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Antibacterial Potentials of some Toothpaste Products against some Oral Bacterial Isolates

Objetive: Manufacturers of toothpastes claim that their products are active against oral microbiome capable of causing tooth decay. The objective of this study was to investigate the manufacturers’ claim using some of the toothpaste products sold in Ado-Ekiti, Nigeria. Material and methods: The antibacterial potentials of five commercialized toothpaste products (designated sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphate, sodium fluoride-eugenol, sodium fluoridesodium laurylsulfate and sodium fluoridepotassium nitrate) were tested against six oral isolates of dental caries and periodontal origin – Staphylococcus aureus, Streptococcus mitis, Streptococcus salivarius, Streptococcus pyogenes and Pseudomonas aeruginosa. The antimicrobial potentials were evaluated using modified agar well diffusion method. Various dilutions of the toothpaste products from 1:1 to 1:16 were tested against each test microorganism. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the toothpastes were determined. Results: sodium fluoride-zinc sulphate, benzyl alcoholsodium fluorophosphate and sodium fluorideeugenol toothpastes showed inhibitory effects on S. aureus, S. mitis and S. salivarius. Sodium fluoride-sodium laurylsulfate and sodium fluoride-potassium nitrate toothpastes showed no inhibitory effect on the organisms except S. pyogenes. Only sodium fluoride-potassium nitrate toothpaste inhibited E. coli while none of the toothpastes inhibited P. aeruginosa. The RESUMO

formulations contain fluoride [4]. Most researchers and public health authorities regard fluoride toothpastes as the method of choice for the prevention of dental caries because they are convenient and culturally accepted, widely used, and are commonly associated with decline in caries prevalence in many countries [5]. Triclosan in toothpastes has been reported to reduce the viability of bacteria in vivo and decreases gingival and plaque index scores [6,7].
The two major diseases that affect the teeth are caries (decay) and periodontitis, with gingivitis as the milder form of gum disease. Untreated gum disease can result to periodontitis [8]. Of the wide variety of oral bacteria, Streptococcus mutans, E. coli, Pseudomonas aeruginosa and Lactobacillus species are among the specific species of bacteria that are believed to cause dental caries [9]. Bacteria classified as periodontal pathogens include Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, P. aeruginosa, E. coli, Staphylococcus species, Klebseilla pneumoniae, Streptococcus species, Tannerella forsythia, Treponema denticola, [10,11]. These bacteria are documented in larger numbers in patients with periodontal diseases and have hardly ever been isolated from healthy subjects [10]. S. mitis occupy hard surfaces in the oral cavity and are part of the oral flora. S. mitis is usually a cause of odontogenic infection and endocarditis and only in some cases has it been recognized as respiratory pathogen [12]. S. salivarius is most often harmless but is considered an opportunistic pathogen [13]. S. pyogenes often starts infection on the surface of the skin or in the throat spreading into deeper areas of the skin, which can potentially lead to life-threatening diseases [14]. S. aureus is however not always pathogenic, it is a common cause of skin infections including abscesses, respiratory infections such as sinusitis. It can cause various skin and soft tissue infections [15], particularly when skin or mucosal barriers have been breached. Antimicrobials and their concentrations in different products of toothpastes may contribute to the effectiveness of the toothpastes. It is therefore pertinent to find out the inhibitory potentials of the different products of toothpastes to investigate their ability to decrease bacterial load in human oral cavity to know if they contribute to dental health. Labels on toothpastes indicate that the products have antimicrobial properties capable of reducing oral microorganisms causing dental caries. Hence the present study aimed to investigate antimicrobial efficacy of different toothpaste products sold in Ado-Ekiti against oral bacteria isolates.

Study Area
The study was conducted in Ado-Ekiti, Ekiti State, Nigeria. Ado-Ekiti lies on latitude 7.62 and longitude 5.22 with coordinates 7° 37' N and 5° 13' E. The city has elevation of 455 m with population of 424,340 [16]. It has an area of 36.7 Km2 [17].

Toothpaste products under study
Five common toothpaste products were investigated. They were designated sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphate, sodium fluoride-eugenol, sodium fluoride-sodium laurylsulfate and sodium fluoride-potassium nitrate toothpastes. Table I shows the composition contained in the labels of each toothpaste, as well as the active ingredients, registered according to the manufacturers.

Identification and Storage of isolates
The tested microorganisms were E. coli, S. aureus, P. aeruginosa, S. pyogenes, S. mitis, and S. salivarius. They were oral isolates of dental caries collected from Ekiti State University Teaching Hospital, Ado-Ekiti and Federal Teaching Hospital, Ido-Ekiti, Ekiti State.

Preparation of inoculums of selected isolates
Isolates of the microorganisms were picked from the slant with an inoculating wire loop and suspended in 5 ml of peptone water -REF M028-500G, LOT 0000338533 (HiMedia Laboratories Pvt. Ltd, Mumbai, India) and incubated overnight at 37 o C to reactivate the organisms. The resulting turbidity was adjusted to 0.5 McFarland turbidity standards [24].

