Antimicrobial Efficacy of Herbal and Chlorhexidine Mouthrinse against Staphylococcus aureus - An in vitro Microbiological Study.

Background: Microorganisms in the oral cavity are still considered serious public health problems and position a costly burden on health-care services worldwide and elsewhere. Mouthrinses have been used in the oral cavity for decades with the intention of reducing the amount of microorganisms. Mouthrinses are used as additives to oral mechanical hygiene. Therapeutic mouthwashes are also prescribed as an alternative to mechanical plaque control for plaque accumulation prevention and for gingival and peri-implant health maintenance. Mechanical control alone has been questioned to eliminate recalcitrant biofilms in the oral cavity because it is known to be very time-consuming and, most significantly, inadequate for good oral hygiene. The aim of this study was to assess the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antimicrobial efficacy of herbal and chlorhexidine (CHX) mouthrinse against Staphylococcus aureus. Materials and
Methods: For MIC (macrobroth dilution method), MBC and antimicrobial effectiveness (zone of inhibition) of an herbal mouthrinse and 0.2% CHX mouthrinse were determined by the agar well diffusion method.
Results: The zone of inhibition of S. aureus was 24 mm for the CHX mouthrinse. The arowash liquid mouthrinse shows that S. aureus does not produce a zone of inhibition.
Conclusion: CHX mouthrinse (0.2%) has a better antimicrobial efficacy against the S. mutans when compared to herbal mouthrinse (arowash liquid). Copyright:

INTRODUCTION

A diverse community of microorganisms, including bacteria, fungi, protozoa, and probably even viruses, forms the usual oral flora.[1]

The oral cavity is occupied by more than 300 species, about 30 of which are routinely found and account for most of the cultivable strains. In addition to these elements, the oral cavity has a variety of sites, each with its own set of environmental circumstances.[2]

The importance of staphylococci as medical pathogens has been known for several years; however, the existence of Staphylococcus species as a component of the resident oral flora is debatable, and there have been few systematic studies of staphylococci distribution in the mouth. Staphylococcus aureus is responsible for numerous infections in the oral cavity, at least in part. Angular cheilitis, several endodontic infections, jaw osteomyelitis, parotitis, and, more recently, a kind of oral mucositis in elderly, highly dependent patients receiving parenteral feeding are among them.[3456789]

S. aureus cells are Gram-positive and spherical in shape. Once viewed under light microscope after Gram staining, they are always in clusters that resemble a bunch of grapes. The name “Staphylococcus” was derived from Greek, meaning bunch of grapes (staphyle) and berry (kokkos).[10] The diameter of the cells ranges from 0.5 to 1.0 μM.[11] The electron transmission microscopy of cells reveals thick cell walls, distinctive cytoplasm membranes, and amorphous cytoplasms.[12]

Many early qualitative studies of S. aureus isolated from a healthy oral cavity, but little is known about the staphylococci's intra-oral dispersion. Most investigations check the oral cavity with swabs, rinses, or plaque scrapings; however, one group discovered staphylococci in plaque from pits and fissures.[13]

In this analysis, there were no strong age-related changes in the incidence of isolation or proportions of staphylococci. Another study examining oral staphylococcal carriage in 307 children <1–5 years of age attending a pedodontics department found that 84% bore staphylococcal species, of which 33% were S. aureus (5% of the S. aureus isolates were Methicillin-resistant S. aureus). The fact that 19% of S. aureus was of concern. S. aureus isolates produced exfoliative toxin and enterotoxin was produced by 40%.[14] A more recent study found that S. aureus was borne by 84% of healthy children in the oral cavity.[15]

Despite the comprehensive literature on S. aureus and coagulase-negative staphylococci, the oral cavity has provided relatively little attention as a reservoir for these species. Therefore, this study aimed to assess the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antimicrobial efficacy of herbal and chlorhexidine (CHX) mouthrinse against S aureus.

MATERIALS AND METHODS

Study design

An in vitro experimental study on microbiological analysis of herbal mouthrinse and 0.2% CHX gluconate mouthrinse against S. aureus.

Test materials

Control mouthrinse: Hexidine mouthrinse (ICPA Health Products Ltd.)

Experimental mouthrinse: Arowash liquid mouthrinse (Cadila Pharmaceuticals Ltd.).

