Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary Staphylococcus aureus.

OBJECTIVE: The aim of the present study was to determine the in vitro maximum inhibitory dilution (MID) of two chlorhexidinebased oral mouthwashes (CHX): Noplak, Periogard, and one polyhexamethylene biguanide-based mouthwash (PHMB): Sanifill Premium against 28 field Staphylococcus aureus strains using the agar dilution method.
MATERIALS AND METHODS: For each product, decimal dilutions ranging from 1/10 to 1/655,360 were prepared in distilled water and added to Mueller Hinton Agar culture medium. After homogenization, the culture medium was poured onto Petri dishes. Strains were inoculated using a Steers multipoint inoculator and dishes were incubated at 37 degrees C for 24 hours. For reading, MID was considered as the maximum dilution of the mouthwash still capable of inhibiting microbial growth.
RESULTS: Sanifill Premium inhibited the growth of all strains at 1/40 dilution and of 1 strain at 1/80 dilution. Noplak inhibited the growth of 23 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Periogard showed inhibited growth of 7 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Data were submitted to Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated (p<0.05). No significant difference was found between Noplak and Periogard (p>0.05). Sanifill Premium was the least effective (p<0.05).
CONCLUSION: It was concluded that CHX-based mouthwashes present better antimicrobial activity against S. Aureus than the PHMB-based mouthwash.

INTRODUCTION

Mouthwashes have been used for centuries19 with the objective of reducing the amount of microorganisms in the oral cavity14. These chemical agents have been widely employed in the fields of Preventive Dentistry and Periodontics2,10,13. Among the microorganisms present in the oral cavity, the reduction in the number of Staphylococcus aureus prior to surgical procedures has been associated with a lower incidence of infective endocarditis and postoperative infections4.

Chlorhexidine gluconate (CHX) is a cationic biguanide with broad-spectrum antimicrobial action, whose effectiveness in decreasing the formation of dental biofilm (plaque) and gingivitis has been demonstrated in clinical studies1,5,6,13. An important characteristic of chlorhexidine is its substantivity or persistence of action, which consists of the ability of this product to bind to oral tissues and remain active for long periods after application1.

The antimicrobial properties of mouthwashes containing CHX and other antimicrobial agents have been assessed in vivo and in vitro, with excellent results for CHX-based solutions3,4,7,11,15,16,20.

Polyhexamethylene biguanide hydrochloride (PHMB) is a polymeric biguanide with broad antimicrobial spectrum against both Gram-positive and Gram-negative bacteria8,9. PHMB has been used for several years as an antiseptic agent in Medicine12. Welk, et al.23 demonstrated in clinical studies that PHMB-based mouthwashes inhibited biofilm formation and reduced contamination in the oral cavity, suggesting that these solutions may be an alternative for prevention of dental biofilm.

The purpose of this study was to determine in vitro the maximum inhibitory dilution (MID) of two mouthwashes containing 0.12% chlorhexidine gluconate and one mouthwash containing 0.35% PHMB and to compare their action against 28 Staphylococcus aureus field strains using the agar dilution method.

MATERIAL AND METHODS

The following mouthrinses were evaluated: Periogard® (Colgate-Palmolive, Ind. Brasileira, Osasco, SP, Brazil), Noplak® (Laboratório Daudt Oliveira Ltda., Rio de Janeiro, RJ, Brazil) and Sanifill Premium® (Facilit Odontológica e Perfumaria, Brazil) (Table 1).

TABLE 1

Formulation of the antiseptic solutions evaluated

Product	Composition
Periogard®	0.12% chlorhexidine gluconate, water, glycerin, ethanol, polysorbate 20, flavoring agents, - sodium saccharin, FD&C Blue n°1
Noplak®	0.12% chlorhexidine gluconate, water, 3.5% ethyl alcohol, hydroxyethylcellulose, sodium cyclamate, glycerin, menthol, hydrogenated castor oil, sodium saccharin, sorbitol, CI-19.140 and 42.090, flavoring agents, demineralized water
Sanifill Premium®	0.05% sodium fluoride (226ppm), sorbitol 70%, sodium benzoate, 0.35% polyhexamethylene biguanide chlorhydrate, methylsilanol, sodium cocoanphoacetate, disodium monophosphate, disodium phosphate, sodium saccharin, CI-19.140 and 42.090, deionized water

Determination of the maximum inhibitory dilution (MID) was performed in duplicate by double serial dilution (from 1/10 through 1/655.360) in test tubes (20x200mm) with 2.0 mL of sterile distilled water. After dilutions were made, 18.0 mL of Mueller Hinton Agar culture medium (Difco®) were added to each tube, and the resulting solutions were poured onto Petri dishes (20×100mm).

The microbial inoculum (~108 UFC/mL) with turbidity equivalent to a 0.5 McFarland standard was prepared in test tubes (15×125 mm) with saline, using 28 young S. aureus field strains previously incubated at 35°C for 24 h. S. aureus strains were collected from the oral and nasal cavities of volunteer undergraduate students.

Microorganisms were seeded using a Steers multipoint inoculator21. The Steers inoculator consists of two metallic plates: one of these plates has 25 wells into which 200 μL of each standardized microbial inoculum were transferred. The other plate has 25 metallic needles that fit into the wells. Using these needles, the inoculi were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. Since the Steers inoculator has 25 wells and 28 strains were evaluated, three inoculi (5.0 μL) were seeded equidistantly from each other, approximately one centimeter from the periphery of each Petri dish, using an automatic pipette.

