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Antimicrobial effect of a natural mouthwash against different microbial species

Hassan Ali shafiee¹, Golnaz Nahvi^1*, Gita Eslami², Hossein Pour Abbasivand³, Farzad Aghdashi⁴, Shahryar Karami⁵, Reza Najafzadeh⁶

1 Department of Orthodontics, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Department of Oral and Maxillofacial Surgery, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Department of Orthodontics, School of dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

6 Post Graduate Student, Endodontics Department, Faculty of Dentistry, Tehran Medical Science, Islamic Azad University, Tehran, Iran

*Email: [email protected], Tel: +982122051856

ABSTRACT

Background: In Iranian traditional remedy, herbal and animal based medications have shown considerable remedic effects on mucosal ulcers and inflammations. The present investigation sought to determine a traditional animal based extract called ANNAS effects as a mouthwash on prevalent human pathogenic bacteria.

Objectives: In this in-vitro experimental study the minimum inhibitory concentrations (MICs) of ANNAS 0.2% and chlorhexidine (CHX) 0.2% as the control group were measured.

Materials and methods: The agar diffusion test (ADT) and the figures of the diameter of the inhibition zone and minimal inhibitor concentration (MIC) and minimal bacterial concentration (MBC) of the three bacterial test groups (Pseudomonas aeruginosa, Staphylococcus aureus and Lactobacillus casei) were also investigated. Data were analyzed using SPSS software using Kruskal-Wallis and Mann-whitney U test.

Results: The inhibitory zone of ANNAS was the same as CHX for Pseudomonas aeruginosa and statistically greater for Staphylococcus aureus and Lactobacillus casei. The MIC of Pseudomonas aeruginosa was significantly less in ANNAS compared to CHX and the same as CHX in Pseudomonas aeruginosa and Staphylococcus aureus. MBC of CHX and ANNAS had no statistical difference for Staphylococcus aureus. ANNAS natural mouthwash might act as an effective antibacterial agent against some bacterial species with fewer side-effects compared to commercially available chemical mouth rinses.

Conclusion: this traditional extract proved the promising findings against microbial agents, it also opens doors to future investigation on its impact on other human pathogens.

Kew words: Mouthwashes, anti-infective agents

INTRODUCTION

Numerous patients fail to obtain acceptable oral hygiene by mechanical oral hygiene protocols and for those patients suffering from mocusitis and mouth pain due to chemo or radiotherapy, mouthwashes may be the only practical way of oral hygiene maintenance (1-3).

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Therefore, the application of the antimicrobial mouthwashes has increased with this perception. Nowadays mouthwashes contribute to reduction of oral bacteria in microbial plaque such as lactobacillus (4-6). In the area of the oral cavity, diminishing the number of Staphylococcus aureus by using a suitable mouthwash before surgical procedures has been related with diminished incidence of infective endocarditis and postsurgical infections (7, 8).

Interestingly, host-derived nutrients can also contribute to promote bacterial competition, as in the case of oral commensal Streptococci and Pseudomonas aeruginosa (9). So, bacterial species such as Pseudomonas aeruginosa need to be controlled by suitable antibacterial agents as they contribute to complicate oral infections refractory to antibiotics treatment. With this regard an ideal antibacterial solution should have some basic criteria such as: low toxicity (10) and broad‑spectrum antibacterial activity (11-13), low risk of topical or systemic allergic reactions (14), no harm to normal flora of the mouth (15), palliative effects on oral ulcers (16). No ideal mouthwash has been introduced up to now that covers all the criteria mentioned above. Regarding antiplaque and anti-gingivitis effects, current evidence suggests CHX is the first choice (17-19) but CHX has been reported to have some local side effects.

These side effects are staining on teeth and dental mucosa, oral mucosal erosion, and taste perturbation (20).Also recent research shows that globally assumption of CHX in dentistry field could be a vulnerable source for the promotion of resistance against CHX per se or cross-resistances against other antimicrobial agents (2, 3, 21, 22).

