the antimicrobials and also the type of sauce used (Grande et al., 2007). Enterocin AS-48 was also shown to interact in a synergistic manner with lactic acid, p-hydroxybenzoic methylester acid (PHBME) and citric acid in a separate study (Antonio et al., 2009). In salads spiked with L. monocytogenes, the application of 30 μg/g of AS-48 in combination with PHBME, Nisaplin or lactic acid led to a significant reduction in L. monocytogenes numbers (Antonio et al., 2009). Amrouche and co-workers showed that the sactibiotic bacteriocin subtlosin was effective at inhibiting L. monocytogenes Scott A and NR30 when combined with ε-poly-L-lysine, zinc lactate or with curcumin (Amrouche et al., 2010). The strain L. monocytogenes Scott A was originally implicated in a listeriosis outbreak in Massachusetts in 1983 (Fleming et al., 1985; Briers et al., 2011) while the strain NR-30 has been reported to display resistance to nisin (Mazzotta and Montville, 1997). Partial synergy against L. monocytogenes Scott A was apparent when subtilosin was used in conjunction with an encapsulated form of curcumin in the study by Amrouche et al., while subtilosin-zinc lactate combinations also exhibited synergy against both NR30 and Scott A. However, when combined with non-encapsulated poly-L-lysine or curcumin, subtilosin displayed merely additive effects against the two L. monocytogenes strains (Amrouche et al., 2010). Finally, a separate study reported that the combination of a cell-bound bacteriocin produced by Lactobacillus curvatus CWBI-B28 with savory essential oil or oregano was effective at reducing L. monocytogenes to below detectable levels in pork meat spiked with the pathogen, after a week of storage (Ghalfi et al., 2007). The oregano essential oil combined with the cell-bound bacteriocin was still effective after 3 weeks of storage, whereas all other combinations led to an increase in L. monocytogenes levels after the same duration of time. Overall, this cell-bound bacteriocin combined with savory essential oil was found to retard the growth of L. monocytogenes by 2 weeks, relative to the use of the cell-bound bacteriocin on its own (Ghalfi et al., 2007).

L. monocytogenes has the ability to form biofilms in addition to growing in a planktonic state, and a recent study demonstrated the effectiveness of the bioengineered derivative of nisin, M21A, in combination with natural food-grade additives (cinnamaldehyde and citric acid) in targeting biofilms of strain F6854 (Smith et al., 2016). This strain has been associated with contaminated turkey frankfurters (Nelson et al., 2004). The study by Smith et al., showed that the bioengineered nisin derivative M21A (0.1 μg/ml) was more effective at inhibiting F6954 biofilms than wild-type nisin, when combined with citric acid (175 μg/ml) or cinnamaldehyde (35 μg/ml) (Smith et al., 2016). A separate study recently reported that the class IIb bacteriocin, enterocin B3A-B3B, was effective when used in combination with nisin at decreasing the MIC needed to disrupt the growth of L. monocytogenes in either a planktonic state or when present as a biofilm (Al-Seraih et al., 2017). Finally, the cell-free supernatant (CFS) containing a bacteriocin from Lb. curvatus ET31 was tested in combination with EDTA and the investigators noted that while the bacteriocin and the EDTA independently were ineffective against biofilms which had already formed, the CFS combined with EDTA was effective at reducing the viability of L. monocytogenes biofilms which had already formed, whilst not fully eliminating the biofilms (Camargo et al., 2016).

Gram negative pathogens in particular have proven to be more problematic to tackle using bacteriocins than Gram positive pathogens primarily because of the outer membrane present in Gram negatives that limits access to the cell membrane. Importantly however, the bioengineered nisin variants S29A and S29G have been shown to display activity against Gram negatives (Field et al., 2012). While nisin A has been shown to be effective against Gram negatives when used in combination with chelating agents such as EDTA, perhaps a more attractive option is combining nisin with natural phenolic compounds such as thymol and carvacrol, which facilitate the permeabilization/disruption of the membrane (Stevens et al., 1991; Helander et al., 1998). However, as concentrations of essential oils which are needed to have antimicrobial activity can compromise the organoleptic qualities of foods, their use as preservatives in high concentrations have been rather limited thus far.

