Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against Staphylococcus aureus: an in vitro study.

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey) and CPC (Cepacol), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37 degrees C for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

INTRODUCTION

Currently, a wide range of options in oral antiseptics and toothpastes is available in the market. These products contain synthetic and/or natural compounds with antimicrobial activity12.

Among these synthetic compounds is cetylpyridinium chloride (CPC), a quaternary ammonium compound included in the group of the cationic surface-active agents18. It acts primarily by penetrating the cell membrane, causing leakage of cell components, disruption of the bacterial metabolism, inhibition of cell growth, and finally, cell death3.

Natural extracts, such as propolis, Malva sylvestris, Punica granatum, Zizyphus joazeiro, Eucalyptus globulus, and Salvadora persica are included in the formulation of commercially available oral hygiene products. The addition of these substances aims to improve the antibacterial action, since these natural extracts have demonstrated effect against a wide range of microorganisms23. The antibacterial action of CPC-based mouthwashes is variable and depends on the product's formulation10.

Staphylococcus aureus is a major human pathogen, responsible for a number of hospital-acquired infections17. This microorganism is able to colonize several locations in the human body, but mouth, hands, and nasopharynges are the main reservoirs for propagation of this germ in the hospital environment15. Therefore, control of S. aureus is extremely relevant for the determination of the antiseptic properties of hygiene products. The increased occurrence of methicillin-resistant S. aureus strains (MRSA), as well as of other strains resistant to different broad-spectrum antimicrobial agents, represents a therapeutic challenging situation5. Among the microorganisms present in the oral cavity, the reduction in the number of S. aureus prior to surgical procedures has been associated with a lower incidence of infective endocarditis and postoperative infections1.

The purpose of this in vitro study was to determine the maximum inhibitory dilution (MID) of CPC-based mouthwashes and other products containing natural extracts in addition to CPC against 28 S. aureus field strains.

MATERIAL AND METHODS

The following CPC-based products were evaluated: Cepacol (Aventis Pharma Ltda., São Paulo, SP, Brazil), Natural Honey CPC + Propolis (Skill Brothers Indústria e Comércio Ltda, São Paulo, SP, Brazil), Natural Honey CPC + Malva (Skill Brothers Indústria e Comércio Ltda.), and Natural Honey CPC+Eucaliptol+Juá+Romã+Propolis (Skill Brothers Indústria e Comércio Ltda.) (Table 1).

TABLE 1

Chemical composition of the antiseptic solutions evaluated

Antiseptic	Composition
Cepacol®	– Cetylpyridinium chloride – Disodium EDTA – Sodium saccharin – Polysorbate 80 – Glycerin – Water – Sodium phosphate monobasic anhydrous	– Eucalyptol – Menthol – Methyl salicilate – Mint oil – Chinese cinnamon flavor – Yellow tartrazine – Ethyl alcohol 96GL
Natural Honey CPC+Propolis	– Natural propolis extract – Sodium fluoride 0.05% (226ppmF) – Cetylpyridinium chloride (CPC) – Sorbitol – Sodium phosphate monobasic – Sodium phosphate dibasic	– Ethanol – Sorbitan monolaurate – Sodium saccharin – CI42.053 green – Mint flavor – Demineralized water
Natural Honey CPC+Malva	– Natural Malva sylvestris extract – Sodium fluoride 0.05% (226ppmF) – Cetylpyridinium chloride(CPC) – Sorbitol – Sodium phosphate monobasic – Sodium phosphate dibasic	– Ethanol – Sorbitan monolaurate – Sodium saccharin – CI42.090 blue – Mint flavor – Demineralized water
Natural Honey CPC + Eucaliptol + Juá + Romã + Propolis	– Natural extracts of pomegranate, propolis, and Zizyphus joazeiro – Eucalyptol – Methyl salicilate – Sodium fluoride 0.05% (226ppmF) – Cetylpyridinium chloride(CPC) – Sorbitol	– Sodium phosphate dibasic – Ethanol – Sorbitan monolaurate – Sodium saccharin – CI 19.140 and 15.985 color – Pomegranate flavor – Demineralized water

MID determination was performed in duplicate by double serial dilution (from 1/10 through 1/655,360) in test tubes (20x200 mm) with 2.0 mL of sterile distilled water. After dilutions, 18.0 mL of Mueller Hinton Agar culture medium (Difco®, USA) were added to each tube, and the resulting solutions were poured onto Petri dishes (20x100 mm).

The microbial inoculum (∼108 cfu/mL) with turbidity equivalent to a #0.5 McFarland standard was prepared in test tubes (15x125 mm) with saline, using 28 S. aureus field strains obtained from nasal and oral cavities. The strains were conserved on a collection cultured on Ni agar medium. The identification of S. aureus was based on the production of catalase and coagulase. Regardless of the results of the coagulase test, all catalase positive Gram-positive cocci were submitted to the API-Staph system (bioMérieux, France) for biochemical identification.

Microorganisms were seeded using a Steers multipoint inoculator27. The Steers inoculator consists of two metallic plates. One plate has 25 wells onto 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 inocula 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 inocula (5 μL) were seeded equidistantly from each other, approximately 1 cm from dish periphery, using an automatic pipette.

The dishes were then incubated overnight at 37°C and readings were performed considering the MID as the greatest dilution of mouthwash capable of inhibiting the growth of all test strains, following the methodology proposed by Roberts and Addy21.

Statistical Analysis

Results were expressed as scores determined from minimum to maximum dilution. Comparisons among the groups were performed by Friedman's nonparametric test. When this test indicated 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%.

RESULTS

The mouthwashes evaluated in this study presented different MIDs (Table 2 and Figure 2).

