Abstract
Aim: To compare the antibacterial efficacy of Manuka honey against E.faecalis and E.coli.
Materials and Methods: Escherichia coli (ATCC-25922) and Enterococcus faecalis (ATCC-29212) were separately inoculated in the nutrient broth and incubated at 37oC for 24-48 hrs. Bacterial samples were kept in contact with each disinfectant solution for varying intervals of time. Once the test time had elapsed 10μL of the bacterial dilutions were plated on Mueller–Hinton agar and incubated for 24-48 hrs at 37°C to estimate the density. Study of the disinfection process with respect to time and Modeling was done.
Results: The mean value of the antimicrobial activity of Manuka honey against E.coli and E.faecalis are 1.55 and 0.36 respectively and are relatively higher. It shows that there is a significant difference among the various root canal disinfectant groups against E. coli and E. feacalis. (p<0.001)
Conclusion: Manuka honey is shown to be a potential root canal disinfectant against gram positive and gram negative bacterial pathogens.
Keywords: Antibacterial efficacy, Death kinetics, Manuka honey
Introduction
During the past 20yrs endodontics has began to appreciate critically the important role of irrigation in successful endodontic treatment. The objective of the endodontic treatment is to remove the infection within the root canal. Over the years research and clinical practices have concentrated on instrumentation, irrigation, medication of root canal system followed by filling of the root canal space, it’s truly said ‘Instruments shape, irrigants clean’. Following cleaning and shaping of the infected root canal the number of bacteria are reduced, but instrumentation only cannot clean all the surfaces of root canal. Bacteria can be found on the root canal walls and lateral canals within the dentinal tubules. Antibacterial disinfectants and intracanal medicaments are needed to kill the remaining microorganisms [1].
History of honey in medicine is related to stoneage paintings in several locations dating 6000 BC. It is indicated that honey has a role as therapeutic agent in oral disease [2]. It has been often assumed that it is entirely due to osmotic effect of its high sugar control. The fact that the antibacterial properties of honey were increased when diluted [3]. Manuka honey is a monofloral honey obtained from the species Leptospermumscorpium and has a long standing reputation in New Zealand for its antiseptic property [4].
Hence, the present study was to compare the antibacterial efficacy of Manuka honey against E.faecalis and E.coli with some of the routine root canal disinfectants used in endodontics. They are Calcium Hydroxide (Ca(OH)2), 5.25% Sodium Hypochlorite (NaOCl), 2% Chlorhexidine (CHX), 0.2% Chlorhexidine and Saline.
Materials and Methods
Direct Contact Test (DCT)
Nutrient broth was prepared and sterilized. Escherichia coli (ATCC-25922) and Enterococcus faecalis (ATCC-29212) were inoculated separately and incubated at 37oC for 24-48 hrs.
One milliliter (mL) of the bacterial suspensions (109CFU/mL−1) was centrifuged in micro tubes. Routinely used root canal disinfectants 5.25% NaOCl, Net Manuka honey, 2% CHX, 0.2% CHX, Ca(OH)2, Honey 1:2 dilution, Honey 1:4 dilution, Saline were added to the sediment (pellet), vortexed and stored for different time intervals before 50 μL aquilots were transferred to 5mL of Nutrient broth. Bacterial samples were kept in contact with each disinfectant solution for varying intervals of time; Group1-Immediately, Group2-5 min, Group 3-10 min, Group 4-15 min, Group 5-20 min, Group 6-25 min, Group 7-30 min. Once the test time had elapsed 10 μL of the bacterial dilutions were plated on Mueller–Hinton agar and incubated for 24-48 hrs at 37°C to estimate the density. All tests were repeated 10 times for each irrigating solution. Each irrigant solution without the bacterial strains was kept as negative control.
Modelling equation of Disinfectants
dx/dt= -Kdx
dx/dt=Death rate
X=microbial density
Kd =death rate constant
Results
Direct contact test of E.coli and E. faecalis with various root canal disinfectants were tabulated in [Table/Fig-1,2].
