Peptides 60 (2014) 32–40

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Antibacterial peptide nisin: A potential role in the inhibition of oral pathogenic bacteria Zhongchun Tong a,b , Longxing Ni c , Junqi Ling a,b,∗ a b c

Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China Department of Conservative Dentistry & Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, China

a r t i c l e

i n f o

Article history: Received 6 June 2014 Received in revised form 20 July 2014 Accepted 21 July 2014 Keywords: Nisin Streptococcus mutans Enterococcus faecalis MTAD MTADN

a b s t r a c t Although the antimicrobial peptide nisin has been extensively studied in the food industry for decades, its application in the oral cavity remains to develop and evaluate its feasibility in treating oral common diseases. Nisin is an odorless, colorless, tasteless substance with low toxicity and with antibacterial activities against Gram-positive bacteria. These biologic properties may establish its use in promising products for oral diseases. This article summarizes the antibacterial efficiency of nisin against pathogenic bacteria related to dental caries and root canal infection and discusses the combination of nisin and common oral drugs. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of cariogenic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin alone for the inhibition of cariogenic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin in combination with sodium fluoride or chlorhexidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin in combination with free d-amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of pathogens related to root canal infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin in combination with MTAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial activity of MTADN against E. faecalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial activity of MTADN against common pathogens related to root canal infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The antibacterial efficiency of sub-minimal inhibitory concentrations of MTADN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin in combination with common antibiotics against E. faecalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nisin alone evaluated as an intracanal medicament . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion: remarks and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction The oral microbial community is the most complex microflora in the human body, and at present, more than 700 bacterial species

∗ Corresponding author at: Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510055, Guangdong, China. Tel.: +86 20 83862621; fax: +86 20 83822807. E-mail addresses: Junqi [email protected], t z [email protected] (J. Ling). http://dx.doi.org/10.1016/j.peptides.2014.07.020 0196-9781/© 2014 Elsevier Inc. All rights reserved.

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have been detected as inhabitants of the oral cavity [40]. In a healthy mouth, the pathogenic bacteria and non-pathogenic bacteria typically maintain a dynamic balance through synergistic and antagonistic actions between microorganisms. However, when the microflora environment of the oral cavity is modified, for example, due to carbohydrate absorption, dental plaque acidification, and saliva reduction, the pathogenic microorganisms proliferate and oral diseases may ensue if the quantity of pathogens exceeds the amount needed to initiate infection [40,45,80]. Dental caries and periodontitis are the two most common oral diseases and

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are closely related with the oral microbial flora [10,22,68,80]. Furthermore, pathogenic bacteria in the root canal system are the major cause of endodontic and periapical infection [23]. Use of antimicrobials is still the most common method used to reduce the over-proliferation of pathogens and to control oral diseases. The common antibiotics are not suitable to be used to treat common oral diseases, because it can cause emergence of multidrug resistant microorganisms [2,14,15]. Antimicrobial peptides, a novel class of antibiotics, originated from bacteria, insects, plants, amphibians, birds, fish, and mammals, and have attracted much attention due to their strong antibacterial activity against a very broad spectrum of microorganisms and low rates of bacterial resistance [2,3,33,57]. Antimicrobial peptides have also received attention in the control of pathogenic microorganisms of the oral cavity, and defensin, histatin, cathelicidin LL-37, lactoferrin, nisin, pleurocidin, and other synthetic peptides showed good antibacterial activity against a few oral pathogenic bacteria in some studies [17]. Nisin, a natural antimicrobial peptide, was isolated from Lactococcus lactis by Mattick and Hirsch in 1947 and is the oldest known and most widely studied lantibiotic [47]. Nisin is a polypeptide with 34 amino acid residues and is a ribosome synthesized and post-translationally modified peptide with one lanthionine, four ␤-methyl-lanthionine rings, and unusual residues such as dehydroalanine and dehydrobutyrine [13,19,39]. Nisin is highly active against Gram-positive bacteria such as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Lactobacillus plantarum, Micrococcus luteus, and Micrococcus flavus, and it is odorless, colorless, tasteless, and has a low toxicity [29,59,70,71]. Nisin was permitted as a safe food additive in over 50 countries around the world, particularly in dairy products, canned foods, plant protein foods, and cured meat. [21]. Furthermore, nisin has demonstrated antibacterial activity against oral pathogenic bacteria such as Streptococcus mutans, Streptococcus sanguinis, Lactobacillus acidophilus, and Enterococcus faecalis [82,88]. The antibacterial activity of nisin is attributed to its interaction with anionic lipids on the cytoplasmic membrane of bacterial cells, resulting in perturbation of the plasma membrane. The pore formed by interaction of nisin–anionic lipids causes an efflux of adenosine triphosphate (ATP), amino acids, preaccumulated rubidium, or the collapse of vital ion gradients, leading to cell death [7,9]. The interaction of nisin and the cytoplasmic membrane occurs by two different mechanisms. In the first mechanism, at the micromolar range, nisin binds to the anionic lipids of membranes and demonstrates a low-affinity permeation; in the second mechanism, at the nanomolar range, nisin interacts with Lipid II in the target membrane of cells by specific recognition, followed by the Lipid II-dependent pathway for pore formation that is composed of four Lipid II molecules and eight nisin molecules [8,31]. The Lipid IImediated pore complex is much more stable than pores formed in the absence of Lipid II [31]. Microbiologists have endeavored to use nisin to inhibit the pathogens related to food manufacturing for the past twenty years, but the application of nisin in the inhibition of oral pathogens is still in the initial stage of development. This review will introduce the potential role of nisin in the oral cavity and highlight the effect of nisin on the cariogenic bacteria and root canal pathogens.

