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ORIGINAL ARTICLE
Year : 2019  |  Volume : 23  |  Issue : 1  |  Page : 31-34  

The antiplaque efficacy of lantibiotic Nisin extract mouthrinse


Department of Periodontology, T P C T's Terna Dental College and Hospital, Nerul, Navi Mumbai, Maharashtra, India

Date of Submission18-May-2018
Date of Acceptance23-Aug-2018
Date of Web Publication3-Jan-2019

Correspondence Address:
Dr. Amitabh Yadav
Building No. 33/04, L.I.G Colony, Vinoba Bhave Nagar, Pipe Road, Kurla West, Mumbai - 400 070, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisp.jisp_326_18

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   Abstract 


Aim: This study aimed to determine the antiplaque efficacy of Nisin Z extract mouth rinse (Test) as compared to 0.2% chlorhexidine gluconate mouthrinse (Positive control) and distilled water (Placebo) and to assess minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Nisin Z against Prevotella intermedia (Pi), Porphyromonas gingivalis (Pg), and Aggregatibacter actinomycetemcomitans (Aa) in vitro. Materials and Methods: Forty-five individuals were randomly divided into three groups (15 in each group). The baseline plaque scores were brought to 0 with scaling and polishing. They were asked not to use any kind of mechanical oral hygiene aid for 4 days and advised to use mouthwashes A, B, or C (Group A: Nisin Z; Group B: distilled water; Group C: chlorhexidine) for 4 days. Following the 4-day plaque re-growth model, Plaque Index was checked on days 1 and 5. Results: In intergroup comparison, a statistically significant difference was seen between Nisin and placebo groups and chlorhexidine and placebo groups, with Nisin and chlorhexidine showing superior antiplaque activity. There was also statistically significant difference between Nisin and chlorhexidine groups, with chlorhexidine showing superior antiplaque activity. In vitro Nisin showed effective inhibition against all three bacterial strains Pi, Pg, and Aa exhibited the highest sensitivity to Nisin with a MIC of 2.5 μg/ml and MBC of 15 μg/ml. Conclusion: Nisin Z mouthrinse was found to be a potent plaque inhibitor, though less effective than chlorhexidine mouthrinse. However, it can serve as a good natural alternative to the gold standard.

Keywords: Dental plaque, mouthrinse, Nisin, probiotic


How to cite this article:
Mitra D, Yadav A, Prithyani S, John LE, Rodrigues S, Shah R. The antiplaque efficacy of lantibiotic Nisin extract mouthrinse. J Indian Soc Periodontol 2019;23:31-4

How to cite this URL:
Mitra D, Yadav A, Prithyani S, John LE, Rodrigues S, Shah R. The antiplaque efficacy of lantibiotic Nisin extract mouthrinse. J Indian Soc Periodontol [serial online] 2019 [cited 2019 May 22];23:31-4. Available from: http://www.jisponline.com/text.asp?2019/23/1/31/248225




   Introduction Top


Periodontitis has been defined as an infectious disease resulting in inflammation within supporting tissues of teeth, progressive attachment loss, and bone loss.[1] The most common etiological factor for causing periodontitis is the dental plaque.[2]

Although mechanical plaque control remains the gold standard treatment modality for maintaining oral hygiene, use of chemotherapeutic agents as an adjunct is often necessary.

The most common antimicrobial agent used is chlorhexidine which is the gold standard. However, due to its local adverse effects especially reversible brown staining of teeth, tongue and resin restorations, and transient impairment of taste perception, prevent its long-term use as a mouthwash.[3] Hence, research for alternative antiplaque agents is necessary.

Bacteriocins are antimicrobial proteins produced by bacteria.[3] They are natural compounds with selective antimicrobial activities in the oral cavity. One of the best known is Nisin.[4] Nisin acts against Gram-negative organisms. It is found in milk and has been utilized as a preservative in food.

Its recent research has shown that when a chelator is added to nisin, it acts against Gram-positive and Gram-negative organisms. Hence, it can be an excellent compound for plaque prevention.[5]

To the best of the authors' knowledge, no study has evaluated the antiplaque efficacy of Nisin Z; hence, we conducted this study to access the effect of a mouthwash containing the antimicrobial peptide Nisin Z as an antiplaque agent and to assess minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Nisin Z against Prevotella intermedia (Pi), Porphyromonas gingivalis (Pg), and Aggregatibacter actinomycetemcomitans (Aa) in vitro.


   Materials and Methods Top


In vitro study design

The Nisin Z extract was tested against three strains: Pg, Pi, and Aa for MICs and MBCs and the values of MIC and MBC were 2.5 and 15 μg/ml, respectively.

pH of the lantibiotic Nisin mouthwash was 5.

Media used

Brain–heart infusion (BHI) agar was used and prepared according to the standard protocol;[5] agar plates were brought to room temperature before use.

Inoculum preparation

To assess MIC, nine dilutions (Nisin Z mouthwash) were done with BHI. In the first tube, 20 μL of drug was added into the 380 μL of BHI broth. For dilutions, 200 μL of BHI broth was put in the other nine tubes separately. Then, from the first tube, 200 μL was put into the first tube containing 200 μL of BHI broth. This was said to be as 10:1 dilution.