This side effects and also the entrance of more contagious pathogens such as Covid 19 encouraged the stewardship programs and search on alternative anti-plaque agents with an acceptable wide spectrum antimicrobial quality and less unwanted effects. In recent decade with increasing knowledge on chemical medicine side effects, widespread attention is attracted toward traditional and herbal remedies in all medical fields including dentistry (23- 25). In Iranian traditional medicine smoke condensate derived from an indirect heating of jennet feces (Anbernesara or Sargin) has a prolonged history of therapeutic application for oral inflammations such as stomatitis and ear infections (otitis) (26, 27). Anbarnesara smoke or shortly ANNAS is highly recommended by Iranian ancient scientists as an effective antimicrobial, antiseptic and flavoring, anti-inflammatory, anti-nociceptive, anti-bronchitis, wound and bone healing and anti-sinusitis component (28-30). Also due to its affordability and availability it could be a suitable alternative for many chemical products. Recently green synthesis of nanoparticles such as Ag driven from Anbarnesara has been reported which has considerable antibacterial and anticancer effects (31-37). Despite the high values of ANNAS, there were few published data on its effects on common pathogenic bacteria. Therefore we aimed to compare the effect of ANNAS as a mouthwash with CHX on Lactobacillus casei which is one of the most common pathogenic oral bacteria. Also we decided to evaluate the effect of ANNAS on two major resistant pathogenic bacterial species; Pseudomonas aeruginosa and staphylococcus aureus.

METHOD AND MATERIALS ANNAS preparation

ANNAS gained from female donkeyʼs feces was heat processed in a sealed container covered by propylene glycol. Following the air condition cooling, components of the Anbarnesara smoke covered the wall of the container. The aformentioned process was reoperated several times so that sufficient smoke sticked to the containerʼs covering. Then 10 ml solution of the propylene glycol was poured to the container and intermigled completely with the sediments on the containerʼs covering. Then the material was transferred into a falcon tube to be centrifuged for 30 min in 10 rpm (Hetich, Germany). The supernatant fluid was poured into another tube in order to separate impurities by a Pasteur pipette (19, 24, 25). The remaining

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containing was nominated ANNAS. The liquid chromatography was applied to measure the liquid concentration and the liquid was diluted to the concentration of 0.2%.

In the current in-vitro experimental investigation, Inhibitory zone diameter (IZD), and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated in different bacterial species. Agar diffusion test methods (agar diffusion test: ADT) and the dilution method for determining MIC and MBC were applied to compare the two mouthwashes in terms of antibacterial activity. Three standard bacterial strains were examined in the study: Lactobacillus casei (ATCC: 314), Pseudomonas aeruginosa (PTCC: 1707) and Staphylococcus aureus (PTCC: 1431). The bacterial specimens were obtained from the microbial bank of Pasteur Institute and Medical School of Shahid Beheshti University (Tehran, Iran). Then the specimens were passaged in medical school of Shahid beheshti university (Department of Microbiology, Medical School, Shahid Beheshti University of Medical sciences, Tehran, Iran) under sterile conditions. Primary identification of Pseudomonas aeruginosa and other organisms were done based on the conventional biochemical tests, including Gram staining, growth on cetrimide agar, growth at 42°C, Kligler iron agar, oxidase, catalase, pigment production, and oxidative-fermentative (9) tests.

Overnight cultures of the microorganisms were used. Cultures were maintained on MHB (Muller Hinton Broth) slants at 47°C.

Dilution method test

MHA (Muller Hinton Agar) and BA (Blood Agar) mediums were used for experimental groups.0.5 ml of MHA medium was poured to 8 tubes. CHX and ANNAS were prepared with different concentrations (from 1 to 1 / 256). The participant bacterial strains were grown at 37˚C for 24 h in MHB and then seeded into 15mL of the MHA, to produce a turbidity of 0.5 on the Mc Farland scale, which corresponds to a concentration of 108 colony forming unit mL^-1. Each experimental tube contained equivalent of 0.5 ml of bacterial suspension with a concentration of 0.5 McFarland and then each tube was incubated for the period of 24 hours.

Then, 0.5ml of the tube contents was calculated in appropriate media for bacterial species.