TABLE 2

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

	CPC+Propolis				CPC+Malva				CPC+Eucaliptol+Juá+Romã+Propolis				CPC*
	i. s.	i. s.	c. d.	c. d.	i. s.	i. s.	c. d.	c. d.	i. s.	i. s.	c. d.	c. d.	i. s.	i. s.	c. d.	c. d.
Dilution	N	%	N	%	N	%	N	%	N	%	N	%	N	%	N	%
1/640	0	0.0	0	0.0	0	0.0	0	0.0	2	7.1	2	7.1	0	0.0	0	0.0
1/320	27	96.4	27	96.4	28	100.0	28	100.0	26	92.9	28	100.0	3	10.7	3	10.7
1/160	1	3.6	28	100.0									25	89.3	28	100.0

(p<0.05)

i.s. Inhibited strains

c.d. Cumulative data

Statistical analysis demonstrated no statistically significant differences (p>0.05) among CPC+Propolis, CPC+Malva, and CPC+Eucaliptol+Juá+Romã+Propolis. However, the MID for Cepacol® was lower than that determined for all other three products (p<0.05).

DISCUSSION

CPC is a cationic compound used in oral antiseptics. It has a broad action against bacteria present in the oral cavity21. Over 99% of the microorganisms associated with biofilm/dental plaque formation and gingivitis are eliminated by solutions containing 0.065% CPC30. A reduction of 39% in biofilm/dental plaque formation has been observed in brushed surfaces, while this percentage is 25% in non-brushed surfaces6,9. Roberts and Addy22 (1981) reported a residual effect for 180 to 300 min following the use of CPC-based oral products.

Mouthwashes containing 0.05% CPC promote reduction in the amount of salivary microorganisms for 3 h following use21. One-minute rinsing with 15.0 mL of CPC-based mouthwash (0.05% CPC) for two weeks resulted in an inhibition of biofilm/dental plaque formation when associated with mechanical cleaning24. Rawlinson, et al.20 (2008) determined the plaque inhibition properties of two formulations of alcohol-free mouthwash with 0.1% or 0.05% CPC. They showed that the use of both CPC mouthwashes resulted in less plaque accumulation compared to the control (placebo).

According to the FDA Plaque Subcommittee, CPC is a safe antimicrobial agent for prevention of biofilm formation and gingivitis, when used in concentrations ranging from 0.05 to 0.1%8. In the present study, the products evaluated had a CPC concentration of 0.05%.

According to Albuquerque Jr., et al.1 (2004), mouthwashes containing CPC are capable of inhibiting S. aureus strains in vitro at a 1/20 dilution. However, in this study, the MID was 1/160 for CPC+Propolis and Cepacol® and 1/320 for CPC+Malva and CPC+Eucaliptol+Juá+Romã+Propolis.

Several natural extracts have been incorporated to the formulation of oral antiseptics13, such as Jupinerus communis, Urtica dioca, Achillaea millefoluim and Salvadora persica 14. In the present experiment, three out of the four mouthwashes evaluated contain natural extracts. Based on the obtained results, addition of these extracts to CPC improved its antimicrobial action, as reported elsewhere10.

Propolis, a substance present in two of the CPC-based solutions evaluated in the present study, is described as a natural antibiotic produced by bees, and has shown activity against S. aureus 26. However, Silici and Kutluca26 (2005) reported only one-weak activity of propolis against the Gram-negative microorganisms Escherichia coli and Pseudomonas aeruginosa.

Malva sylvestris, found in one of the products evaluated in the present study, has been shown to have antiinflammatory action11, as well as antimicrobial activity against E. coli and P. aeruginosa 2. However, according to Coelho de Souza, et al.7 (2004), extracts from aerial portions of M. sylvestris do not demonstrate activity against S. aureus, S. epidermidis, E. coli, Micrococcus luteus, and C. albicans. Still according to the same study, only a slight activity was observed against Saccharomyces cerevisiae. Extract from the seed of Malva moschata, a plant found in Scotland, is active against S. aureus, S. epidermidis, Proteus mirabilis, and E. coli. Nevertheless, extracts obtained from leaves, roots, and seeds may present different effects due to the different concentrations of the active principle in the solutions produced from each of these parts of the plant16.

Another natural ingredient added to one of the CPC-based products evaluated is Zizyphus joazeiro extract, which has shown antimicrobial activity against Gram-positive microorganisms25.

Eucalyptus, another source of natural extract present in one of the solutions evaluated in the present study, is a genus comprising approximately 600 species of trees, native from Australia. Eucalyptus globulus is the most commonly cultivated species in subtropical and Mediterranean regions, and its natural extract has shown effectiveness against S. aureus, E. coli, P.aeruginosa, and C.albicans, Eucalyptus maculata, and Eucalyptus viminalis were able to inhibit the growth of several Gram-positive bacteria (S. aureus, MRSA, Bacillus cereus, E. faecalis, Alicyclobacillus acidoterrestrus and Propionibacterium acnes) and one yeast (Trichophyton mentagrophytes). On the other hand, these extracts showed little effectiveness against Gram-negative bacteria (E. coli and P. putida). Nevertheless, Eucalyptus botryoides and Eucalyptus nitens extracts inhibited the growth of both Gram-positive and Gram-negative microorganisms28.

Punica granatum (pomegranate) extract is another compound reported as being active against S. aureus, P. aeruginosa, C. albicans, C. krusei, C. parapsilosis and C. tropicalis 4.

CONCLUSIONS

According to the proposed methodology and based on the obtained results, it may be concluded that the addition of natural extracts to CPC enhanced its antimicrobial effect.