Inhibition of cariogenic microorganisms Nisin alone for the inhibition of cariogenic microorganisms Dental caries is an infectious disease caused by dental plaque biofilm, which is composed of Lactobacillus spp., Streptococcus spp., Actinomyces spp., and many others [69]. A few studies have

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indicated that the initial colonizer on the surface of the teeth is mostly non-mutans streptococci and Actinomyces in which acidification is mild and infrequent. However, when more sugar is supplied, acidification becomes moderate and frequent. This enhances the acidogenicity and acidurance of some non-mutans bacteria, and mutans streptococci, Lactobacillus spp., aciduric strains of nonmutans streptococci, Actinomyces, bifidobacteria, and yeasts may become dominant through acid-induced selection [80]. The microorganisms related to dental caries are mostly Grampositive, and the antimicrobial peptide nisin generally has an inhibitory effect on Gram-positive bacteria, which establishes the possibility for nisin application in prevention of dental caries. In a previous study, nisin was considered to be a caries-preventive agent due to its inhibition of nine common cariogenic-relevant bacteria: S. sanguinis, Streptococcus gordonii, S. mutans, Streptococcus sobrinus, L. acidophilus, Lactobacillus casei, Lactobacillus fermenti, Actinomyces viscosus and Actinomyces naeslundii. The results of MIC and MBC showed that S. sanguinis, L. casei and L. fermenti were the more sensitive to nisin, and the two Actinomyces spp. were less sensitive to nisin [82]. The antibacterial activity and stability of nisin is affected by the pH value in the environment, and nisin has high antibacterial activity and stability at low pH values [12,66]. In general, cariogenic bacteria, such as S. mutans, S. sobrinus and L. acidophilus, can produce more acid products by carbohydrate intake, thereby keeping the pH of the dental plaque low, conducive to the antibacterial activity and stability of nisin [18,80]. Furthermore, the antibacterial activities of nisin are not influenced by enzymes, proteins and other inorganic components in the saliva of the human cavity, though nisin is rapidly degraded and inactivated by digestive enzymes such as trypsin and pancreatin after entering the gastrointestinal (digestive) tract [32,82]. In morphologic observation, S. sanguinis, L. fermenti, L. acidophilus and S. mutans showed significant damage after nisin treatment (Fig. 1B, D, F and I). In light of these inspiring results, nisin has considerable potential to be used as an antibacterial agent to prevent dental caries. Nisin in combination with sodium fluoride or chlorhexidine Fluoride and chlorhexidine are the most common anti-caries agents and have been used as the primary components in toothpaste and mouthwash, thus playing an important role in the prevention of dental caries [4,81,92]. At present, nisin is not regarded as a common anti-caries agent, but it has potential advantages in the prevention of dental caries and needs to be studied further [88]. In regard to antibacterial activity, chlorhexidine and nisin are not significantly different. Chlorhexidine and nisin have similar bactericidal manners by which they damage the cell membranes and have a wide range of antibacterial activity against Gram-positive bacteria [7,9,52]. As a result, no synergy was found in the antibacterial activity of combined nisin and chlorhexidine against S. mutans [86]. As for the adverse effects, nisin may be superior to chlorhexidine. Chlorhexidine might lead to potential hypersensitivity and discoloration of the teeth and tongue, alerting dentists [1,58]. Nisin is odorless, colorless, tasteless, has a low toxicity, and is approved as a safe food preservative by the Food and Drug Administration (1988) in the USA [21]. At present, to the best of our knowledge, nisin appears to have no side effects with recurrent use. With regard to antibacterial activity, fluoride has a lower impact compared to chlorhexidine and nisin, but many studies consider fluoride to be the most effective caries-preventive agent [41,48,81]. The anticaries properties of fluoride not only depend on its antibacterial activity but also occur mostly through its remineralization and inhibition of demineralization [67]. In the evaluation of a fluoride and nisin combination, the two drugs displayed a great synergetic effect on the cariogenic pathogen S. mutans as well as

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Fig. 1. Morphologic observations of cariogenic bacteria after treatment with nisin, CHX and NaF. (A) and (B) S. sanguinis untreated and treated with 20,000 U/ml nisin; (C) and (D) Lactobacillus fermenti untreated and treated with 20,000 U/ml nisin; (E) and (F) Lactobacillus acidophilus untreated and treated with 20,000 U/ml nisin; (G) and (I) Streptococcus mutans untreated and treated with 20,000 U/ml nisin; (H) S. mutans treated with 20 g/L NaF; (K) S. mutans treated with 20,000 U/ml nisin and 0.1 g/L CHX; (L) S. mutans treated with 20,000 U/ml nisin and 20 g/L NaF; (J) is a local magnification of (L). The white arrows represent cell lyses; black arrows represent cell membranes rupture. S. sanguinis, L. fermenti, L. acidophilus and S. mutans after exposure to nisin showed holes and ruptures on cell surfaces (B, D, F, I). Some S. mutans cells fell to pieces after exposure to a combination of nisin and CHX, but cells with a normal shape were still clearly visualized in each field (K). The majority of S. mutans cells had dissolved into pieces when exposed to a combination of nisin and NaF (L). The modified images from Tong et al. [82,86].