Nonetheless, a recent study utilized nisin A, and its bioengineered derivatives nisin S29A and nisin M21V independently and in combination with the essential oils carvacrol, trans-cinnamaldehyde, thymol, as well as the preservative citric acid, to evaluate the efficacy of such combinations against the Gram negative food-borne pathogens Escherichia coli O157: H7 and Cronobacter sakazakii (Campion et al., 2017). E. coli O157: H7 is an enterohaemorrhagic strain and has been associated with outbreaks in the US, Canada, UK and Japan (Besser et al., 1993; Bach et al., 2002; Vidovic and Korber, 2016). C. sakazakii has been associated with contaminated infant milk formula and is implicated in cases of enteritis, meningitis and septicaemia (Gurtler et al., 2005; Drudy et al., 2006; Iversen and Forsythe, 2007; Yan et al., 2012). Campion et al., noted that extended lag phases of C. sakazakii and O157: H7 were apparent when 30 μM of the bioengineered nisin variants were combined with 0.035% trans-cinnamaldehyde, 0.03% carvacrol and 0.015% thymol, when compared to corresponding combinations of nisin A-essential oils. In addition, a 4-log reduction of C. sakazakii and a 3-log reduction in viable counts of O157: H7 was particular noteworthy when 60 μM of the nisin variants were combined with 0.03% carvacrol, in comparison to corresponding nisin A-carvacrol combinations (Figure 1D). Importantly, the study also showed that when stored at room temperature, sub-lethal concentrations of nisin variants in combination with carvacrol were successful in fully inactivating O157: H7 in apple juice, again when compared to nisin A-carvacrol combinations. Similarly, the commercial product Nisaplin at concentrations of 10 mg/ml, in combination with 30 mM citric acid, elicited >3 log decreases in C. sakazakii viable counts in infant formula after 3 h of incubation (Campion et al., 2017). The increased stability of nisin at lower pH conditions compounded by the ameliorated diffusion properties of essential oils in acidic conditions could explain the effective combinations in low-pH drinks such as apple juice (Delves-Broughton, 1990; Burt, 2004; Campion et al., 2017). As thymol and carvacrol disrupt Gram negative outer membranes (which renders them more sensitive to nisin), while trans-cinnamaldehyde disrupts the transmembrane ATPase, combinations of such oils with nisin may prove to be the most effective option to control Gram negative food-borne pathogens (Helander et al., 1998; Gill and Holley, 2006a,b).

With regards to other studies investigating bacteriocin synergy against Gram negative food-borne pathogens, Moon et al., reported that a bacteriocin 4.5 kDa in size from Pediococcus acidilactici K10 in combination with the organic acids lactic acid, succinic acid and acetic acid interacted synergistically against E. coli O157: H7 both in vitro and in situ. This bacteriocin from P. acidilactici K10 in combination with 0.35% lactic acid or 0.25% acetic acid was evaluated in a ground beef sample at 4°C and it was noteworthy that a 2.8-log reduction of O157: H7 was observed with lactic acid combinations (Moon et al., 2002). Thus, P. acidilactici and organic acid combinations may have potential as food bio-preservatives. Branen and Davidson showed the efficacy of nisin combined with EDTA against enterohaemorrhagic E. coli strains (Branen and Davidson, 2004) while a separate study demonstrated the efficacy of 250 IU/ml of nisin combined with 500 μg/ml lactoferrin at preventing E. coli O157: H7 growth (Murdock et al., 2007). Finally, Ananou et al., observed synergistic effects when enterocin AS-48 was combined with agents that disrupted the outer membrane of a pathogenic E. coli O157: H7 isolate (Ananou et al., 2005).

A summary of studies involving bacteriocins in combination with various stressors with a view to targeting food-borne pathogens is found in Table 3.

While antibiotics are unlikely to be used in foods, several studies have nonetheless investigated their efficacies in combination with bacteriocins against food-borne pathogens in laboratory conditions. Studies of this nature can be useful with regards to providing insights into the mechanisms of synergistic interactions, especially when bacteriocins are combined with antibiotics with known modes of action. Bacillus cereus is an important food-borne pathogen and several strains have the ability to cause food poisoning, often resulting in vomiting and diarrhea (Schoeni and Wong, 2005). In a relatively recent study, it was shown that the two-component lantibiotic, lacticin 3147, interacted synergistically with the antibiotic polymyxin B against B. cereus 8079 and B. cereus 5247 (Draper et al., 2013). Interestingly however, lacticin 3147-polymyxin E combinations resulted in indifferent (1.0 < FIC < 2.0) and antagonistic effects (FIC > 2.0), in contrast to the above-mentioned synergistic interactions with polymyxin B against the same targets, showing that a single amino acid change in the polymyxin backbone can lead to profound differences in terms of interactions with lacticin 3147 against specific target strains. The lantibiotic nisin and class IIa bacteriocin, pediocin PA-1, have also been combined with the antibiotic polymyxin E to target L. monocytogenes and E. coli isolates which had exhibited resistance to pediocin and polymyxin E respectively (Naghmouchi et al., 2011). Polymyxin and nisin combinations at concentrations of 0.6 μg/ml and 15.6 μg/ml; 4.7 μg/ml and 62.5 μg/ml; and 9.3 μg/ml and 32 μg/ml, respectively, resulted in the inhibition of polymyxin-resistant E. coli, pediocin PA-1-resistant L. monocytogenes and nisin-resistant L. monocytogenes variants by 74, 97, and 94% respectively, relative to the controls. While nisin A and pediocin PA-1 combinations were synergistic against L. monocytogenes and its resistant variants, they were ineffective against E. coli or its resistant variants. Nonetheless, nisin-polymyxin combinations at concentrations of 7.8 μg/ml and 0.3 μg/ml, respectively, and polymyxin independently at a concentration of 0.21 μg/ml decreased the growth of log phase E. coli cells by approximately 94 and 31% respectively. Pediocin PA-1-polymyxin E combinations at concentrations of 25 μg/ml and 4.7 μg/ml, respectively, reduced the growth of L. monocytogenes in the exponential and stationary phases by 90% and 78%, respectively. Thus, the study indicated that resistant L. monocytogenes and E. coli isolates can be managed by using combinations of nisin/polymyxin E or pediocin PA-1/polymyxin E respectively (Naghmouchi et al., 2011). The same group also evaluated the activity of colistin in combination with pediocin PA-1/AcH or nisin with a view to targeting E. coli O157: H7, Yersinia enterocolitica ATCC 9610, P. aeruginosa ATCC 27853 and Salmonella choleraesuis ATCC 14028. Significantly, 1.56 μg/ml of pediocin PA-1/AcH or 1.7 μg/ml of nisin in combination with colistin elicited a marked reduction in the concentration of colistin needed to inhibit O157: H7 (Naghmouchi et al., 2013).