The dishes were then incubated at 37°C for 24 hours, and readings were performed considering the MID as the greatest dilution of mouthwash capable of inhibiting growth of all evaluated strains, following the methodology by Wade and Addy22.

FIGURE 1

Graph depicting MID values obtained for each solution evaluated

Statistical analysis

Results were expressed as scores determined from minimum to maximum dilution and comparison between all groups was performed using Kruskal-Wallis nonparametric test. When this test showed significant difference between the groups, Dunn's multiple comparison test, which allows two-by-two comparison between groups, was applied. Significance level was set at 5% (p<0.05).

RESULTS

The mouthwashes evaluated in this study presented different MIDs (Table 2). No significant difference was found between Noplak® and Periogard® (p>0.05). Sanifill Premium® was the least effective agent (p<0.05).

TABLE 2

Percentage of S aureus strains inhibited (from a total of 28 strains) by each dilution of the mouthwashes tested

	Sanifill Premium				Noplak				Periogard
	Inhibited strains		Cumulative data		Inhibited strains		Cumulative data		Inhibited strains		Cumulative data
Dilution	No.	%	No.	%	No.	%	No.	%	No.	%	No	%
1/640	0	0.0	0	0.0	23	82.1	23	82.1	7	25.0	7	25.0
1/320	0	0.0	0	0.0	5	17.9	28	100.0	21	75.0	28	100.0
1/160	0	0.0	0	0.0
1/80	2	7.1	2	7.1
1/40	26	92.9	28	100.0

DISCUSSION

Chlorhexidine gluconate mouthwashes are available in the market at concentrations ranging from 0.12% and 0.2%. Smith, et al.20, using the plaque formation index, compared in vivo the dental biofilm-inhibiting properties of mouthwashes containing 0.12% and 0.2% chlorhexidine gluconate. Both formulations presented better results than other mouthwashes without CHX. The CHX-based mouthwashes evaluated in this study contain 0.12% CHX.

Albuquerque Jr, et al.4 assessed the MID of a mouthwash containing 0.12% chlorhexidine gluconate against 25 S. aureus strains, and found that all of them were inhibited up to a 1/80 dilution of the mouthwash. In the present study, the S. aureus strains were inhibited by Periogard® and by Noplak® up to a 1/320 dilution. This difference in the results of both studies may be related to the different sources of the microorganisms. Albuquerque Jr, et al.4 used S. aureus strains collected from the oral cavity, while in the present study the microorganisms were retrieved from the oral and nasal cavities of the patients.

A study by Herrera, et al.11 demonstrated that four mouthwashes containing the same active ingredient (0.12% chlorhexidine) but different formulations demonstrated statistically significant differences in their antimicrobial activity, both in vitro and in vivo. For the in vitro antimicrobial activity test, 20 selected bacterial species were evaluated. The in vivo test consisted of salivary bacterial count. The samples were cultured both aerobically and anaerobically. The formulation with alcohol was more active than those without alcohol, except for the formulation with chlorhexidine and cetylpyridinium chloride, which presented better antimicrobial activity. The addition of other active components should be further evaluated because chlorhexidine is highly cationic and may be inactivated by anionic substances11.

Our results demonstrated that the CHX-based mouthwashes (Noplak® and Periogard®) were more effective in inhibiting bacterial growth in comparison to the PHMB-based product (Sanifill Premium®).

The antimicrobial activity of mouthwashes containing PHMB has been evaluated in several in vivo studies17,18,23. In one of these works, Rosin, et al.17, using a method that measured bacterial counts on the tooth surface and oral mucosa, demonstrated that a mouthwash with 0.04% PHMB was more capable of inhibiting biofilm/dental plaque formation, compared to a negative control (placebo); however, the PHMB-based product was not as efficient as a mouthwash containing 0.12% chlorhexidine. This result is in agreement with our study, in which the PHMB-based mouthwash was not as effective as the CHX-based mouthwashes.

In another in vivo study in which a method to measure bacterial counts on tooth surface and mucosa was used, Rosin, et al.18, observed that a mouthwash containing 0.12% PHMB was more capable of inhibiting biofilm/dental plaque, in comparison to a negative control (placebo), but no statistically significant difference was observed between the PHMB-based mouthwash and a product with 0.12% chlorhexidine. Regarding the ability to reduce the number of intraoral bacteria, the chlorhexidine-based product performed better than the PHMB-based mouthwash. In our study, the evaluated mouthwash contains 0.35% PHMB, and presented lower MID values than CHX-based mouthwashes containing 0.12% CHX.

An in vivo study23 using a method to determine bacterial counts on tooth surface and mucosa demonstrated that a solution containing 0.12% chlorhexidine was more capable of inhibiting and reducing biofilm/dental plaque than a mouthwash containing 0.2% PHMB. However, the PHMB-based mouthwash presented similar results to the chlorhexidine solution in reducing the number of bacteria present on the oral mucosa.

The mouthwashes evaluated by Rosin, et al.17,18 and Welk, et al.23 had a lower concentration of PHMB than the product tested in the present study (Sanifill Premium®). Despite its higher concentration of PHMB, Sanifill Premium® showed lower MID than the chlorhexidine-based mouthwashes.

CONCLUSIONS

The mouthwashes containing 0.12% chlorhexidine (Noplak® and Periogard®) presented higher MID values than that containing 0.35% PHMB (Sanifill Premium®) against salivary S. aureus. It was concluded that CHX-based mouthwashes have better antimicrobial activity than the PHMB-based mouthwash.