on biofilm [86]. The antibacterial action of fluoride takes place in the cytoplasm. Fluoride can prevent bacterial glycolysis by directly affecting the intracellular glycolytic enzyme enolase, and fluoride can also inhibit membrane-bound ATPases and glucose transport by the protonmotive force and the phosphotransferase system [28,44,78]. Nisin exerts its bactericidal activity by pore formation on the surface of the cell, and the pore will help more fluoride ions enter the cytoplasm to better exert the antibacterial roles of fluoride [7,31,77,86]. Morphologic observations also showed that S. mutans cells were seriously destroyed and that the majority of cells had dissolved into pieces after treatment with nisin and fluoride combination, but a few cells with a normal shape were still clearly visualized after treatment with the nisin and chlorhexidine combination (Fig. 1K and L)[86]. Furthermore, the nisin and fluoride combination may generate a stronger bactericidal effect on S. mutans biofilm than the two drugs alone [86]. At present, no study has indicated the role of nisin in remineralization. Nisin and fluoride combination may make up for their respective shortcomings and has the potential to be used as an effective drug combination in the prevention of dental caries. Nisin in combination with free d-amino acids d-amino acids are one of the most striking features of a peptidoglycan composition. d-AAs were recently found to play very important roles in regulating and disassembling bacterial biofilms [11,38]. In one study, d-cysteine, d-aspartic acid, and d-glutamic acid were found to significantly improve the antibacterial activities of nisin against S. mutans in a test of 18 d-amino acids, and the combination of the three d-amino acids generated an even more

significant effect. Furthermore, their combination also improved the bactericidal activities of nisin against the S. mutans biofilm (Fig. 2)[89]. The improvement in the antibacterial activity of nisin may be related to the structure of the peptidoglycan. The restoration of the pore, formed as a result of the nisin treatment, may need more normal d-AA components in the peptidoglycan, but the addition of the different d-amino acids may affect the restoration of the peptidoglycan due to mismatch. Additionally, the penetration of the exogenous d-amino acids into cells by the pores may affect the normal amino acid metabolism in bacteria. Therefore, nisin in combination with d-amino acid may be a viable option for the prevention of dental caries. No antimicrobial agent is absolutely perfect. Antimicrobial peptides are also considered antimicrobial agents, and microorganisms may develop adaptive stress responses when exposed to nisin [61,62]. Nisin is a low drug-resistant antimicrobial peptide, but nisin resistance may occur in some bacteria. Nisin resistance proteins (NSRs) are found in some bacteria such as Streptococcus agalactiae, L. lactis, and S. aureus [35,36,42]. NSRs could proteolytically cleave nisin by removal of the C-terminal tail of nisin and thus decrease nisin affinity for the cell membrane [77]. At present, two-component systems, the NsrRS and LcrRS, were also found in S. mutans and help S. mutans develop resistance to nisin and nukacin ISK-1 [34]. Therefore, the effect of nisin on cariogenic bacteria still needs to be further studied. Inhibition of pathogens related to root canal infection Pathogenic bacteria and their products are considered to be the major cause of pulpal and periapical infection [79]. At the

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Fig. 2. Confocal laser scanning microscopic images of the existing S. mutans biofilm (24 h biofilm) on the surfaces of tooth pieces after 1 h of challenge with (A) BHIS (negative control); (B) 125 mg/L nisin; (C) 125 mg/L nisin in combination with three d-AA (d-Cys, d-Asp, and d-Glu at their respective concentration of 40 mM). Cells with intact membranes are stained green, whereas cells with damaged cell membranes are stained red. Three-dimensional S. mutans biofilms were captured and scanned along the Z axis at 20 layers from bottom to top using the oil lens of the Carl Zeiss CLSM and ZEN software. The bactericidal activities of nisin against S. mutans in biofilm were significantly improved by three d-AA at their respective concentration of 40 mM, and few viable cells were visualized after exposure to nisin and three d-AA (C). The modified images from Tong et al. [89].

primary root canal infection, the ratio of Gram-positive bacteria and Gram-negative bacteria is close to 1:1, and at secondary infection, Gram-positive bacteria may dominate in the root canal [46,54]. At present, nisin is typically considered to be combined with other medicaments as an application in root canal therapy. Though sodium hypochlorite and chlorhexidine are common root canal medicaments, nisin and the two drugs are not an ideal combination [49,50]. Sodium hypochlorite is an alkaline solution, and nisin has low antibacterial activity in alkaline conditions; chlorhexidine and nisin have similar antibacterial mechanisms of attacking the cell membrane and may not generate a synergic effective. Our previous studies investigated the combination of nisin and the intracanal irrigant MTAD [83,84,87,88]. Nisin in combination with MTAD MTAD, a common intracanal irrigant, is composed of 3% doxycycline, 4.25% citric acid, and 0.5% polysorbate 80 detergent and has many advantages, for example, the removal of the smear layer, tissue-dissolving action, biocompatibility, and little erosive change on the dentin surface [51,72]. The bactericidal activity of MTAD remains to be improved because doxycycline is bacteriostatic rather than bactericidal, and tetracycline was overused in the past [65,74]. Nisin demonstrated good antibacterial activity against many drug-resistant pathogenic bacteria, and thus, nisin was considered a substitute for or to be used in combination with doxycycline in MTAD to improve antibacterial activity [16,24,53,60]. Antibacterial activity of MTADN against E. faecalis Nisin significantly improved the antibacterial activity of MTAD against E. faecalis ATCC 29212. E. faecalis did not develop a drugresistance to MTADN (MTAD in combination with nisin) and MTAN (substituting doxycycline with nisin) because the MBC/MIC of MTADN and MTAN were less than 32. For the antibacterial activities, MTADN is superior to MTAD and MTAN, attributed mainly to the synergetic action of nisin and doxycycline, which has been demonstrated through the evaluation of the fractional inhibition concentration (FIC). The MIC of nisin and doxycycline alone did not decrease the E. faecalis survival rate, but their combination generated a significant inhibitory effect on E. faecalis, and this inhibitory effect even occurred at 1/2 MIC of nisin and doxycycline combination. Furthermore, morphologic observations revealed that E.