From 10:1 diluted tube, 200 μL was put to second tube to make 10:2 dilution. The serial dilution was repeated up to 10:9 dilution for each drug. From the maintained stock cultures of required organisms, 5 μL was taken and was put into 2 ml of BHI broth. In each serially diluted tube, 200 μL of the above culture suspension was added. The tubes were incubated for 24 h and observed for turbidity.[6]

The tubes showed no visible signs of growth/turbidity (MIC and higher dilutions), and from these tubes, a loopful was inoculated onto sterile Mueller–Hinton agar plates by spread plate method. The plates were incubated overnight at 37°C. The least concentration that did not show any growth of tested organisms was considered as the MBC value of the Nisin against the same tested bacterial species.

In vivo study

This trial was a randomized, double-blinded, longitudinal study. A total of 45 periodontally healthy individuals (13 males and 32 females) between the age group of 21 and 23 years (mean age: 22 years) were selected. Mouthwashes were labeled as A, B, and C by one examiner and another examiner conducted the study.

  • Mouthwash A (test): Nisin Z mouthwash
  • Mouthwash B (placebo control): Placebo that constituted distilled water
  • Mouthwash C (positive control): A commercially available 0.2% chlorhexidine (CHX) mouthwash (ICPA Health care products, Ankleshwar, Gujarat, India).


Nisin Z mouthwash contained Nisin Z powder and ethylenediaminetetraacetic acid powder. It was prepared at the Biotechnology Department of a reputed institution in Navi Mumbai. The solubility of the mouthwash was 1 mg per 100 ml of water.

Inclusion criteria

Systemic healthy individuals with at least 24 scoreable teeth (excluding third molars or crowned teeth) were included in the study.

Exclusion criteria

  1. Individuals who wore appliances or prostheses (fixed or removable)
  2. Individuals on antibiotics or other medications in the last 3 months, or had undergone treatment for periodontal problems in the last 6 months were not included in the study. Uncooperative individuals were excluded from the study.


A total of 45 students, studying in Terna Dental College, Nerul, Navi Mumbai, India, who fulfilled the inclusion criteria, were selected. The study was approved by the Ethical Committee of the institution and was conducted in accordance with the Declaration of Helsinki.

The participants were randomly assigned into three equal groups namely, Groups A, B, and C (15 participants per group).

The duration of the study was 5 days.

Thorough oral prophylaxis was done for all the participants by the examiner 2 to bring the baseline plaque score to 0. A two-tone plaque-disclosing agent (Dento Plac, India) was used to check that all deposits had been removed. Participants were asked to refrain from using any mechanical cleaning aid or chewing gum for the next 4 days. In its place, participants were instructed to use assigned mouthwash twice a day.

At a specific time during day, the participants were instructed to rinse twice a day for 1 min with 10 mL of the allocated rinse. On day 5, the participants were recalled; Plaque Index (PI) (Turesky modification of Quigley Hein Plaque Index) was recorded.

Adverse effects such as burning sensation, altered taste, and desquamation of gingival epithelium, if any, were also evaluated on the 5th day.

Statistical analysis

After the indices were calculated and the mouthwashes were decoded, further testing was done with SPSS 17 software (SPSS Inc, IBM Corporation, Chicago, U.S.). The mean was calculated for each mouthwash. Analysis of variance (ANOVA) was done. Differences between the mouthwashes and distilled water were determined via the Bonferroni multiple comparison test.


   Results Top


A total of 45 individuals, 3 males and 32 females, completed the study. In all groups, PI showed significant (P < 0.01) increase from baseline to the 5th day.In vitro Nisin showed effective inhibition against all the three bacterial strains Pi, Pg, and Aa exhibited the highest sensitivity to Nisin with a MIC of 2.5 μg/ml and MBC of 15 μg/ml.

The in vivo results demonstrated that the mean PI values were the highest for the distilled water mouthwash (2.65) and the least for CHX mouthwash (1.90). The mean PI value for distilled water (2.65) was higher than the Nisin Z group (2.12). ANOVA showed a statistically significant difference between the PI scores (P = 0.001).

Differences between the individual mouthwashes and distilled water, determined via the Bonferroni multiple comparison test, showed significantly less plaque re-growth in case of both CHX (P = 1.90) and Nisin Z (P = 2.12) as compared to distilled water.

The Nisin Z group had higher PI values than the CHX group and the results were statistically significant.

[Table 1] shows results by ANOVA test and [Table 2] shows results by Bonferroni multiple comparison test. No complications were reported after the use of mouthwashes. In addition, there was no evidence of gingival epithelial desquamation on examination.
Table 1: Results of Plaque Index and comparison between groups using one-way ANOVA

Click here to view
Table 2: Comparison between groups using Bonferroni multiple comparison test

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   Discussion Top


Nisin is a type A lantibiotic (ribosomally synthesized bacteriocins) found naturally in milk and has been used as a food preservative.[7] Nisin in combination with a chelator has a broad-spectrum activity. Nisin kills the microorganisms by forming pores in bacterial cell wall. It has dual mode of action. They bind to lipid II inhibiting cell wall synthesis and they employ lipid II as a docking molecule to initiate a process of membrane insertion and pore formation leading to rapid cell death.[8]

Since Nisin is a probiotic, it may also be used as an antiplaque agent.[6] Hence, this study was done to assess the antiplaque effects of a novel Nisin Z mouthwash and to evaluate whether the mouthwash had any adverse effects.