With respect to studies involving other Gram negative food-borne pathogens, lacticin 3147 was reported to exhibit synergy when combined with polymyxin B against C. sakazakii DPC6440 with FIC values of 0.25 (Draper et al., 2013). The lantibiotic also exhibited synergistic activity against the same strain when combined with polymyxin E with corresponding FIC values of 0.062 against DPC6440. Interestingly however, indifferent and antagonistic effects were obtained when lacticin 3147 was combined with polymyxin B or polymyxin E against the target strains Salmonella Typhimurium UK1 and LT2, with FIC values greater than 1.125 in all cases (Draper et al., 2013). A separate study by Rishi and co-workers demonstrated the efficacy of β-lactam antibiotics combined with nisin with a view to inhibiting the food-borne pathogen Salmonella enterica serovar Typhi (Rishi et al., 2014). Nisin-β-lactam synergistic combinations were assessed by conducting FIC and time-kill assays and with the exception of three strains, synergy was observed with all combinations against the clinical Salmonella strains in the in vitro study by Rishi and co-workers with nisin-cefotaxime and nisin-ceftriaxone proving to be the most effective combinations (Rishi et al., 2014). Singh et al., in a similar study, also evaluated the effectiveness of nisin in combination with standard antibiotics against multi-drug resistant strains of Salmonella and any such synergistic interactions were evaluated by FIC determinations using the checkerboard assay as well as time-kill assays and radial diffusion assays (Singh et al., 2013). Furthermore, scanning electron microscopy (SEM) and mouse trials assessing the combinatorial interactions were also conducted to validate synergistic effects observed with in vitro assays. Decreases in the numbers of Salmonella in various organs of infected mice were observed as a consequence of the antimicrobial combination. Using FIC values and time-kill assays, nisin-cefotaxime and nisin-ceftriaxone combinations yielded synergistic effects whereas nisin-EDTA and nisin-ampicillin combinations yielded additive effects against serovar Typhimurium. Significant alterations in the outer membrane of the target cells, elicited by the antimicrobial combinations were apparent and bacteriocin-β-lactam combinations caused greater log decreases of Salmonella in the spleen, intestine and liver of mice, relative to treatment with the antimicrobials independently (Singh et al., 2013). More specifically, the combination of nisin (at concentrations of 25 mg/Kg body weight and 50 mg/Kg body weight) with ceftriaxone (also at concentrations of 25 mg/Kg and 50 mg/Kg) resulted in 2.83, 3.11, 2.6, and 3.1-log decreases in Salmonella respectively in the spleen. Identical concentrations of nisin and cefotaxime were also combined, resulting in 2.06, 2.49, 2.11, and 2.44-log decreases respectively in the spleen as well. In contrast, 25 mg/Kg nisin and 50 mg/Kg nisin administered independently resulted in a mere 0.16 and 0.3-log decrease respectively in Salmonella in the spleen. Similarly, combinations of nisin (25 mg/Kg body weight and 50 mg/Kg body weight) combined with identical concentrations of ceftriaxone proved to be effective, resulting in decreases in Salmonella in the liver ranging from 2.75 to 3.5-log units. Corresponding combinations of nisin and cefotaxime resulted in reductions in Salmonella in the liver ranging from 2.27 to 3.26-log units. These values were higher than the 0.42 to 0.67-log unit reductions achieved by nisin independently. Following on from this study, the same group attempted to elucidate the mechanism of these synergistic interactions (Singh et al., 2014). Essentially, the ability of nisin and the β-lactams to target the cell membrane was evaluated by conducting membrane permeabilizing assays in combination with pulse labeling techniques. The results showed that the bacteriocin-β-lactam combination affected membrane permeability, as confirmed by the uptake of 1-N-phenylnapthylamine (NPN) by the treated cells. This uptake of NPN as a consequence of membrane permeabilization, as well as interference with DNA, RNA and protein synthesis was dependent on both the dose of the antimicrobials, as well as the duration of exposure to the antimicrobials in combination. Significantly, results of in vivo assays involving mouse trials corroborated synergistic effects seen in in vitro assays in the study (Singh et al., 2014). Thus, nisin-cefotaxime and nisin-ceftriaxone synergistic effects against Salmonella were predominantly due to permeabilization of the membrane, as well as DNA, RNA, protein synthesis inhibition, and immune-modulatory activity (Singh et al., 2014).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.