[Table/Fig-1]:
Effect of root canal disinfectants on E.coli
| Groups | N | Mean | SD | p-value |
|---|---|---|---|---|
| 1 | 1 | 0.00 | 0.00 | p<0.001 |
| 2 | 3 | 1.07 | 0.01 | p<0.001 |
| 3 | 3 | 1.59 | 0.01 | p<0.001 |
| 4 | 3 | 0.48 | 0.01 | p<0.001 |
| 5 | 3 | 1.55 | 0.01 | p<0.001 |
| 6 | 3 | 1.04 | 0.01 | p<0.001 |
| 7 | 3 | 0.67 | 0.58 | p<0.001 |
| Total | 19 | 1.01 | 0.51 | p<0.001 |
[Table/Fig-2]:
Effect of root canal disinfectants on E.faecalis
| Groups | N | Mean | SD | p-value |
|---|---|---|---|---|
| 1 | 3 | 1.00 | 0.200 | p<0.001 |
| 2 | 3 | 0.49 | 0.025 | p<0.001 |
| 3 | 3 | 0.53 | 0.051 | p<0.001 |
| 4 | 3 | 0.12 | 0.020 | p<0.001 |
| 5 | 3 | 0.36 | 0.020 | p<0.001 |
| 6 | 3 | 0.18 | 0.026 | p<0.001 |
| 7 | 3 | 0.95 | 0.020 | p<0.001 |
| Total | 21 | 0.52 | 0.032 | p<0.001 |
Death kinetics analysis of E. coli and E. faecalis showed reduction in the density of pathogens at various time intervals. The observation data from various time intervals shows that 5.25% NaOCl, 2% CHX, 0.2% CHX, Net Manuka honey are bactericidal while Ca(OH)2, Honey 1:2 dilution, Honey 1:4 dilution are bacteriostatic based on the death rate constant values.
Mean and Standard deviation were estimated from the sample for each study group. The mean values were compared by one-way ANOVA. In the present study, the level of significance was set at p = 0.05. The obtained p-value is less than 0.001and there was a significant difference among the groups.
Discussion
Direct contact test was performed in this study to quantitatively measure the effect of physical direct contact between the tested material and bacteria [5].The microorganisms used in this study were facultative bacteria, which are predominant in persistent periapical lesions of teeth subjected to periapical surgery. E.faecalis is a robust microorganism that may infect root canal and are more likely to be found in root canal failure cases than in cases of primary infection. E.coli is sometimes recovered from root canals and represents a standard organism used in antimicrobial testing [6,7].
5.25%NaOCl, 2% CHX, 0.2% CHX are widely used in endodontics as root canal irrigants at different concentrations, but some of them are causing irritation to the periapical tissues. To overcome the side effects of root canal irrigants like sodium hypochlorite, chlorhexidine previous studies have investigated the effects of natural compounds mainly extracted from teas, cranberrie or propolis for antimicrobial purpose [8,9].
The antibacterial properties of honey have recently reviewed in different aspects. In ripe honey the glucose oxidase is inactive and it contains less peroxide amount which is not sufficient to inhibit bacterial growth. When it is heated and diluted peroxide is formed which is having antibacterial property. The non peroxide antibacterial activity is not sensitive to heat and light and remains intact after storage of honey for longer periods. Sugar is the main constituent of honey. The osmotic effect of these sugars exerts an antimicrobial action. Honey contains an enzyme lysozyme, which is a well known antibacterial agent [10]. The antibacterial flavanoid pinocembrim is present in honey, but its concentration and contribution to honey’s non peroxide antibacterial activity is small. In New Zealand Manuka honey, several aromatic acids with antibacterial activity have been isolated [11]. Another investigation claimed, the low ph and high osmolarity of honey was responsible for the antibacterial activity [12].
In some studies, they have isolated volatile substances with antibacterial activity [13,14] but their quantitative contribution to the antibacterial action of honey was not examined. Some authors postulated that non peroxide activity of honey is by organic solvents, but the exact chemical nature of these substances is not known [15,16]. The chemical identity, the quantitative contribution and origin of the different honey antimicrobial substances remain to a greater extent unknown [17].
