A Comparative Evaluation of the Antimicrobial Effects of Different Mouthrinses against Oral Pathogens: An In Vitro Study¹

INTRODUCTION

Streptococcus mutans (S. mutans) is a gram-positive bacterium. It is a significant contributor to the development of cariogenic plaque.^1,2Lactobacillus is a genus of gram-positive bacterium associated with caries progression and represent one of the main parts of the lactic acid bacteria group.³ However, a few number of children with no caries were positive for lactobacilli.^4–6Lactobacillus genus includes more than 80 species, some of which having been found in the oral cavity such as Lactobacillus fermentum and Lactobacillus casei groups.^6–12 In clinical practice, the use of preventive measures is critical to decrease the risk for dental caries in highly susceptible individuals. It is believed that early prevention of bacterial growth can eliminate its colonization which aid in prevention of destruction of the tooth structure.¹³ Practicing oral hygiene by teeth brushing as a mechanical plaque control measure is desirable and commonly used.¹⁴ Besides that, many anti-plaque agents have been in use as supplementary aids.¹⁴ It is believed that using mouthrinses could act as an effective and harmless method for delivery of antimicrobial agents that prevent bacterial adhesion, colonization, and disturb the bacterial growth.¹⁴ Chlorhexidine is the gold standard mouthrinse due to its antimicrobial action.^15,16 However, despite its side effects such as teeth and soft tissue discoloration, taste alteration, and supragingival calculus formation, its continued use is supported.^16–18 It was reported that the existence of plaque covering the teeth surfaces increased the chlorhexidine side effects and authors reinforced the importance of plaque removal before chlorhexidine mouthrinse usage.¹⁹ Nowadays, patients are more worried about the side effects of synthetic chemical products on their oral health. Moreover, growing connection between oral health and herbal remedy offers an enduring approach of health repair in a most valuable way.²⁰ To the best of our knowledge, there are no investigations concerning the antimicrobial effects of the combination of naturally derivative compounds such as herbal mix mouthrinse and semi-natural compounds such as cranberry extract mixed with chlorhexidine digluconate mouthrinse against oral pathogens. Therefore, the aim of this in vitro comparative study was to assess the antimicrobial effects of different natural and semi-natural mouthrinses on isolates of S. mutans, L. fermentum, and L. casei that were obtained from the saliva of the 7- to 10-year-old Saudi children and their reference strains ATCC 25175, ATCC 9338, and ATCC 334, respectively. The null hypothesis was there is no significant difference in the antimicrobial effects between tested and control mouthrinses against S. mutans, L. fermentum, and L. casei that were isolated from the saliva of the 7- to 10-year-old Saudi children.

MATERIALS AND METHODS

Twenty children who met the inclusion criteria were selected from the Dental University Hospital, King Saud University, after study approval by the Institutional Review Board. A consent was obtained from parents or legal guardian.

