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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 164-168

Antibacterial activity of melatonin against prime periodontal pathogens: An in vitro study


1 Department of Periodontology, Jagadguru Sri Shivarathreshwara Dental College and Hospital, Mysuru, Karnataka, India
2 Public Health Dentistry, Jagadguru Sri Shivarathreshwara Dental College and Hospital, Mysuru, Karnataka, India
3 Department of Microbiology, Jagadguru Sri Shivarathreshwara Medical College and Hospital, Mysuru, Karnataka, India
4 Department of Pharmaceutics, Jagadguru Sri Shivarathreshwara College of Pharmacy, Mysuru, Karnataka, India

Date of Submission29-Jun-2020
Date of Decision20-Aug-2020
Date of Acceptance28-Dec-2020
Date of Web Publication17-Apr-2021

Correspondence Address:
Dr. Aruna Ganganna
Department of Periodontology, Jagadguru Sri Shivarathreshwara Dental College and Hospital, Mysuru 570015, Karnataka.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_225_20

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  Abstract 

Aim: Accumulated data make it clear that by introducing large amount of antibiotics into the ecosystem, we have provided an environment conducive to antibiotic resistance and periodontal microbes are no different. Therefore, in the quest of finding a suitable drug as an alternate to antibiotic, we investigated the antibacterial activity of melatonin against predominantly Gram-negative periodontal pathogens in vitro. Melatonin with varied functions has driven its usage enormously; therefore, identifying its action against periodontal pathogens has driven this laboratory investigation. Materials and Methods: American type culture collection (ATCC) strains of Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were used to determine minimum inhibitory and bactericidal concentrations (MIC and MBC) of melatonin using macro broth dilution method. MIC values were determined at 24, 48, and 72 h and sensitive MIC values were sub-cultured to determine MBC value at 24 h. Qualitative data were obtained and it was recorded as “sensitive” or “resistant” at respective concentrations. Results: When tested over a dilution range of 0.2–100 µg/mL, melatonin significantly inhibited microbial growth. At 48 h, the MIC value against A. actinomycetemcomitans, P. gingivalis, and F. nucleatum was 6.25, 0.8, and ≤0.2 µg/mL, respectively. The MBC value determined at 24 h demonstrated significant bactericidal activity against the pathogens. Conclusion: Melatonin exhibited bactericidal activity against prime periodontal pathogens even at low concentrations in comparison to previously documented evidences suggesting greater potency of the drug. Hence, renewed effort to find and develop new class of drug which can inhibit periodontal pathogens, rather than just improvements on already-existing drugs can potentially prevent the development of resistant strains in periodontal microenvironment.

Keywords: Antibiotic resistance, minimum inhibitory concentration, periodontal pathogens


How to cite this article:
Ganganna A, Rudariah CB, Rao R, Prakash VM. Antibacterial activity of melatonin against prime periodontal pathogens: An in vitro study. J Int Oral Health 2021;13:164-8

How to cite this URL:
Ganganna A, Rudariah CB, Rao R, Prakash VM. Antibacterial activity of melatonin against prime periodontal pathogens: An in vitro study. J Int Oral Health [serial online] 2021 [cited 2021 Jun 17];13:164-8. Available from: https://www.jioh.org/text.asp?2021/13/2/164/313837


  Introduction Top


The discovery of penicillin by Sir Alexander Fleming in 1945 emerged as a life savior and transformed the field of medicine. This discovery extended the individual life span by changing the outcome of devastating infections, worldwide. Downside, overzealous usage and constant evolution of microbes raised an alarm of antibiotic resistance threatening the health benefits that were once achieved with these drugs. Hence, CDC drove aggressive action for combatting antibiotic resistance by initiating development of newer antibiotics/antibiotic as alternatives to fight the global threat.