Dold and Witzenhainen coined the term inhibine number to describe the degree of dilution to which the honey will retain its antibacterial activity.
The present work proved that Manuka honey was active against gram positive and gram negative bacterial pathogens. Besides hydrogen peroxide, which is produced on dilution in most “conventional” honeys by the endogenous enzyme glucoseoxidase several other “non peroxide factors” were discussed to be responsible for the unique antibacterial activity of Manuka honey on dilution but the chemistry beyond the phenomenon remained unclear for decades. Nevertheless, the so called “unique manuka factor” (UMF) was introduced some years ago for marketing purposes, leading to the classification of premium products based on microbiological assays. A UMF of 10 used in this study has the same antibacterial activity to a 10% solution of phenol (Allen et al., 1991).
Bilal et al., found that honey exhibited good antimicrobial activity against gram positive and gram negative bacteria [18]. Raied T ahe-Al-Naama reported that 100% honey concentration showed higher zone of microbial inhibition when compared to that of 50% honey concentration [19].
In a study conducted by Litik Mittal et al., 50% and 100% honey concentrations showed greater antimicrobial activity against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25853 and least antimicrobial activity against Pseudomonas aeriginosa ATCC 27853 and concluded that 50% honey and 100% honey had significant antimicrobial action against broad spectrum of bacteria [20].
The results of this study showed decreased antibacterial activity on dilutions. So the increased antibacterial activity of net Manuka honey suggest that osmotic disturbance, leakage of cellular materials, floral origin, phytochemical component could be the possible mechanism behind the antibacterial activity of net Manuka honey used in this study. Ahmadi et al., reported that 5%, 10%, 20%, 50% and 100% honey concentrations prepared in sterile solutions showed significant antibacterial activity against Streptococcus mutans PTCC 1683 and Lactobacillus PTCC 1643 and concluded that greater than 20% honey concentrations had significant antibacterial activity on Streptococcus mutans and 100% honey concentration on Lactobacillus [21]. The role of H2O2 and nonperoxide are not promising in this study as they showed decreased antibacterial activity of Manuka honey on dilutions and this should be confirmed in further studies and research on this particular statement.
Manuka honey can be used as an alternative endodontic disinfectant to sodium hypochlorite and chlorhexidine as it shows good antimicrobial properties and as a substitute in patients who are allergic to NaOCl [22]. This study also shows that non peroxide and hydrogen peroxide components alone does not play active role in the antibacterial property of honey. The known safe use of honey without toxic effects suggests that honey could be used to treat infections arising from bacterial pathogens [23].
Conclusion
This study suggests Manuka honey to be a potential root canal disinfectant against gram positive and gram negative bacterial pathogens in the search for a biocompatiable agent, but clinical trials need to be carried out to what extent this is true.
Financial or Other Competing Interests
None.
References
- [1].Valera Menlze M. Invitro evaluation of the effectiveness of irrigants and intracanal medicament. Int Endod J. 2004;37:311–19. doi: 10.1111/j.0143-2885.2004.00799.x. [DOI] [PubMed] [Google Scholar]
- [2].Molan PC. The potential of honey to promote oral health and wellness. J General Dentistry. 2001;49(6):584–89. [PubMed] [Google Scholar]
- [3].Sackell WG. Honey as a carrier of intestinal diseases. Bull Colarado State Univ Agric Exp. 1919;252:1–18. [Google Scholar]