RESULTS

The study included 20 children, 9 (45%) females and 11 (55%) males with age (mean ± standard deviation) 8.15 ± 1.09 years and range 7–10 years as well as DMFT 7.65 ± 1.84 and range 5–11. We used robust analysis of variance based on M estimation and Kruskal–Wallis to analyze the MIC (Table 1) and the MBC (Table 2). In regard to zone of inhibition test, a total of 144 samples (number/percent); 72/50% saliva isolate samples and 72/50% reference strain samples were analyzed regarding the six experimental and control mouthrinses each 24/16.67% and the three oral pathogens each 48/33.33%. The effects of type of strains, mouthrinses, and oral pathogens on zone of inhibition were assessed (Table 3). We found that type of strains, mouthrinses, oral pathogens, and their interactions explain 94.5% of the variance in the zone of inhibition. This indicates that the model explains the variability in the zone of inhibition well. Compared with distilled water (Table 3), herbal mix, cranberry, cranberry mixed with chlorhexidine, chlorhexidine with alcohol (+), and chlorhexidine mouthrinses were associated with a significant increase in the zone of inhibition 34.354 (p = 0.001), 34.255 (p = 0.001), 34.219 (p = 0.001), 10.801 (p = 0.001), and 9.386 (p = 0.001), respectively (Figs 1A and 2). The S. mutans was associated with an increase of 1.554 (p = 0.022) in the zone of inhibition compared to L. casei (Fig. 1B). There was no significant effect of L. fermentum on the zone of inhibition 1.053 (p = 0.120) compared to L. casei. There was evidence that the effect of mouthrinses on the zone of inhibition varied by type of strains (Table 3). Compared with the reference strain samples, saliva isolate samples were associated with an increase in the zone of inhibition of 5.417 (p = 0.001) in herbal mix mouthrinse and 1.991 (p = 0.006) in cranberry mouthrinse with a decrease in the zone of inhibition of 1.851 (p = 0.011) in chlorhexidine with alcohol (Fig. 3A). There was no evidence that chlorhexidine (p = 0.286) or cranberry mixed with chlorhexidine (p = 0.848) varied by type of strains. We also observed significant interaction effects between mouthrinses and oral pathogens on the zone of inhibition (Table 3). In chlorhexidine, there was a significant increase in the zone of inhibition of 8.346 (p = 0.001) in S. mutans compared to L. casei. Additionally, in chlorhexidine with alcohol (+), there was a significant increase in the zone of inhibition of 4.632 (p = 0.001) in S. mutans compared to L. casei (Fig. 3B). However, in cranberry mixed with chlorhexidine, there was a significant decrease in the zone of inhibition of 10.327 (p = 0.001) in S. mutans and a decrease of 5.679 (p = 0.001) in L. fermentum compared to L. casei (Fig. 3B). For each oral pathogen, the MIC and MBC of mouthrinses were assessed for saliva isolate samples. The Kruskal–Wallis test demonstrates that the mouthrinse has a significant effect on the MIC and MBC in S. mutans, L. fermentum, and L. casei (p < 0.05). The robust analysis of variance demonstrates that the MIC and MBC were significantly higher in the cranberry mixed with chlorhexidine group than in chlorhexidine with alcohol (+) (p = 0.0012) (Fig. 4 and Tables 1 and 2). The R² value was moderate 0.593; it indicates that this model explains the variability in the MIC and MBC to be moderate. The MIC and MBC were significantly lower in the S. mutans and L. fermentum groups than in L. casei (p < 0.05) (Fig. 4). Figure 5 shows the distribution of the MIC and MBC by oral pathogens. Most of L. fermentum were inhibited in their growth in 0.000001 MIC and 0.00001 MBC compared to other oral pathogens. However, most of L. casei were inhibited in their growth in 0.001 MIC and 0.01 MBC compared to other oral pathogens. The inference of the study is that using different mouthrinses proposed in this research showed antimicrobial effects against the tested oral pathogens, and possibly the tested mouthrinses will lead for future formulation of natural or semi-natural pharmaceutical mouthrinses. Moreover, using different mouthrinses proposed in this research provide objective data for future research to challenge production of more effective caries-preventive agents.

