Abstract
Background:
Oral diseases continue to represent a major health problem worldwide. Various bacteria and fungi are found to be the possible pathogens responsible for the oral disease.Objectives:
The present in vitro study was conducted to compare the antimicrobial efficacy of mouthrinse (MR) prepared from Brazilian propolis with that of two commercially available MR, Hexidine (chlorhexidine mouthrinse) (CHX), S-Flo (0.2% sodium fluoride) against common oral pathogens i.e. Streptococcus mutans, Candida albicans and Escherichia coli.Materials and Methods:
The antimicrobial efficacy of three MR was measured against freeze-dried culture of S. mutans (MTCC 890), E. coli (MTCC 44) and C. albicans (MTCC 1637).The antimicrobial activity was determined by the agar well diffusion method and the zone of inhibition produced by the mouthwashes against the test organisms was measured. The data were analyzed using SPSS software, version 20 and the one-way ANOVA test was used to compare in vitro antimicrobial activity of various MR.Results:
The antimicrobial efficacy of Hexidine against S. mutans and E. coli was higher compared to that of C. albicans. The Brazilian Propolis (P) MR showed antimicrobial action against S. mutans and C. albicans. The antimicrobial efficacy of P against C. albicans was higher compared to that of CHX. The S-Flo MR (0.2% sodium fluoride) and the negative control group did not show any zone of inhibition against all three test stains.Conclusions:
The results of the present study indicate that Brazilian Propolis demonstrated in vitro anti-microbial efficacy against S. mutans and C. albicans.Keywords
Candida albicans Escherichia coli Propolis Streptococcus mutans
1. Background
Oral diseases continue to be a major health problem worldwide. Various bacteria and fungi are found to be the possible pathogens responsible for the oral diseases (1). Dental caries and periodontal diseases are among the most important global oral health problems. Streptococcus mutans is one of the main opportunistic pathogens for dental caries (2), which plays a central role in demineralization of the tooth enamel (3). There are several virulence factors that make these bacteria cariogenic, including adhesion, acidogenicity and acid tolerance (4). In addition, other microflora like Escherichia coli and Candida albicans are also associated with active carious lesions. Candidaalbicans is the most common yeast isolated from the oral cavity and a common cause of oral thrush, endocarditis, septicemia, vaginitis and infection of skin, nails and lungs. It is by far the most common of the fungal species most commonly isolated from infected root canals, showing resistance to intercanal medication (5). Poor oral hygiene is one of the reasons for accumulation of these microbes and their harmful activities (3).
According to Carranza, plaque control is the most effective way for preventing the development of the oral diseases. Most of the oral microbial diseases can be prevented by effectively removing plaque deposition from the teeth surfaces by the use of the mechanical aids, such as a toothbrush (6). Although people try to maintain their oral hygiene, many of them cannot remove plaque, and therefore mouth rinses are used to complete the process of plaque removal (7). Although mouth rinses have been used for centuries for medicinal and cosmetic purposes, but it is only in recent years that the rationale behind the use of chemical ingredients has been subject to scientific research and clinical trials (8, 9). The most common commercially used mouth rinses are chlorhexidine (CHX), essential oils, triclosan and sodium fluoride (S-Flo).
The chemical plaque control agents such as CHX can be useful adjuncts for plaque control. Nevertheless, they can only be used as supplement and not as substitute to the mechanical plaque control. They have been found to be effective in the control of plaque and gingivitis, as demonstrated in the various studies (9). Topical fluoride promotes remineralization and inhibits demineralization of enamel during caries process. Another effect of fluoride includes the inhibition of glycolysis, coupled with a reduction in the production of extracellular polysaccharide (10, 11).
There are multiple natural compounds like xylitol, green tea, mint and propolis that had been tested as mouth rinses. Propolis, sometimes called as bee glue, is a natural resinous substance collected by honey bees (Apis mellifera L.) from plant buds and bark exudates. Propolis is a very complex mixture and its chemical constituents vary according to its source. Bioflavanoids are the key contributors to the antimicrobial properties of propolis (12).
The potential of propolis, as a natural antibiotic, has long attracted the scientific interest (12). Propolis has been found effective in inhibiting several species of microbes, such as bacteria, viruses and fungi, which explains its antimicrobial action (13, 14).
2. Objectives
The present in vitro study was conducted to assess the antimicrobial efficacy of MR prepared from Brazilian propolis, as compared to S-Flo MR and CHX MR against common oral pathogens like S. mutans, C. albicans and E.coli.