faecalis had serious damage after treatment with the combination of MTAD and nisin (Fig. 3)[88]. MTADN still has a strong antibacterial effect against E. faecalis during stress states. E. faecalis at the starvation and the alkalization states, similar to the normal physiologic state, was more sensitive to MTADN and MTAN than MTAD, and no significant differences were found between the survival rates of the three states of E. faecalis with treatment with MTAD, MTADN, or MTAN [87]. A few studies indicated that starvation improved bacterial resistance to antimicrobials by triggering the synthesis of stress proteins [25,30]. However, even under starvation and alkalization conditions, E. faecalis remains sensitive to MTADN. E. faecalis can survive at a pH of 4.0–11, and 4.25% citric acid keeps MTAD, MTAN and MTADN at an acidic environment with a pH of approximately 2. The acid resistance of E. faecalis requires H+ -adenosine triphosphatase activation on the cell membrane by an energy source [56]. Therefore, energy deficiency resulting from starvation is unfavorable for E. faecalis resistance to the lethal acid at a pH of 2, and the pH 2 acid favors the antibacterial activity of nisin. Furthermore, it is difficult for E. faecalis at the alkalization state to respond immediately to a sudden attack mediated by lethal acid. Hence, E. faecalis is still sensitive to MTAD, MTAN, and MTADN when in the starvation and the alkalization states. The above studies focused on the lab strain E. faecalis ATCC 29212. The addition of nisin also improved the antibacterial efficacy of MTAD against E. faecalis strains isolated from infected root canals [84]. In an MBC assay, ten E. faecalis root canal isolates displayed varying sensitivity to MTADN, although their sensitivities were greater than that of E. faecalis ATCC 29212. Bacteria from biofilm growth are generally more resistant to antimicrobial agents compared to planktonic bacteria [75]. In the in vitro root canal model of E. faecalis isolate contamination, MTADN showed the highest antibiofilm activity out of MTAD, MTAN, and MTADN. Confocal laser scanning microscopy revealed that MTADN was able to kill biofilmgrown E. faecalis after a 5-min. challenge, which could be consistent with the active time of intracanal irrigants, but a few viable bacteria were found in the root canal model through MTAD irrigation [84]. This result indicated that nisin improved the antibiofilm activity of MTAD. Antibacterial activity of MTADN against common pathogens related to root canal infections Endodontics and periradicular infection are caused by diverse microorganisms [73]. MTAD, MTAN, and MTADN have

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Fig. 3. The morphologic modification of E. faecalis ATCC 29212 after 24 h of treatment with MATD, MTAN and MTADN. (A) Untreated control; (B) MTAD treatment; (C) MTAN treatment; and (D) MTADN treatment. All images were observed at 60,000× magnification. The black arrows indicate hole formation, and the white arrows indicate intracellular exposure. After MATD treatment, E. faecalis maintained its original shape, but the surface was dented. The E. faecalis surface showed a hole after MTAN treatment. E. faecalis was seriously destroyed after MTADN treatment. A larger hole was formed, and the intracellular contents were exposed. The modified images from Tong et al. [88].

demonstrated a similar antibacterial activity against four common Gram-negative bacteria related to root canal infection, namely Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Peptostreptococcus, and completely killed these pathogens after a 1-minute challenge. However, for Gram-positive bacteria from root canal infections such as L. fermenti, Lactobacillus paracasei, A. viscosus, A. naeslundii, S. gordonii, and E. faecalis, MTAN and MTADN showed greater and more rapid bactericidal activity compared to MTAD. This finding may be because nisin has a strong bactericidal effect on Gram-positive bacteria [87]. In the morphologic observations, MTADN generated an obvious destruction of A. naeslundii and L. paracasei but did not produce marked damage when administered to P. gingivalis (Fig. 4) [87]. Nisin added to MTAD did not affect the antibacterial activity of MTAD against Gram-negative bacteria. Gram-negative bacteria have an outer membrane that hinders the binding of the nisin molecule to Lipid II in the cell membrane, thus inhibiting the effect of nisin. However, this does not indicate that nisin does not have an antibacterial role against Gram-negative bacteria. Stevens et al. showed that ethylene diaminetetraacetic acid (EDTA) or citrate can destabilize the outer membrane and thus may help nisin exert its inhibitory effect on some Gram-negative bacteria in root canal infections [76].

The antibacterial efficiency of sub-minimal inhibitory concentrations of MTADN There is no doubt that nisin improves the antibacterial activity of MTAD. However, due to rinsing and buffering during the root canal procedure, the concentrations of the drugs gradually decrease and reach sub-minimum inhibitory concentrations (sub-MIC); therefore, it is necessary to evaluate pathogens in the root canal when challenged with nisin in combination with MTAD at sub-MIC levels. In the post-antibiotic effect (PAE) and post-antibiotic sub-MIC effect (PASME) assays, no significant difference was found between MTAD and MTADN because the MIC of MTADN was at a 1:8192 dilution, and at such a low concentration, nisin hardly exerts its role [83,88]. This result does not indicate that the addition of nisin does not improve the antibacterial role of MTAD. In their original concentrations, MTADN is still significantly superior to MTAD according to many criteria [83,84,87,88]. Pathogens in the root canal generally confront the challenges of calcium hydroxide intracanal dressing after irrigation. E. faecalis showed the least resistance to alkaline after treatment with MTADN, and this indicated that nisin in combination with doxycycline could improve the calcium hydroxide intracanal dressing to better inhibit the pathogenic bacteria [83]. Low concentrations of antibiotics can act as stress inducers or cues/coercions on receiver bacteria and may cause diverse