DISCUSSION

The null hypothesis was rejected, as there was a significant difference in the antimicrobial effects between tested and control mouthrinses against S. mutans, L. fermentum, and L. casei that were isolated from the saliva of the 7- to 10-year-old Saudi children. S. mutans and lactobacilli are key bacterial species implicated in the etiology of caries.^30,31 In a study conducted by Wen et al., they suggested that the relations between S. mutans and lactobacilli are species specific, and this could affect the cariogenic potential of the community.³¹ In our study, compared with distilled water, herbal mix, cranberry, cranberry mixed with chlorhexidine, chlorhexidine with alcohol (+), and chlorhexidine mouthrinses were associated with a significant increase of 34.354, 34.255, 34.219, 10.801, and 9.386, respectively, in the zone of inhibition. This result could be due to the component of the tested rinses. Herbal mix mouthrinse was made mainly from PPE and myrrh resin extract. Pomegranate (Punica granatum L.) is an old fruit that has been widely used for therapeutic purposes.³² It exhibits antibacterial, anti-inflammatory, and anticarcinogenic effects.³² Pomegranate peel extract mouthrinse has notable antimicrobial activity against S. mutans and can be used to prevent dental caries.³³ Moreover, an in vitro study was conducted by Mehta et al. to evaluate the efficacy of pomegranate peel, lotus leaf, guava leaf, and coffee extracts on oral microorganisms reported that pomegranate peel and lotus have the maximum efficacy against S. mutans and S. mitis.³⁴ Another study conducted by Kote et al. found that there was a significant reduction in the number of colony-forming units of streptococci and lactobacilli after pomegranate rinse.³⁵ A recent clinical study conducted by Singla and his colleagues to evaluate and compare the antibacterial efficacy of different mouthrinses prepared from pomegranate, grape seed, and guava extracts against oral streptococci reported that there is a significant reduction in the colony-forming unit of oral streptococci after the usage of pomegranate and guava extracts mouthrinses for 7 days.³⁶ In respect to myrrh, Eid et al. reported the capability of myrrh plant extract to inhibit the growth of S. mutans with a clear zone of 11 mm around the disk.³⁷ Another recent study was conducted by Park and Yoon to explore the antimicrobial activity of plant-derived essential oils against S. mutans, Porphyromonas gingivalis, and L. rhamnosus showed that myrrh oil had antimicrobial effect against the aforementioned three bacterial strains with the highest antimicrobial activity at 18.34 mm zone of inhibition against S. mutans.³⁸ Yemeni myrrh was also evaluated by Almekhlafi et al., and the result showed a notable antimicrobial activity of myrrh mouthrinse against Staphylococcus aureus, S. mutans, and Candida albicans.³⁹ Some investigators continue trying to find safe compounds that could prevent and obstruct biofilm formation.⁴⁰ In this regard, it was reported by Bonifait and Grenier that the high-molecular-weight polyphenols extracted from cranberries could limit dental caries by preventing biofilms formation and inhibit the production of organic acids by cariogenic bacteria.⁴⁰ In our study, S. mutans was associated with an increase of 1.554 in the zone of inhibition compared to L. casei. Lactobacilli display a wide range of antibiotic resistance naturally.⁴¹ D’Aimmo reported that lactobacilli are resistant to nalidixic acid, aztreonam, cycloserine, kanamycin, metronidazole, polymyxin B, and spectinomycin and susceptible to rifampicin, bacitracin, clindamycin, erythromycin, novobiocin, and penicillin.⁴² In specific, L. casei demonstrated high resistance to aztreonam, cycloserine, polymyxin B and vancomycin.⁴² In our study and in comparison with the reference strain samples, saliva isolate samples were associated with an increase in the zone of inhibition of 5.417 in herbal mix mouthrinse, an increase of 1.991 in cranberry mouthrinse, and a decrease in the zone of inhibition of 1.851 in chlorhexidine with alcohol. This could be a good sign that indicates the susceptibility of oral pathogens isolated from Saudi children saliva to active compounds of herbal extracts and proanthocyanidins and flavonols constituents of cranberry. The effects of the extracts of flavonols, anthocyanins, and proanthocyanidins from the cranberry on virulence factors involved in S. mutans biofilm development and acidogenicity showed inhibition of the surface-adsorbed glucosyltransferases and F-ATPases activities, and the acid production by S. mutans.⁴³ Philip et al. reported that a highly purified polyphenol-rich cranberry extract was able to significantly disrupt acidogenicity, metabolic activity, exopolysaccharide/microbial biovolumes, and structural organization of S. mutans biofilms without affecting bacterial viability.⁴⁴ In regard to pomegranate, the antimicrobial agents (tannins) form high-molecular-weight complex that can aid in microorganisms lysis and obstruct bacterial adherence orally.⁴⁵ In our study, chlorhexidine with and without alcohol mouthrinses exhibited a significant increase in the zone of inhibition against S. mutans compared to L. casei. This is supported by other studies stated that chlorhexidine is considered a potent agent against S. mutans. However, L. casei has shown some resistance to chlorhexidine.⁴⁶ Besides that, this study showed that the MIC and MBC were significantly higher in the cranberry mixed with chlorhexidine group than in chlorhexidine with alcohol (p = 0.0012), and this could be due to the decreased concentrations of cranberry (0.3%) and chlorhexidine (0.06%) that were used in the semi-natural rinse. The findings in our study should be interpreted in the context of some limitations including its in vitro setting. Our in vitro methodology does not reflect the complex polymicrobial, ecological, and environmental influences encountered in the oral cavity. In vitro studies lack reproduction of oral environment. Nevertheless, in vitro studies can provide valuable data of some variables with no interference from other factors. However, in spite of these limitations, the research does designate a number of positive links between in vitro effect and clinical effect. Well-designed clinical trials are warranted to demonstrate whether the beneficial properties shown in our study can translate into efficient and valuable therapeutic approaches for caries prevention.

CONCLUSION

Within the limitations of this in vitro study, it can be concluded that herbal mix and cranberry mouthrinses could be effective natural alternative to chlorhexidine mouthrinse with or without alcohol in improving oral health.

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