4. Results
The results for the antimicrobial efficacy of various MRs against three different organisms were as presented in Table 1.
Comparison of Zone of Inhibition Produced by Test Solutions Against Three Microorganisms According to the Microorganism a
| Compounds | Microorganisms | P Value b | Post Hoc Test | ||
|---|---|---|---|---|---|
| Streptococcus mutans | Candida albicans | Escherichia coli | |||
| Chlorhexidine | 16.30 ± 0.48 | 7.10 ±1.10 | 11.80 ± 1.03 | < 0.001 | C. albicans < E. coli < S. mutans |
| Propolis extract | 11.00 ± 0.67 | 11.30 ± 1.16 | 0 | < 0.001 | E. coli < S. mutans < C. albicans |
| Sodium fluoride | 0 | 0 | 0 | NA c | NA |
| Normal saline | 0 | 0 | 0 | NA | NA |
4.1. Streptococcus mutans
The CHX followed by propolis had a significantly larger zone of inhibition than S-Flo and the control group (Figure 1).
Mean Zone of Inhibition Produced by the Four Test Solutions Against Streptococcus mutans
4.2. Candida albicans
Propolis extract MR (11.30 ± 1.160) and CHX (7.10 ± 1.101) showed significantly (P < 0.05) larger zones of inhibition in comparison to S-Flo (0) and the control group (0), whereas propolis had a significantly (P < 0.05) greater zone of inhibition than CHX (Figure 2).
Mean Zones of Inhibition Produced by Four Test Solutions Against Candida albicans
4.3. Escherichia coli
Only CHX MR showed zone of inhibition (11.80 ± 1.033) against E. coli. The results for antimicrobial efficacy of individual MR against the three organisms are depicted in Table 2 and Figure 3.
Comparison of the Zone of Inhibition Produced by Four Test Solutions Against Microorganism According to the Mouthwash a
| Microorganisms | Compounds | P Value b | Post Hoc Test | |||
|---|---|---|---|---|---|---|
| Chlorhexidine | Propolis Extract | Sodium Fluoride | Control | |||
| Streptococcus mutans | 16.30 ± 0.48 | 11.00 ± 0.67 | 0.00 | 0.00 | 0.000 (< 0.05) | Chlorhexidine > Propolis Extract > Sodium Fluoride, and Control |
| Candida albicans | 7.10 ± 1.10 | 11.30 ± 1.16 | 0.00 | 0.00 | 0.000 (< 0.05) | Propolis Extract > Chlorhexidine > Sodium Fluoride = Control |
| Escherichia coli | 11.03 ± 1.03 | 0.00 | 0.00 | 0.00 | 0.000 (< 0.05) | Chlorhexidine > Propolis Extract, Sodium Fluoride, and Control |
Mean Zones of Inhibition Produced by Four Test Solutions Against Escherichia coli
4.4. Chlorhexidine
Chlorhexidine MR showed a significantly (P < 0.05) higher zone of inhibition against S.mutans (16.30 ± 0.48) in comparison to E. coli (11.80 ± 1.03) and C. albicans (7.10 ± 1.10). Mean zone of inhibition produced by CHX MR was significantly higher against E. coli in comparison to C. albicans.
4.5. Propolis Extract
Propolis extract MR showed a significantly (P < 0.05) larger zone of inhibition against C. albicans (11.30 ± 1.16) in comparison to S. mutans (11.0 ± 0.67) and E. coli. Mean zone of inhibition produced by propolis extract was significantly higher against S. mutans, in comparison to E. coli.
4.6. Sodium Fluoride
Sodium fluoride MR did not show any zone of inhibition against all three test organisms, demonstrating no antimicrobial activity.
5. Discussion
Dental plaque harbors the microorganisms and is primarily responsible for the occurrence of various dental diseases (2). The plaque formation is a multistep process which consists of a sequential colonization of the bacteria on the tooth surface, beginning from the attachment of the first species onto the tooth surface, followed by the evolution into the highly evolved species (2, 3).
Most of the oral diseases are a result of the activity of various microorganisms present in the plaque, which need to be kept in check. Therefore, agents which can inhibit these microorganisms have represented an area of interest in research (15). These substances act by either killing the microorganisms, or by disrupting their cell walls, or by inhibiting their enzymatic activity. They also prevent bacterial aggregation, slow multiplication and release of endotoxins (16). Several clinical studies have demonstrated the inhibitory effects of antimicrobial MRs on oral bacteria (17).