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Fig. 4. Morphologic modification of the three pathogenic bacteria by FE-SEM after MTAD and MTADN treatment. (A) and (D) Actinomyces naeslundii ATCC 12104; (B) and (E) Lactobacillus paracasei ATCC 25598; (C) and (F) Porphyromonas gingivalis ATCC 33277. (A, B and C) MTAD treatment; (D, E and F) MTADN treatment. The black and white arrows indicate the membrane ruptures of A. naeslundii and L. paracasei, respectively. MTADN did not produce marked damage when administered to P. gingivalis. The modified images from Tong et al. [87].

bacterial biologic responses [6]. MTAD, MTAN, and MTADN, at subMIC levels, induced different expression levels for some virulence and stress genes in E. faecalis. The inductions of MTAD and MTADN for the relevant genes are similar because the concentration of nisin is very low at the sub-MIC level of MTADN. However, MTAN had a different level of induction for some genes of E. faecalis due to its high MIC and the role of nisin alone (Fig. 5). The different levels of induction may induce various stress states in E. faecalis, thereby affecting bacterial pathogenicity [83]. Nisin in combination with common antibiotics against E. faecalis E. faecalis ranks among the leading causes of nosocomial infection worldwide and is also frequently isolated from secondary and persistent root canals [23,54,64]. E. faecalis has both an intrinsic

and acquired resistance to antibiotics and is thus often considered a test strain to evaluate the antibacterial activity of antimicrobials by many endodontic researchers [20,26,27,63,83]. In a previous study, 18 antibiotics, including linezolid and imipenem, were evaluated for antibacterial activity against three different E. faecalis strains, namely ATCC 29212, OG1RF, and a root canal isolate strain. The results of the study showed that the E. faecalis strains were not completely killed by the 18 antibiotics alone, and the MBCs of 3, 5, and 9 antibiotics against OG1RF, ATCC 29212, and the root canal isolate were more than 1024 mg/L. However, 200 U/ml nisin can significantly improve the antibacterial activities of the 18 antibiotics, with the exception of sulfapyridine, metronidazol, and polymyxin, while aiding the activities of penicillin, chloramphenicol, vancomycin, and linezolid to eradicate E. faecalis ATCC 29212 at low concentrations. As observed by transmission

Fig. 5. RT-PCR was used to evaluate the effects of sub-MIC of MTAD, MTADN, and MTAN on stress and virulence-associated genes in E. faecalis. 16S rRNA was used as an internal control. The asterisk represents up-regulation of genes expression and a statistically significant difference between the relative quantities (RQ) of gene expression of E. faecalis with sub-MIC levels of the drug group and the control. The expression of clpC and clpP was up-regulated in response to the sub-MIC levels of each drug. Transcriptions of ace, clpX, cylB, efaA, and gelE were significantly up-regulated and sprE transcription was down-regulated after treatment with sub-MIC amounts of MTAN. However, transcription of dnaK, gls24, and groEL was insignificantly induced by the three drugs. Furthermore, sub-MIC amounts of MTAD and MTADN significantly induced the transcription of sprE approximately 5-fold. The modified images from Tong et al. [83].

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Fig. 6. Morphologic changes of E. faecalis ATCC 29212 observed by transmission electron microscopy after 12 h of treatment with antibiotics in combination with nisin. (A) Control; (B) 1024 mg/L penicillin; (C) 2000 U/ml nisin; (D) 1024 mg/L penicillin and 2000 U/ml nisin; (E) 1024 mg/L chloramphenicol; and (F) 1024 mg/L chloramphenicol and 2000 U/ml nisin. The control E. faecalis exhibited normal sphericity (A). After 12 h of treatment with penicillin alone, the majority of E. faecalis still maintained their original shapes (B). The shapes of a few E. faecalis cells were destroyed after treatment with nisin alone (C). Under the combination of penicillin and nisin, many E. faecalis cells in the observation area lost their original morphology, instead showing distinct cellular disruption (D). Furthermore, upon treatment with chloramphenicol alone, E. faecalis cells did not show overt signs of cellular disruption, but a few cells were subjected to severe damage by the combination of chloramphenicol and nisin (E and F). The modified images from Tong et al. [85].

electron microscopy, penicillin or chloramphenicol with the addition of nisin generated more damage than either antibiotic alone (Fig. 6). Furthermore, in an antibiofilm assay, a great deal of E. faecalis cells survived after treatment with penicillin, ciprofloxacin, and chloramphenicol, but the addition of nisin can significantly improve the bactericidal activities of the three antibiotics against E. faecalis cells in biofilm. These studies indicated that nisin can significantly improve the antibacterial and antibiofilm activities of many common antibiotics, especially antibiotics with different antibacterial mechanisms [85]. Therefore, the combination of nisin and antibiotics has been considered for potential use as an inhibitor against drug-resistant pathogens. Nisin alone evaluated as an intracanal medicament Nisin is not only considered to be an intracanal irrigant but was evaluated alone as an intracanal dressing. Turner et al. compared

the antibacterial effect of nisin and calcium hydroxide as intracanal dressings. In the in vitro root canal infection model by E. faecalis or S. gordonii, nisin and calcium hydroxide were sealed for 7 days, resulting in the same effectiveness with significantly superior results compared to the sterile water control group. No root canal sample showed any growth of the two bacteria after nisin and calcium hydroxide were sealed in the root canal. Nisin also inhibited pathogens in the dentinal depth layer. In the evaluation for dentinal tubules, a calcium hydroxide dressing can reduce S. gordonii by 39% and E. faecalis by 45%; nisin reduced S. gordonii by 49% and E. faecalis by 48% [90]. Though nisin can effectively inhibit E. faecalis and S. gordonii, nisin alone may be an inappropriate intracanal medication, as root canal infections generally are caused by a mixture bacteria, including Gram-negative bacteria that have a low sensitivity to nisin. Nisin has good antibacterial efficacy against multidrug-resistant Gram-positive pathogens, and this suggests that nisin can be