Test organism

S. aureus (ATCC 25293).

Review board clearance

Before the commencement, the study was submitted for approval and clearance was obtained from Scientific Review Board, Saveetha University, Chennai. Approval from authorities The study was carried out in the Department of Microbiology, Sri Muthukumaran Medical College and Hospital, Chennai. Permission to conduct the study was obtained from the Dean and Head of the Department (Microbiology) of the college.

Scheduling

The study was scheduled from May 1, 2013, to May 31, 2013.

Procedure for antimicrobial activity by the zone of inhibition (agar well diffusion method)

For each test organism, 0.5 McFarland turbidity-adjusted inoculum was prepared in brain heart infusion broth.

Using a sterile cotton swab, a lawn inoculum was made in the blood agar for S. mutans, in the Mueller-Hinton agar for S. aureus and Enterococcus faecalis, and in sabouraud dextrose agar for Candida albicans strains, respectively.

With the help of the sterile Pasteur pipette (blunt end), two wells measuring 8 mm in diameter were made in the media (one in each half) and 80 μl of undiluted control and test solutions were dispensed in each well.

Petri dishes were incubated upright at 37°C for 24 h to grow the microorganisms.

The diameter of zones of inhibition of both the test solution as well as the control solution was measured in mm.[16]

Procedure for minimum inhibitory concentration (by macro broth dilution method)

For test solution

Twelve sterile test tubes were taken for each test organism and numbered 1–12.

Serial doubling dilutions of the Arowash liquid solutions were prepared in Brain Heart Infusion broth to achieve dilutions of 100%–0.02% as follows.

1 ml of Arowash liquid solution was pipetted in one test tube (100% concentration) 1 ml of brain heart infusion broth was taken in each 12 test tubes.

Then, 1 ml of Arowash liquid solution was added in 1st test tube.

After mixing well, 1 ml was transferred from 1st test tube (50%) to 2nd test tube (25%), this process was continued till the last (12) tubes. 1 ml was discarded from the 12th test tube.

Similarly, a set of 12 dilutions was prepared for each organism to be tested 1 ml of Streptococcus mutans inoculum was added in each test tube with varying dilutions of Arowash liquid solution (1–12 test tubes).[17]

The procedure was repeated for E. faecalis, S. aureus, and C. albicans. The tubes were incubated aerobically at 37°c for 24 h.

After 24 h of incubation, the highest dilution which was showing no turbidity, was taken as the MIC for that organism by visual inspection.

Procedure for minimum bactericidal concentration

A loopful of content from test tubes with no visible turbidity in MIC test were inoculated on appropriate culture media plates (i.e., S. mutans on blood agar, S. aureus, E. faecalis on nutrient agar and C. albicans on sabouraud dextrose agar).[18]

The tubes were incubated aerobically at 37°c for 24 h.

Culture plates were observed for growth.

The highest dilution of Arowash liquid solution that did not produce any growth was considered MBC of Arowash liquid for that particular organism.

RESULTS

Table 1 describes the estimation of MIC of control and experimental mouthrinses. Among the control mouthrinses, negative control mouthrinse did not inhibit the growth of any bacterial strain used, positive control mouthrinse inhibited the growth of strain used, and 0.2% CHX mouthrinse did not inhibit the growth of strain used. At 50% concentration, the experimental mouthrinse inhibits the growth of S. aureus.

Table 1

Estimation of minimum inhibitory concentration of control and experimental mouthrinses

Strain	Control mouthrinse			Experimental mouthrinse (%)
	Negative	Positive	CHX	50	25	12.5	6.25	3.12	1.56	0.78	0.39	0.19	0.09	0.04	0.02
Staphylococcus aureus	−	+	−	−	+	+	+	+	+	+	+	+	+	+	+

+: Positive bacterial growth, −: Negative bacterial growth, CHX: Chlorhexidine

Table 2 describes the MBC for experimental mouthrinse. The MBC for S. aureus was >50%.

Table 2

Minimum bactericidal concentration for experimental mouthrinse

Strain	50%	25%	MBC value
Staphylococcus aureus	+	+	>50%

+: Positive bacterial growth, −: Negative bacterial growth

Table 3 describes the zone of inhibition for control and experimental mouthrinse against S. aureus. The zone of inhibition for control mouthrinse was 24 mm and there was no zone of inhibition observed for the experimental mouthrinse.

Table 3

Zone of inhibition for control and experimental mouthrinse against Staphylococcus aureus

Mouthrinse	Staphylococcus aureus
Control mouthrinse (mm)	24
Experimental mouthrinse (mm)	00

Table 4 describes the zone of inhibition for control and experimental mouthrinses against oral bacterial strains. The control mouthrinse showed sensitivity to S. aureus, whereas the experimental mouthrinse showed resistance to S. aureus.