The plates were kept at room temperature for 2 hours for pre-diffusion of the materials and then incubated at 37˚C for 24 h. For medium preparation, 15 ml specific culture medium was poured equally in each 8-cm plate and 3 wells with equal diameter and depth (6 mm diameter and 5 mm depth) were created by the Pasteur pipette. Then after cooling, 0.5 ml of the prepared bacterial suspension was cultured. Each well was filled with 50 micro liters of each filtered material (ANNAS or CHX) to prevent any contamination and after 30 min incubated at temperature 37°C. A total number of 20 plates were employed; the experimental plates were randomly divided into two test groups and microorganisms were tested ten times.

During the experiment plates were monitored to rule out cross contamination with other microorganisms. After 48 hours, IZD for each material was measured. The diameter of bacterial growth inhibition zones was measured with a millimeter ruler with accuracy of 0.5 mm in two perpendicular locations for each sample by an independent observer. Positive and negative controls were prepared, maintaining the plates with and without inoculums, for the same period and under identical incubation conditions. All assays were carried out under aseptic conditions. The experimental groups were compared in terms of inhibition zone diameter and minimum inhibitory concentrations of various species. SPSS software version 21 was used for statistical analysis of the results. Kruskal-Wallis non-parametric analysis meanwhile non parametric Mann-whitney U test were used to analyze different variables of mouth rinses in each bacterial species.

RESULTS ADT test

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Mean diameter of inhibition zone of ANNAS 0.2 % and CHX 0.2% mouthwashes in three bacterial groups is shown in Table 1. The results of Mann-whitney U test showed ANNAS had greater diameter of inhibition zone for Staphylococcus aureus. Also diameter of inhibition zone for staphylococcus aureus was greater than two other bacterial groups but this difference is not significant according to standard deviation (Table 1). The inhibitory zone of ANNAS was the same as CHX for Pseudomonas aeruginosa (p= 6720/ ) and statistically greater for Staphylococcus aureus (p= 0030/ ) and Lactobacillus casei (p= 0120/ ).

Table 1. Values of IZD in different experimental groups.

Antibacterial agent

Bacterial species

IZD Mean (mm) Standard

Deviation

Minimum (mm)

Maximum (mm)

ANNAS

Lactobacillus casei 19/833 0/9832 19/0 21/0

Pseudomonas aeruginosa

917 /

18 0/9174 18 /0 20 /0

staphylococcus aureus

833 /

23 0/9832 23 /0 25 /0

CHX

Lactobacillus casei 18/250 0/6124 17 /5 19 /0 Pseudomonas

aeruginosa

167 /

19 0/9832 18/0 20/0

500 /

20 0/8367 19/0 21/0

MIC test

According to Kruskal-Wallis test, no significant differences were found between ANNAS 0.2% and CHX 0.2% in terms of MICs in Pseudomonas aeruginosa (p= 0560/ ) and Staphylococcus aureus (p= 0560/ ). Whereas MIC of ANNAS for Lactobacillus casei was significantly more than CHX (p = 0020/ ). MIC of ANNAS on Pseudomonas aeruginosa was significantly less than two other bacterial species in ANNAS groups. In CHX groups MIC of Lactobacillus casei was significantly less than two other bacterial species (p= 0.020), (Table 2 and Figure 1).

Table 2. Mean values of MBC in different experimental groups.

Antibacterial agent

Bacterial species

Mean MIC p-value MIC Mean MBC p-value MBC

ANNAS

Lactobacillus casei

00 / 13

041 / 0

83 / 10

161 / 0 Pseudomonas

aeruginosa

33 /

6 6/83

17 /

9 10/83

CHX

Lactobacillus casei

50 / 5

002 / 0

00 / 7

002 / 0 Pseudomonas

aeruginosa

50 /

7 6/00

50 /

15 15/50

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Figure 1. Mean MIC of ANNAS 2% and CHX 2% in different bacterial groups.

MBC test

According to Kruskal-Wallis test, no significant differences were found between ANNAS 0.2% and CHX 0.2% in terms of MBCs for Staphylococcus aureus (p= 5750/ ). Whereas MBC of ANNAS was significantly more than CHX for Pseudomonas aeruginosa (p= 0040/ ) and Lactobacillus casei (p= 0030/ ). MBC of CHX for Lactobacillus casei was significantly less than two other species (Table 2 and Figure 2).