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considered for use in intracanal medicaments applied to persistent root canal infections or root canal retreatments. The application of nisin in root canal therapy is still in the experimental stages. At present, there is not an optimal therapy for persistent periapical periodontitis. We wish to further study the antimicrobial peptide nisin to provide a strategy for the therapy of persistent root canal infections. Conclusion: remarks and perspectives The antimicrobial peptide nisin has been extensively studied over the past decades in the food industry but remains in the initial research phase for application in the oral cavity. The biologic properties of nisin suggest that nisin might be a suitable and effective drug in the prevention of dental caries and root canal therapies. Recently, nisin’s cytotoxicity has been evoked by some researchers [5,37,43,55,91]. Nisin may be effect on simian virus 40transfected human colon (SV40-HC), Vero monkey kidney (Vero) cells, human T-cell lymphoma Jurkat cells, Molt-4 cells and freshly cultured human lymphocytes [5,37,55]. However, local application in oral cavity may not cause hemolysis reaction because nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity [43]. Furthermore, if nisin was accidentally swallowed, it will rapidly degraded and inactivated by digestive enzyme. Therefore, nisin may be relatively safe in the local application in oral cavity. However, these in vitro studies have not completely favored the effectiveness of nisin for oral diseases, and nisin application in the oral cavity requires the support of in vivo studies. Acknowledgements This work is supported by Guangdong Natural Science Foundation (S2013040014932) and Guangdong Medical Scientific Research Fund (A2013225). References [1] Al-Tannir MA, Goodman HS. A review of chlorhexidine and its use in special populations. Spec Care Dentist 1994;14:116–22. [2] Angebault C, Andremont A. Antimicrobial agent exposure and the emergence and spread of resistant microorganisms: issues associated with study design. Eur J Clin Microbiol Infect Dis 2013;32:581–95. [3] Aoki W, Kuroda K, Ueda M. Next generation of antimicrobial peptides as molecular targeted medicines. J Biosci Bioeng 2012;114:365–70. [4] Autio-Gold J. The role of chlorhexidine in caries prevention. Oper Dent 2008;33:710–6. [5] Begde D, Bundale S, Mashitha P, Rudra J, Nashikkar N, Upadhyay A. Immunomodulatory efficacy of nisin—a bacterial lantibiotic peptide. J Pept Sci 2011;17:438–44. [6] Bernier SP, Surette MG. Concentration-dependent activity of antibiotics in natural environments. Front Microbiol 2013;4:20. [7] Breukink E, de Kruijff B. Lipid II as a target for antibiotics. Nat Rev Drug Discov 2006;5:321–32. [8] Breukink E, Ganz P, de Kruijff B, Seelig J. Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry. Biochemistry 2000;39:10247–54. [9] Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S, et al. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 2003;278:19898–903. [10] Carinci F, Scapoli L, Girardi A, Cura F, Lauritano D, Nardi GM, et al. Oral microflora and periodontal disease: new technology for diagnosis in dentistry. Ann Stomatol (Roma) 2013;4:170–3. [11] Cava F, de Pedro MA, Lam H, Davis BM, Waldor MK. Distinct pathways for modification of the bacterial cell wall by non-canonical d-amino acids. EMBO J 2011;30:3442–53. [12] Cerrutti P, Terebiznik MR, de Huergo MS, Jagus R, Pilosof AMR. Combined effect of water activity and pH on the inhibition of Escherichia coli by nisin. J Food Prot 2001;64:1510–4. [13] Cheigh CI, Pyun YR. Nisin biosynthesis and its properties. Biotechnol Lett 2005;27:1641–8. [14] Chon SY, Doan HQ, Mays RM, Singh SM, Gordon RA, Tyring SK. Antibiotic overuse and resistance in dermatology. Dermatol Ther 2012;25:55–69.