Periodontal infections, being congregation of diverse microbiota requires adequate level of antimicrobial activity to revert from disease to health. Though more than 600 bacteria species populate the oral cavity about 10–15 species have been identified as potential periodontal pathogens.[1]Porphyromonas gingivalis, Aggregatibacter actinimycetemcomitans, and Fusobacterium nucleatum are some of the well-studied and documented oral microbes capable of periodontal soft tissue invasion leading to tooth loss. These are gram negative anaerobic organisms with inherent toxin producing abilities and liberate proteinases, cyto-lethal distending toxin, collagenases, hemolysin, hydrogen sulfide, epithelia-toxin, and ammonia to take charge of the host.[2]

When antibiotic resistance is virtually seen in almost all species, oral microbes are no different. Hence, melatonin as an antibiotic alternative against potent periodontal pathogens is being evaluated. Melatonin is known to bind to copper, iron, and zinc reducing its cytoplasmic pool and the unavailability of these metal ions leads to bacterial cell lysis. It is an endogenous molecule released from the pineal gland and possesses significant anti-microbial activity. It is known to inhibit the uptake of linoleic acid which is an important growth factor for tumor cells thereby exhibiting anti-tumorigenic effect.[3] Being a versatile molecule, it is used in managing sleep disorders,[4] sepsis management,[5] bacterial infections,[6] oral diseases,[7]and many more. Melatonin is known to exhibit circadian rhythm with a secretory peak at night and, therefore, called the “chemical of darkness.” The beneficial effects of melatonin are manifested through its pleiotropic properties as an immunomodulator, antioxidant, and cyto-protector which in turn is mediated by ubiquitous distribution of melatonin receptors.[8]

Interestingly, when the whole world cringed to fight the pandemic, corona virus disease-19 (COVID-19), Zhang et al. discussed the role of melatonin as an adjuvant in the treatment of COVID 19 which was further supported by Herrera et al.[9],[10] With such varied functions and beneficial effects, the potential use of melatonin in oral and periodontal diseases are encouraging. Hence, it is important to identify the minimum inhibitory/bactericidal concentration of this drug on periodontal pathogens. These evaluations can be quite useful in determining the appropriate concentrations of active ingredient required during drug development and its adequate release at the site of action.

Kilinçel et al.[11] reported the MIC values of melatonin with other antifungal drugs against candida strains and concluded that MIC values of melatonin to be much lower than other antifungal drugs. He also stated that combination of melatonin with Fluconazole made an effective combination against candida strains. Tekbas et al.[2] studied the antibacterial activity of melatonin on primary nosocomial pathogens, usually antibiotic resistant and concluded that the values can be effectively translated to in vivo use due to its significant inhibitory action. However, Wang et al. and Konar et al. reported contradicting results, stating limited in-vitro antimicrobial properties of melatonin.[12],[13]

The existing difference in behavior of periodontal pathogens and rest of the oral pathogens warrants the determination of MIC values. Melatonin, if found effective can offer therapeutic benefit by enhancing the effects of SRP, thereby diminishing the need for expensive and invasive surgical interventions. Hence, our laboratory investigation aims to identify the MIC and MBC values of melatonin against prime potential periodontal pathogens P. gingivalis, F. nucleatum, and A. actinomycetemcomitans.


  Materials and Methods Top


Preparation of melatonin solution

The laboratory investigation was carried at Maratha Mandal research institute, Belgaum. Melatonin was obtained from Sigma-Aldrich, as melatonin decomposes in ethanol solution on standing, fresh solution of melatonin was prepared wherein 10 mg of melatonin powder was dissolved in 1mL of ethanol.

Bacterial strain

Bacterial strains Pg-American type culture collection (ATCC)® 33277™, F. nucleatum- ATCC®25886™ and A. actinomycetemcomitans -ATCC® 29522™ were purchased from LGC Promo-chem, Bangalore and were stored at −80ºC. At the time of reviving, the strains were thawed at 37ºC in water bath and centrifuged at 500rpm for 10 mins. After centrifugation supernatant was removed and deposits were sub-cultured on blood agar. The plates were incubated in anaerobic jar with 80% nitrogen, 80% hydrogen, and 10% carbon-di-oxide at 37ºC for 48–72 h and the obtained growth was confirmed by gram staining to check for purity.

Minimum inhibitory concentration by macro broth dilution method

MIC of melatonin was performed according to Antimicrobial susceptibility testing protocol by Schwalve, Moore, and Goodwin, Crc Press 2007. Ciprofloxacin was used a control antibiotic.