In the present study, CHX MR was found to be significantly (P < 0.001) more efficacious against S. mutans and E. coli than all other tested MR. Similar results have been reported by Malhotra et al. (18), which showed that Hexidine (0.12% chlorhexidine gluconate) MR had the best antimicrobial efficacy against all the tested microorganisms, with laboratory-manufactured propolis MR showing an equivalent efficacy only against S. mutans. According to the study conducted by Aneja et al. (19), Hexidine MR emerged as the most effective MR, with a maximum mean diameter of the inhibition zone against S. aureus, followed by S. mutans, S. cerevisiae, and minimum against C. albicans. Similar results have been reported in the studies conducted by Nakamoto et al. (20), which showed that CHX gluconate has an inhibitory effect on the growth of C. albicans.
Chlorhexidine is a cationic bisbiguanide with antimicrobial properties, which depends on its concentration, acting as bacteriostatic at low concentrations and bactericidal at high concentrations. It has the capacity to inhibit all known microbes in the oral cavity, therefore contributing to its broad-spectrum activity and being considered the gold-standard for plaque control.
The efficacy of Brazilian propolis used in the present study was found to be more substantial against S. mutans and C. albicans, as compared to E. coli. Although the efficacy of the propolis extract was lower than that of CHX MR for S. mutans, the efficacy of propolis extract was superior to that of CHX for C. albicans. Similar results have also been demonstrated by Bruschi et al. (21) and Ugur and Arslan (22). According to the study conducted by Ugur and Arslan, the most sensitive microorganism to propolis were Shigella sonnei, in the gram-negative group, S. mutans, in the gram-positive group, while the least sensitive microorganism was C. albicans (22). However, the study conducted by Malhotra et al. (18) showed that laboratory manufactured propolis MR has an equivalent efficacy against S. mutans when compared to CHX MR.
Duailibe et al. (23) observed that propolis extract possesses in vivo antimicrobial activity against S. mutans. This can be attributed to the following properties: antibacterial activity due to components like the flavonoids, which are considered to be the principal components for its biological activities (24) and anticariogenic effects (25). The different varieties of flavonoids have been reported in different types of propolis, which contribute to its cariostatic activity. The variation in cariostatic activity is due to the varied composition of the flavonoids as a result of the different sources from which propolis is obtained (25-27).
Propolis, due to its availability and no reported side-effects up to present date, can be a useful option. The effectiveness of propolis is different according to the region from which it is obtained, due to a change in the chemical composition from area to area (24). It has a varied effect on the bacteria, being more effective against gram-positive bacteria (28-30). Although the antimicrobial properties of propolis have been evaluated by several investigations, it is difficult to compare the results of different studies due to the different methods used (31). However, the results of the study conducted by Elbaz et al. (4) reveal that the New Zealand propolis lozenges had a potent antimicrobial activity.
The efficacy of the S-Flo MR was lower than CHX and propolis, against all the tested microorganisms. Most of the in vivo studies have shown a cariostatic effect of fluoride gels or MR at 1% or 2% fluoride concentration (32). Consequently, the observed lower efficacy of S-Flo could also be attributed to the lower percentage of fluoride (0.2% of NaF) used in this study.
Meurman (32) studied the ultrastructure, growth, and adherence of S. mutans ATCC 27351 to hydroxyapatite, after treating bacterial suspensions for 1 hour with 0.1% CHX gluconate, 0.1% S-Flo, and a combination of the two. The fluoride treated specimens appeared the same as the controls, while the ultrastructure was mostly normal. Treatment with fluoride alone did not cause alterations in the ultrastructure or reduction in adsorption of S. mutans. Nevertheless, the study conducted by Gamal El-Din et al. (11) showed the presence of a statistically significant difference between the effect of both types (S-Flo and Propolis) on mean S. mutans count in oral cavity of girls and also, a statistically significant difference between their effect on the change in S. mutans count in both sexes during the follow-up period.
This testing method functioned only as a screening method and does not prove similar efficacy when used as a MR. There is a definite reduction in the level of bacteria and other pathogenic microorganisms in saliva (31, 33, 34) and mucosa (17, 35), which has been warranted by multiple studies assessing the efficacy of the antimicrobial MR for the prevention of the oral disease. However, antimicrobial efficacy was checked in vitro, and therefore it cannot be assumed that the results of antimicrobial efficacy could be proportional or transferable to the oral cavity and translated into clinical effectiveness. Consequently, from the overall results obtained, it is evident that various MRs listing CHX and propolis extract as the active ingredient demonstrate different antimicrobial activities.
Acknowledgements
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