39

[15] Cohen ML. Epidemiology of drug resistance: implications for a postantimicrobial era. Science 1992;257:1050–5. [16] Collins B, Cotter PD, Hill C, Ross RP. The impact of nisin on sensitive and resistant mutants of Listeria monocytogenes in cottage cheese. J Appl Microbiol 2011;110:1509–14. [17] da Silva BR, de Freitas VA, Nascimento-Neto LG, Carneiro VA, Arruda FV, de Aguiar AS, et al. Antimicrobial peptide control of pathogenic microorganisms of the oral cavity: a review of the literature. Peptides 2012;36:315–21. [18] de Soet JJ, van Loveren C, Lammens AJ, Pavicic MJ, Homburg CH, ten Cate JM, et al. Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans. Caries Res 1991;25:116–22. [19] de Vos WM, Kuipers OP, van der Meer JR, Siezen RJ. Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol 1995;17:427–37. [20] Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J Endod 2006;32:527–31. [21] FDA (Food Drug Administration). Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed Regist 1988;53:11247–51. [22] Featherstone JD. The continuum of dental caries—evidence for a dynamic disease process. J Dent Res 2004;83. Spec No C: C39–42. [23] George M, Ivancakova R. Root canal microflora. Acta Med (Hradec Kralove) 2007;50:7–15. [24] Ghiselli R, Giacometti A, Cirioni O, Dell’Acqua G, Mocchegiani F, Orlando F, et al. RNAIII-inhibiting peptide and/or nisin inhibit experimental vascular graft infection with methicillin-susceptible and methicillin-resistant Staphylococcus epidermidis. Eur J Vasc Endovasc Surg 2004;27:603–7. [25] Giard JC, Hartke A, Flahaut S, Benachour A, Boutibonnes P, Auffray Y. Starvation-induced multiresistance in Enterococcus faecalis JH2-2. Curr Microbiol 1996;32:264–71. [26] Giardino L, Ambu E, Savoldi E, Rimondini R, Cassanelli C, Debbia EA. Comparative evaluation of antimicrobial efficacy of sodium hypochlorite MTAD, and Tetraclean against Enterococcus faecalis biofilm. J Endod 2007;33:852–5. [27] Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. [28] Hamilton IR. Biochemical effects of fluoride on oral bacteria. J Dent Res 1990;69. Spec No: 660–7; discussion 682–3. [29] Hampikyan H. Efficacy of nisin against Staphylococcus aureus in experimentally contaminated sucuk, a Turkish-type fermented sausage. J Food Prot 2009;72:1739–43. [30] Hartke A, Giard JC, Laplace JM, Auffray Y. Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis. Appl Environ Microbiol 1998;64:4238–45. [31] Hasper HE, de Kruijff B, Breukink E. Assembly and stability of nisin–lipid II pores. Biochemistry 2004;43:11567–75. [32] Jarvis B, Mahoney RR. Inactivation of nisin by alpha-chymotrypsin. J Dairy Sci 1969;52:1448–9. [33] Jenssen H, Hamill P, Hancock RE. Peptide antimicrobial agents. Clin Microbiol Rev 2006;19:491–511. [34] Kawada-Matsuo M, Oogai Y, Zendo T, Nagao J, Shibata Y, Yamashita Y, et al. Involvement of the novel two-component NsrRS and LcrRS systems in distinct resistance pathways against nisin A and nukacin ISK-1 in Streptococcus mutans. Appl Environ Microbiol 2013;79:4751–5. [35] Kawada-Matsuo M, Yoshida Y, Zendo T, Nagao J, Oogai Y, Nakamura Y, et al. Three distinct two-component systems are involved in resistance to the class I bacteriocins Nukacin ISK-1 and nisin A, in Staphylococcus aureus. PLoS One 2013;8:e69455. [36] Khosa S, AlKhatib Z, Smits SH. NSR from Streptococcus agalactiae confers resistance against nisin and is encoded by a conserved nsr operon. Biol Chem 2013;394:1543–9. [37] Kindrachuk J, Jenssen H, Elliott M, Nijnik A, Magrangeas-Janot L, Pasupuleti M, et al. Manipulation of innate immunity by a bacterial secreted peptide: lantibiotic nisin Z is selectively immunomodulatory. Innate Immun 2013;19:315–27. [38] Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R, Losick R. d-amino acids trigger biofilm disassembly. Science 2010;328:627–9. [39] Kuipers OP, Beerthuyzen MM, de Ruyter PG, Luesink EJ, de Vos WM. Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem 1995;270:27299–304. [40] Kuramitsu HK, He X, Lux R, Anderson MH, Shi W. Interspecies interactions within oral microbial communities. Microbiol Mol Biol Rev 2007;71:653–70. [41] Lam A, Chu CH. Caries management with fluoride agents. N Y State Dent J 2012;78:29–36. [42] Liang X, Sun Z, Zhong J, Zhang Q, Huan L. Adverse effect of nisin resistance protein on nisin-induced expression system in Lactococcus lactis. Microbiol Res 2010;165:458–65. [43] Maher S, McClean S. Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem Pharmacol 2006;71:1289–98. [44] Marquis RE. Antimicrobial actions of fluoride for oral bacteria. Can J Microbiol 1995;41:955–64. [45] Marsh PD. The significance of maintaining the stability of the natural microflora of the mouth. Br Dent J 1991;171:174–7. [46] Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279–94.

40

Z. Tong et al. / Peptides 60 (2014) 32–40

[47] Mattick AT, Hirsch A. Further observations on an inhibitory substance (nisin) from lactic streptococci. Lancet 1947;2:5–8. [48] McGrady MG, Ellwood RP, Pretty IA. Why fluoride? Dent Update 2010;37(5958):601–2. [49] Mohammadi Z. Chlorhexidine gluconate, its properties and applications in endodontics. Iran Endod J 2008;2:113–25. [50] Mohammadi Z. Sodium hypochlorite in endodontics: an update review. Int Dent J 2008;58:329–41. [51] Mohammadi Z. MTAD: a review of a promising endodontic irrigant. N Y State Dent J 2012;78:47–53. [52] Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288–302. [53] Mohammadi Z, Shalavi S, Yazdizadeh M. Antimicrobial activity of calcium hydroxide in endodontics: a review. Chonnam Med J 2012;48:133–40. [54] Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7. [55] Murinda SE, Rashid KA, Roberts RF. In vitro assessment of the cytotoxicity of nisin, pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays. J Food Prot 2003;66:847–53. [56] Nakajo K, Komori R, Ishikawa S, Ueno T, Suzuki Y, Iwami Y, et al. Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalis. Oral Microbiol Immunol 2006;21:283–8. [57] Parachin NS, Mulder KC, Viana AA, Dias SC, Franco OL. Expression systems for heterologous production of antimicrobial peptides. Peptides 2012;38:446–56. [58] Pemberton MN, Gibson J. Chlorhexidine and hypersensitivity reactions in dentistry. Br Dent J 2012;213:547–50. [59] Periago PM, Moezelaar R. Combined effect of nisin and carvacrol at different pH and temperature levels on the viability of different strains of Bacillus cereus. Int J Food Microbiol 2001;68:141–8. [60] Piper C, Draper LA, Cotter PD, Ross RP, Hill C. A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species. J Antimicrob Chemother 2009;64:546–51. [61] Poole K. Bacterial stress responses as determinants of antimicrobial resistance. J Antimicrob Chemother 2012;67:2069–89. [62] Poole K. Stress responses as determinants of antimicrobial resistance in Gramnegative bacteria. Trends Microbiol 2012;20:227–34. [63] Portenier I, Waltimo T, Orstavik D, Haapasalo M. The susceptibility of starved, stationary phase, and growing cells of Enterococcus faecalis to endodontic medicaments. J Endod 2005;31:380–6. [64] Richards MJ, Edwards JR, Culver DH, Gaynes RP. Nosocomial infections in combined medical-surgical intensive care units in the United States. Infect Control Hosp Epidemiol 2000;21:510–5. [65] Roberts MC. Tetracycline therapy: update. Clin Infect Dis 2003;36:462–7. [66] Rollema HS, Kuipers OP, Both P, de Vos WM, Siezen RJ. Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering. Appl Environ Microbiol 1995;61:2873–8. [67] Rosin-Grget K, Lincir I. Current concept on the anticaries fluoride mechanism of the action. Coll Antropol 2001;25:703–12. [68] Scapoli L, Girardi A, Palmieri A, Testori T, Zuffetti F, Monguzzi R, et al. Microflora and periodontal disease. Dent Res J (Isfahan) 2012;9:S202–6. [69] Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007;369:51–9. [70] Severina E, Severin A, Tomasz A. Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens. J Antimicrob Chemother 1998;41:341–7.