Serial dilutions of the drug were done with Thio-glycollate broth (TGB), with initial tube containing 20 µL of drug into 380 µL of TGB. For dilutions, 200 µL TGB was added to next 9 tubes separately. From the initial tube 200 µL was transferred to first tube containing 200 µL of TGB. This was considered as 10:1 dilution. From 10:1 diluted tube, 200 µL was transferred to the second tube to make 10:2 dilution and serial dilutions were repeated up to 10:9 dilution of drug. Five microliters of ATCC microbial stock culture adjusted to 0.5 Mc Farland’s was added into 2mL of TGB. Two hundred microliters of the above prepared culture suspension was added to tubes containing serially diluted drug. The tubes were incubated for 48–72 h in anaerobic jar at 37ºC and observed for turbidity.

MBC test

Once the MIC was determined, the tubes with sensitive MIC values were sub cultured on blood agar plates and incubated in anaerobic jar with 80% nitrogen, 80% hydrogen, and 10% carbon-di-oxide at 37ºC for 24 h to check for any growth on the plate. The colonies were counted and tabulated. However, MIC and MBC values extracted were qualitative and did not demand statistical interpretation.


  Results Top


Antimicrobial activity of melatonin against P. gingivalis, A. actinomycetemcomitans, and F. nucleatum was tested over a range of 0.2–100 µg/mL. As the growth rate of non-sporing anaerobic bacteria varies in broth, media readings were taken at the end of 24, 48, and 72 h and the results are tabulated in [Table 1].
Table 1: Minimum inhibitory concentrations against A. actinomycetemcomitans, P. gingivalis, and F. nucleatum

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MBC values were recorded at the end of 24 h and reduction in the number of colonies with increasing concentration of the drug was observed [Table 2]. MBC value of melatonin against A. actinomycetemcomitans was 6.25 µg/mL and for P. gingivalis and F. nucleatum it was 1.6 µg/mL. There was 2–3 times increase in the MBC value of P. gingivalis and F. nucleatum from their respective MIC values at 24 h. However, no such difference was observed against A. actinomycetemcomitans wherein, MIC and MBC values were 6.25 µg/mL.
Table 2: Minimum bactericidal concentrations against A. actinomycetemcomitans, P. gingivalis, and F. nucleatum

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  Discussion Top


In-vitro anti-bacterial activity of melatonin against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans was determined in the present study. Microbial strains used were potent periodontal pathogens; hence, targeting these organisms can facilitate a disease-free periodontium. Additionally, refraining from the use of conventional antibiotics can prevent resistant mechanisms, hence using melatonin seems logical and encouraging.

Microbes causing periodontal disease do not restrict themselves to oral cavity but invade distant sites leading to systemic imbalance. Among the pathobionts, members of the early colonizing green complex, bridging orange complex members, and late colonizing red complex species have a pivotal role to play.[14] Therefore, mechanical debridement primarily aiming at plaque disruption is supplemented with systemic antimicrobials to effectively address microbial invasiveness, virulence, and also prevent disease recurrence.

Presence of a double membrane around Gram-negative organisms with diverse lipids creates permeability barrier rejecting the entry of certain drugs and antibiotics. However, melatonin being more lipophilic crosses cell membrane easily. P. gingivalis, with the help of proteinase–adhesin complex degrades hemoglobin, thereby utilizing heme for the production of metabolic energy. However, melatonin by its affinity to metal ions binds to iron, copper, and zinc, making it unavailable for bacterial utilization and survival.[15],[16]P. gingivalis and A. actinomycetemcomitans induces hypercitrullination by activation of NOS and calcium-dependent peptidyl-arginine deiminases activity. At physiologic concentrations, melatonin is known to inhibit NOS activity, besides its NO and peroxy-nitrite scavenging activity.[17],[18]

Our study showed that MIC value against A. actinomycetemcomitans (6.25 µg/mL at 24 h) remained the same even after prolonged incubation (72 h). However, MIC increased for P. gingivalis and F. nucleatum at the end of 72 h ((from ≤0.2 to 0.8 µg/mL and 1.6 µg/mL) in-contrast to the study by Tekbas et al.[2] where MIC decreased over time and was attributed to the antioxidant effects of DMSO which was used as a solvent. It was also stated that DMSO as a solvent prevented alcohol mixture precipitation at higher concentration of drug. In contrast, we used ethanol as solvent, the drug dilutions used were much lesser and, therefore, the problem of precipitation almost seemed negligible. Another reason for difference in the MIC values among F. nucleatum could be slow growth rate of organism in TGB.