Figure 2. Mean MBC of ANNAS 2% and CHX 2% in different bacterial groups.

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DISCUSSION

It has been a long period that microorganisms such as Lactobacillus casei are designated as major contributors in dental caries (38). Also some bacterial species such as Pseudomonas aeruginosa and Staphylococcus aureus may cause some hospital-acquired refractory infections that need special treatment protocols or medicaments due to their multidrug resistant nature (39-41). In a recent review it was alarmingly anticipated that the death toll related to antimicrobial resistance will show an increasing trend world widely from 700,000 to 10 million in the year 2050 (18, 42, 43). In this light, resistance toward antibiotics has attracted global interest. Although CHX is widespreadly used as the best current mouthwash, some research alarms the risk of resistance toward CHX and accompanying cross-resistance to antibiotics (21). Nowadays with the demand for more efficient antimicrobial agents with less side effects seen in chemical products, traditional medicine has gain the spotlight and ancient medicine seems to have promising chance in combat of human with pathogenic bacterial species (44-46). Nowadays with increasing interest in herbal than chemical products, authors are reporting plant-derived oils with low-toxicity antibacterial effects compared to the commercial mouthwashes against oral pathogens (47).

One of the well-known compounds in Iranian traditional medicine is Anbarnesara with a long history in treatment of inflammatory and infective diseases (28, 48). In recent years few researchers have investigated the use of ANNAS in dentistry field. Some studies have reported cytotoxic effects for CHX in bacteriocidal concentrations (49, 50). According to our study results, because of greater inhibitory zone of ANNAS in Lactobacillus casei and staphylococcus aureus it could be regarded as a suitable alternative for CHX for dental caries and periodontal diseases prevention and microbial plaque control with fewer side effects.

Although, further research is recommended to investigate the ANNAS impact on tissue and cellular level and also in in-vivo conditions.

Mouthwashes are an easy and efficient method for patients who undergo head and neck radiation due to their oral pain and mucositis (51). Most commercially available mouthwashes like CHX consist of alcohol due to its bacterial growth inhibitory effects despite the mucusal irritation it causes (52). So, antimicrobial solutions that could be beneficial in the absence of alcohol are extremely useful for patients under chemotherapy or radiotherapy. For this reason ANNAS mouthwash could be a suitable non-chemical and non-alcoholic mouthwash with antibacterial effect comparable to CHX and less side effects. Among the common microorganisms in the oral cavity, Staphylococcus aureus is reported to be associated with cross-infection and disseminating effects to other body organs (53). So different researches have been conducted to improve antibacterial effects of mouthwashes against Staphylococcus aureus (54, 55). Our result demonstrated promising effects of ANNAS mouthwash on Staphylococcus aureus with same MIC and MBC as CHX and greater diameter of inhibition zone. The result of current study shows ANNAS has same inhibitory zone for Pseudomonas aeruginosa in comparison with CHX 2%. This preliminary result may be valuable in future research to investigate ANNAS antibacterial effect against Pseudomonas aeruginosa which is a designated human pathogen, contributing to a number of hospital-acquired refractory infections.

CONCLUSION

According to the initial results of this study ANNAS has promising advantages as a mouthwash for plaque control and diminishing the number of oral pathogens with less side- effects in comparison with the common chemical antimicrobial solutions. The current available evidence is still inconclusive but reveals that ANNAS mouthwash antimicrobial potentials is comparable to and even better than CHX. Also because of non-alcoholic and

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natural palliative contents it could be suitable alternative mouthwash for patients suffering from mucusitis and mouth pain due to chemo or radiotherapy. These findings are preliminary and further high‑quality in-vivo investigations with sufficient sample sizes are strongly recommended. Also because the initial investigations of ANNAS on microbial species is promising, we highly recommend to design studies for investigation of ANNAS on other human pathogenic species such as contagious viruses as Covid-19.

FUNDING There was no financial support.

ACKNOWLEDGMENT

The authors are deeply thankful to all colleagues for their kind help.

CONFLICTS OF INTEREST The authors declare that they have no competing interests.

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