[71] Sheldon BW, Schuman JD. Thermal and biological treatments to control psychrotrophic pathogens. Poult Sci 1996;75:1126–32. [72] Singla MG, Garg A, Gupta S. MTAD in endodontics: an update review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:e70–6. [73] Siqueira Jr JF, Rocas IN. Diversity of endodontic microbiota revisited. J Dent Res 2009;88:969–81. [74] Smilack JD. The tetracyclines. Mayo Clin Proc 1999;74:727–9. [75] Smith AW. Biofilms and antibiotic therapy: is there a role for combating bacterial resistance by the use of novel drug delivery systems? Adv Drug Deliv Rev 2005;57:1539–50. [76] Stevens KA, Sheldon BW, Klapes NA, Klaenhammer TR. Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl Environ Microbiol 1991;57:3613–5. [77] Sun Z, Zhong J, Liang X, Liu J, Chen X, Huan L. Novel mechanism for nisin resistance via proteolytic degradation of nisin by the nisin resistance protein NSR. Antimicrob Agents Chemother 2009;53:1964–73. [78] Sutton SV, Bender GR, Marquis RE. Fluoride inhibition of proton-translocating ATPases of oral bacteria. Infect Immun 1987;55:2597–603. [79] Takahashi K. Microbiological pathological, inflammatory, immunological and molecular biological aspects of periradicular disease. Int Endod J 1998;31:311–25. [80] Takahashi N, Nyvad B. The role of bacteria in the caries process: ecological perspectives. J Dent Res 2011;90:294–303. [81] Ten Cate JM. Novel anticaries and remineralizing agents: prospects for the future. J Dent Res 2012;91:813–5. [82] Tong Z, Dong L, Zhou L, Tao R, Ni L. Nisin inhibits dental caries-associated microorganism in vitro. Peptides 2010;31:2003–8. [83] Tong Z, Huang L, Ling J, Mao X, Ning Y, Deng D. Effects of intracanal irrigant MTAD combined with nisin at sub-minimum inhibitory concentration levels on Enterococcus faecalis growth and the expression of pathogenic genes. PLoS One 2014;9:e90235. [84] Tong Z, Ling J, Lin Z, Li X, Mu Y. The effect of MTADN on 10 Enterococcus faecalis isolates and biofilm: an in vitro study. J Endod 2013;39:674–8. [85] Tong Z, Zhang Y, Ling J, Ma J, Huang L, Zhang L. An in vitro study on the effects of nisin on the antibacterial activities of 18 antibiotics against Enterococcus faecalis. PLoS One 2014;9:e89209. [86] Tong Z, Zhou L, Jiang W, Kuang R, Li J, Tao R, et al. An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans. Peptides 2011;32:2021–6. [87] Tong Z, Zhou L, Kuang R, Lv H, Qu T, Ni L. In vitro evaluation of MTAD and nisin in combination against common pathogens associated with root canal infection. J Endod 2012;38:490–4. [88] Tong Z, Zhou L, Li J, Jiang W, Ma L, Ni L. In vitro evaluation of the antibacterial activities of MTAD in combination with nisin against Enterococcus faecalis. J Endod 2011;37:1116–20. [89] Tong Z, Zhang L, Ling J, Jian Y, Huang L, Deng D. An in vitro study on the effect of free amino acids alone or in combination with nisin on biofilms as wells as on planktonic bacteria of Streptococcus mutans. PLoS One 2014. [90] Turner SR, Love RM, Lyons KM. An in-vitro investigation of the antibacterial effect of nisin in root canals and canal wall radicular dentine. Int Endod J 2004;37:664–71. [91] Vaucher RA, Teixeira ML, Brandelli A. Investigation of the cytotoxicity of antimicrobial peptide P40 on eukaryotic cells. Curr Microbiol 2010;60:1–5. [92] Zimmer S, Jahn KR, Barthel CR. Recommendations for the use of fluoride in caries prevention. Oral Health Prev Dent 2003;1:45–51.

Antibacterial peptide nisin: a potential role in the inhibition of oral pathogenic bacteria.

Although the antimicrobial peptide nisin has been extensively studied in the food industry for decades, its application in the oral cavity remains to ...
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