Another study by Zhou et al.[19] used melatonin in the dilution range of 3.13–1600 μg/mL, wherein, MIC and MBC were 100 and 1600 μg/mL, respectively. The dilution range used in our study was lower but showed good bactericidal action indicating appreciable potency of the drug even at lower concentrations which can be translated favorably to clinical use. It has been stated that antibacterial agents are regarded as bactericidal if MBC is no more than four times the MIC. Our study showed the MIC and MBC values to be same for A. actinomycetemcomitans, but for P. gingivalis and F. nucleatum the difference was not more than four times the concentration of MIC.[20] Considering these findings it can be stated that melatonin displays excellent bactericidal role among the prime periodontal pathogens.

Limitations

Although most published literature highlight the use of MIC value to determine the antibacterial activity, it cannot be fully explained without the knowledge of pharmacodynamics and kinetics of the drug. It is important to understand that there is no such thing as “true MIC” for a strain and the obtained MIC value is just a value of probability. Additionally, the biological and assay variations such as strain to strain difference, inoculum preparation, pH, media, temperature, technical skills, storage conditions of prepared MIC trays may lead to over/underestimation of the antibacterial activity of the compound in Antimicrobial Susceptibility Tests and thus alter the clinical decision.


  Conclusion Top


Melatonin inhibited the growth of three primary periodontal pathogens. The antibacterial activity of melatonin is further strengthened by this in-vitro study. More importantly, we found potency of the drug at lower concentrations than previously mentioned studies. The presence of peripheral melatonin receptors on periodontal tissues especially on periodontal fibroblasts projects a bigger picture of its potential to be considered as an in vivo antibiotic and anti-inflammatory drug to maintain periodontal health and equilibrium.

Clinical relevance

Determining MIC is a basic laboratory procedure to know the activity of an antimicrobial agent against an organism. By identifying the appropriate drug with its effective concentration, it is possible to improve the patient outcome and also prevent evolution of drug-resistant microbial strains. Lower MIC value indicates that less of the drug is required to inhibit the organism and more effective could be the antimicrobial agent. With phylogenetic diversity of oral microorganisms, it is imperative to identify an appropriate drug against the causative organism before executing any therapeutic regimen. Currently, the use of melatonin against oral organisms as alternate to antibiotic protocol may initiate further trials to unveil its role in periodontal regeneration.

Additionally, the inhibitory concentrations mimicking a biofilm environment can be investigated to render better understanding. Within the limits of this in-vitro study, inhibitory concentrations of melatonin against one subtype of the prime three periodontal pathogens are investigated. However, further studies with multiple subspecies are warranted to validate the results of the present study.

Acknowledgement

The authors acknowledge JSS Academy of Higher Education and Research for funding this project.

Financial support and sponsorship

JSS Academy of Higher Education and Research REG/DIR(R)/URG/54/2011–12/10312.

Conflict of interest

There are no conflicts of interest.

Ethical Policy and Institutional Review Board Statement

The present study is ethically approved by institutional review board. (JSSDCH IEC Research No:58/2019, dated 25/10/2019)

Declaration of patient consent

Not applicable.

Data availability statement

Data will be provided on valid request basis by contacting corresponding author mail.

 
  References Top

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2.
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Xu L, Zhang W, Kwak M, Zhang LJ, Lee PCW, Jin J-O Protective effect of melatonin against polymicrobial sepsis is mediated by the anti-bacterial effect of neutrophils. Front Immunol2019;10:1371.  Back to cited text no. 5
    
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Zhang R, Wang X, Ni L, Di X, Ma B, Niu S, et al. COVID-19: Melatonin as a potential adjuvant treatment. Life Sci 2020;250:117583.  Back to cited text no. 9
    
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Kilinçel Ö, Çaliskan E, Sahin I, Öztürk CE, Kiliç N, Öksüz S The effect of melatonin on antifungal susceptibility in planktonic and biofilm forms of candida strains isolated from clinical samples. Med Mycol 2019;57:45-51.  Back to cited text no. 11
    
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