Table 4

Zone of inhibition for control and experimental mouthrinse

Mouthrinse	Staphylococcus aureus
Control mouthrinse	Sensitive
Experimental mouthrinse	Resistant

DISCUSSION

A mouthwash is a medicinal liquid that is retained in the oral cavity and swished around to remove microorganisms from the mouth by perioral musculature action.[19] Hippocrates suggested a mixture of salt, alum, and vinegar, according to legend.[20] The Talmudic answer, which dates back over 1800 years, advised using “dough water” and olive oil.[21] Pedanius Dioscorides, a Greek physician, created a mouthwash with decoct from olive tree leaves, milk, wine, and oil, pomegranate peelings, nutgalls, and vinegar. Using traditional procedures and botanicals, this is how ancient mouthwashes were made.[22] Urine was found to be a major active element in the 18th century due to the presence of ammonia, which protected the mouth cavity from oral infections, particularly sulfur-producing microbes.

The usage of a different sort of mouthwash is still a point of contention. Herbal mouthwashes have been more popular in recent years as people have become more aware of the benefits of complementary and alternative medicine. It is also due to the widespread belief that alternative therapies have fewer adverse effects.[20] Herbal components or substances vary in content and chemical structure, according to research. The designated products lacked labels that provided a thorough history of the chemicals' composition, and they were also found to be polluted with additional natural pollutants and heavy metals, making them unfit for future use.[23]

In the dentistry sector, CHX is one of the most regularly recommended antiseptics. It has been found to reduce plaque, gingival irritation, and bleeding and has a long-lasting antibacterial activity with a broad spectrum of action. Its use is thought to be a powerful adjuvant to mechanical oral hygiene (brushing and flossing), especially in circumstances when they cannot be done properly.[24] The antimicrobial activity of CHX, on the other hand, has been examined extensively utilizing in vitro culture methods, which limit the identification and cultivation of all microorganisms in the environment.[25]

The most common side effects associated with CHX gluconate oral rinses are: (1) An increase in staining of teeth and other oral surfaces; (2) an increase in calculus formation; and (3) an alteration in taste perception. Oral irritation and local allergy-type symptoms have been spontaneously reported as side effects associated with the use of CHX gluconate rinse. The following oral mucosal side effects were reported during placebo-controlled adult clinical trials: aphthous ulcer, grossly obvious gingivitis, trauma, ulceration, erythema, desquamation, coated tongue, keratinization, geographic tongue, and short frenum. Each occurred at a frequency of <1.0%.[26]

The present in vitro study was conducted to estimate the MIC, MBC, and zone of inhibition of an herbal mouth rinse and a commercially available mouthrinse (0.2% CHX gluconate) against S. aureus. The mouthrinses used in the present study were experimental mouthrinse (arowash liquid [combination of A. catechu extract, G. glabra extract, and clove oil]) and control mouthrinse (hexidine [0.2% CHX mouthrinse]). The strain used in the present study was internationally accepted S. Aureus (ATCC 25293).

MIC for the experimental mouthrinse was found to be 50% for S. aureus. The current experimental mouthrinse is a combination of clove oil, acacia catechu, and Glycyrrhiza glabra. So far, studies have only reported the MIC for any one of the above-mentioned components.

In the current study, the MBC value for experimental mouthrinse was found to be >50% for S. aureus. The study by Geetha et al.[27] and Sedighinia et al.[28] had also reported similar results.

In the current study, the zone of inhibition for control mouthrinse (Hexidine) for S. aureus was found to be 24 mm, which was lower than the study conducted by Sedighinia et al.[28] The reason for this difference in values is because of variation in product formulation.

In the current study, the experimental mouthrinse does not inhibit the zones in S. aureus, whereas in the study conducted by Aneja and Joshi.[29] shows that clove oil extract does not inhibit the zones for S. aureus. Again, the difference could be attributed to the difference in product formulation, thus concluding that a combination is better when compared to a single ingredient.

CONCLUSION

The result from this study claims that CHX gluconate mouthrinse (0.2%) has a better antimicrobial efficacy against the S. aureus when compared to herbal mouthrinse (arowash liquid). Further, studies are required to better evaluate this extract as a root canal irrigants. In vivo clinical testing is essential to confirm in vitro study results to be done in a larger sample size.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.