Nisin Z mouthwash was compared with distilled water and 0.2% CHX solution. The study design (4-day plaque re-growth study) is a standard method for testing plaque-reducing effect.[9],[10] The advantage of a 4-day plaque regrowth study design is that it avoids the effect of the adjunctive mechanical oral hygiene techniques because they are not permitted during the trial period.[11]

In all the groups, PI showed significant (P < 0.01) increase from day 0 to the 5th day. The present study showed significant and comparable reduction in PI in the Nisin Z and CHX group as compared to distilled water (P < 0.01).

As inferred by observed changes in biofilm architecture, biofilms seemingly dispersed and sloughed into smaller aggregates after a 1 min exposure to 10 and 50 μg/ml of Nisin. At 50 μg/ml and after a 5 min exposure, biofilms exhibited cell death, and membrane damage indicated by the live/dead signal quantification. However, at 10 μg/ml, significant bacterial killing was not observed regardless of the treatment time. The antimicrobial action of Nisin may be either bacteriostatic or bactericidal depending on multiple factors, such as Nisin concentration, bacterial concentration, physiological state of the bacteria, and the prevailing conditions (Delves-Broughton et al., 1996). Thus, our data support the premise that Nisin acts as a fast-acting antibiofilm agent with both biofilm-static and biofilm-killing properties.

Difference in plaque scores between Nisin and CHX was statistically significant, with CHX having superior plaque-inhibiting activity. Nisin Z mouthrinse was found to be a potent anti-plaque agent, though less efficacious than CHX mouthrinse.

Nisin Z can serve as a long-term antiplaque agent for maintenance or for the patients who complained of side effects due to CHX mouthrinse.

At the end of the study, no adverse effects of the Nisin Z mouthwash were seen in any of the participants.

Limitations of this study

The limitations of this study include small sample size, short duration, and anti-gingivitis efficacy of the Nisin Z extract mouthwash was not established due to shorter period of study. More studies using a longer period, larger sample size, and a crossover model can be performed for further research.


   Conclusion Top


Nisin Z mouthrinse was found to be a potent natural antiplaque agent, though less efficacious than CHX mouthrinse. It can serve as a good alternative for the patients who are on maintenance therapy or who want to avoid the side effects of CHX.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Flemmig TF. Periodontitis. Ann Periodontol 1999;4:32-8.  Back to cited text no. 1
    
2.
Brogden KA, Guthmiller JM. Polymicrobial Diseases. Washington DC: ASM Press; 2002.  Back to cited text no. 2
    
3.
Tagg JR, Dajani AS, Wannamaker LW. Bacteriocins of gram-positive bacteria. Bacteriol Rev 1976;40:722-56.  Back to cited text no. 3
    
4.
Nisin HA. In Advances in Applied Microbiology. Vol. 27. New York, USA: Academic Press; 1981. p. 85-123.  Back to cited text no. 4
    
5.
Schwalbe R, Steele-Moore L, Goodwin AC, editors. Antimicrobial Susceptibility Testing Protocols. New York, USA: CRC Press; 2007.  Back to cited text no. 5
    
6.
Nuryshev MZ, Stoyanova LG, Netrusov AI. New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective opportunities and prospects. J Microb Biochem Technol 2016;8:290-5.  Back to cited text no. 6
    
7.
Zou Y, Lee HY, Seo YC, Ahn J. Enhanced antimicrobial activity of nisin-loaded liposomal nanoparticles against foodborne pathogens. J Food Sci 2012;77:M165-70.  Back to cited text no. 7
    
8.
Wiedemann I, Breukink E, van Kraaij C, Kuipers OP, Bierbaum G, de Kruijff B, et al. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J Biol Chem 2001;276:1772-9.  Back to cited text no. 8
    
9.
Rosin M, Welk A, Kocher T, Majic-Todt A, Kramer A, Pitten FA, et al. The effect of a polyhexamethylene biguanide mouthrinse compared to an essential oil rinse and a chlorhexidine rinse on bacterial counts and 4-day plaque regrowth. J Clin Periodontol 2002;29:392-9.  Back to cited text no. 9
    
10.
Addy M, Willis L, Moran J. Effect of toothpaste rinses compared with chlorhexidine on plaque formation during a 4-day period. J Clin Periodontol 1983;10:89-99.  Back to cited text no. 10
    
11.
Bhadbhade SJ, Acharya AB, Rodrigues SV, Thakur SL. The antiplaque efficacy of pomegranate mouthrinse. Quintessence Int 2011;42:29-36.  Back to cited text no. 11
    



 
 
    Tables

  [Table 1], [Table 2]



 

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