- [4].English HK, Pack AR, Molan PC. The effects of Manuka honey on plaque and gingivitis. A Pilot Study. J Int Acad Periodontology. 2004;6:63–7. (Pub Med) [PubMed] [Google Scholar]
- [5].Weiss EL, Shalhav M, Fuss Z. Assessment of antibacterial activity of endodontic sealers by a direct contact test. Endod Dent Traumatology. . 1996;12:179–84. doi: 10.1111/j.1600-9657.1996.tb00511.x. [DOI] [PubMed] [Google Scholar]
- [6].Basrani B, Tjaderhane L, Snatos JM, Pascon E, Grad H, Lawrence HP, et al. Efficacy of chlorhexidine and calcium hydroxide – containing medicaments against E.faecalis invitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003;96:618–24. doi: 10.1016/s1079-2104(03)00166-5. [DOI] [PubMed] [Google Scholar]
- [7].Heling I, Chandler NP. Antimicrobial effect of irrigant combination within dentinal tubules. Int Endod J. 1998;31:8–14. [PubMed] [Google Scholar]
- [8].Labrecque J, Bodet C, Chandad F, Grenien D. Effects of high molecular weight cranberry fraction on growth biofilm formation and adherence of porphyromonosgingivalis. J Antimicrob Chemotherapy. 2006;58:439–43. doi: 10.1093/jac/dkl220. [DOI] [PubMed] [Google Scholar]
- [9].Gregoire S, Singh AP, Vorsa N, Kooh Influence of cranberry phenolics on glucansysthesis by glucosyltransferasean streptococcus mutans acidogenicity. J Appl Microbiol. 2007;103:1960–68. doi: 10.1111/j.1365-2672.2007.03441.x. [DOI] [PubMed] [Google Scholar]
- [10].Bogdanor S. Characterisation of antibacterial substances in honey. Lebensmittel –Wissenschaft and Technologies. 1984;17:74–6. [Google Scholar]
- [11].Russel KM, Molan P, Wilkins AL, Holland PT. Identification of some antibacterial constituent of New Zealand Manuka Honey. Journal of Agricultural and Food Chemistry. 1989;38:10–3. [Google Scholar]
- [12].Yatsunami K, Echigo T. Antibacterial activity of honey and royal jelly. Honeybee Sciences. 1984:125–30. [Google Scholar]
- [13].Toth G, Lemberkovics E, Kutasi-szabo The volatile components of some hugarian honeys and their antimicrobial effects. American bee Journal. 1987;127:496–97. [Google Scholar]
- [14].Obaeiseki–Ebor EE, Afonoya TCA, Onyekweli AO. Preliminary report on the antimicrobial activity of honey distillate. Journal of Pharmacy and Pharmacology. 1983;35:748–49. doi: 10.1111/j.2042-7158.1983.tb02884.x. [DOI] [PubMed] [Google Scholar]
- [15].Echigo T, Takenaka T. Production of organic acids in honeys by honey bees. Journal of Agricultural Chemical Society of Japan. 1974;48:225–30. [Google Scholar]
- [16].Bogdanov S. Naturre and origin of the antibacterial substances in honey. Lebensmitte/ Wissenschaft and Technologies. 1997;30:748–53. [Google Scholar]
- [17].Antibacterial substances in honey. Swissbee Research Centre. 1997 [Google Scholar]
- [18].Bilal AN, Molan PC, Sallal AK. Antimicrobial activity of honey on selected microorganisms. A Preliminary study. Biomed Res. 1. 998;9:51–4. [Google Scholar]
- [19].Raied Taha Al Naama. Evaluation in vitro inhibitory effect of honey on some microbial isolate. Journal of Bacteriology Res. 2009;1(6):64–7. [Google Scholar]
- [20].Mittal Litik, Kakkar Pooja, Verma Arun, Dixit Kuldeep Kumar, Mehrotra Mridul. Anti microbial activity of honey against various endodontic microorganisms – An in vitro study. J Int Den Med Res. 2012;5(1):9–13. [Google Scholar]
- [21].Fatemeh Ahmadi – Motamayel, Seyedeh Sare Hendi, Mohammad Yusof Alikhani, Zahra Khamverdi. Antibacrial activity of Honey on cariogenic bacteria. Journal of Dentistry. 2013;10(1):10–5. [PMC free article] [PubMed] [Google Scholar]
- [22].Kaufaman AX, Ketlas Hypersensitivity to NaOCl. J Endod. 1989;15:224–26. [Google Scholar]
- [23].Adetuys FO, Ibrahim TA, Jude-Ojei, Ogundahunsi GA. Total phenol, tocopherol and antibacterial quality of honey apimellifera sold in Owo community, Ondo State, Nigeria. African Journal of Biotechnology. 2009;8(7):1305–09. [Google Scholar]