Determination of antibacterial susceptibility of tested microorganisms
The antibacterial activity of different concentrations of the tothpastes were determined by modified agar well diffusion method [19,25]. Mueller-Hinton agar -REF M173-500G, LOT 0000341708 (HiMedia Laboratories Pvt. Ltd, Mumbai, India) and brain heart infusion agar plates were prepared aseptically following the manufacturer's instructions. The agar plates were seeded with 0.5 ml of the standard inoculum of each microorganism and allowed to dry for 1 hour. Brain heart infusion agar was used for streptococcus strains.
A sterile 6 mm cork borer was used to punch one central and five wells of 6 mm diameter at equidistance in each of the plates. The holes were labeled with dilutions to be inserted and 0.2 ml of the toothpaste dilutions (1/1, 1/2, 1/4, 1/8, and 1/16) were introduced into each of five wells, while 5µg ciprofloxacin disc -LOT SC09/P (Abtek Biologicals Ltd, Liverpool, United Kingdom) was placed on the agar surface to serve as positive control. Sterile distilled water (0.2 ml) was introduced into the central wells to serves as negative controls. The plates were incubated at 37 ºC for 24 hours. The tests were interpreted as valid by growth of microorganisms to the tip of distilled water wells and the microorganisms were inhibited by ciprofloxacin. The antibacterial activities of the toothpastes were evaluated by measuring the diameter of zones of inhibition minus the diameter of the well (6 mm). The experiments were done in triplicates and the mean zones of inhibition were recorded.

*Determination of minimum inhibitory concentration (MIC)
MIC values were determined by the broth tube dilution method [19,20]. The toothpaste was diluted and added to the brain heart infusion broth supplemented with 2 grams / liter of glucose (0.2 %) with 2 times the final concentration and the tested concentrations were from 1000 mg / ml to 15.6 mg / ml. One drop of the standardized inoculum of each bacterium was used in each test. A tube containing brain-heart infusion broth and inoculated with S. aureus was included in the test to be sure that the prepared broth supports the growth of microorganisms. Furthermore, a tube containing toothpaste dilution and broth that is not inoculated with S. aureus, and another tube containing only the broth that is not inoculated with S. aureus were included in the test to show the sterility and clarity of the broth [25]. A drop of 0.2 % phenol red was added to all the tubes [20] and incubated at 37 oC for 24 hours for bacterial cultures. With satisfactory controls, the tubes were checked for evidence of growth -change in color from red to yellow and / or turbidity at the supernatant [20] after incubation. The lowest concentration of the toothpaste dilution that inhibited the growth of the tested microorganism was recorded as the MIC [19].

Determination of minimum bactericidal concentration (MBC)
Minimum bactericidal concentration was determined from MIC. From the tubes showing no visible growth, 0.1 ml of the samples were inoculated on the sterile brain heart infusion agar using streak plate method. The plates were incubated at 37 ºC for 24 hours. The least concentration that did not show any growth of the tested microorganism was recorded as the MBC [19,25].

RESULTS
The tested microorganisms grew to the edges of the wells (0.0 mm) containing distilled water while several zones of inhibition were observed against the bacteria tested with ciprofloxacin (table II). The microorganisms were inhibited by toothpastes according to the concentration used. Wider zones of inhibition were recorded at higher concentrations than at lower concentrations of toothpaste, that is, they were dose dependent. Based on the diameter of inhibition at various dilutions, sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphate, and sodium fluoride-eugenol toothpastes showed comparable antibacterial potential against S. aureus, S. mitis and S. salivarius. S. mitis was most inhibited by sodium fluoride-eugenol toothpaste. Sodium fluoride-zinc sulphate toothpaste inhibited S. aureus, S. mitis, S. salivarius and S. pyogenes, with S. aureus and S. salivarius being inhibited until the 1: 8 dilution, which corresponds to the concentration 125 mg / ml and S mitis and S. pyogenes were inhibited until the 1: 2 dilution, which corresponds to the concentration 500 mg / ml (Table III). Benzyl alcohol-sodium fluorophosphates toothpaste B inhibited S. aureus, S. mitis, S. salivarius and S. pyogenes, with S. aureus and S. pyogenes being inhibited until the 1: 8 dilution (125 mg / ml). The toothpaste inhibited S. mitis until 1: 2 dilution (500 mg / ml) and inhibited S. salivarius until 1: 4 dilution which correspond to the concentration 250 mg / ml. E. coli and P. aeruginosa were not affected by the toothpaste (Table III). Sodium fluoride-eugenol toothpaste inhibited S. aureus, S. mitis, S. salivarius and S. pyogenes, with S. aureus and S. mitis inhibited up till 1: 8 dilution (125 mg / ml), S. salivarius and S. pyogenes inhibited till 1: 2 dilution (500 mg / ml). E. coli and P. aeruginosa were not affected by the toothpaste (Table IV). Sodium fluoride-sodium laurylsulfate toothpaste inhibited only S. pyogenes with bacterium inhibited till 1: 2 dilution (500mg / ml) (

DISCUSSION
There are several toothpastes available in different markets whose manufacturers claim to have antimicrobial potential. The present study was carried out to assess the antibacterial potential of five different brands of toothpaste sold in Ado-Ekiti. The in vitro agar diffusion method was used because it is easier to directly measure the antibacterial potential of each toothpaste dilution. The toothpaste showing the largest zone of inhibition had the strongest antimicrobial properties.
Antimicrobial effects related to toothpastes have been demonstrated in the literature [26]. The toothpastes tested in this study have components which are expected to sufficiently control the oral microbiome, when present in adequate concentrations. The results of the present study indicated that the different toothpastes exhibited varied effectiveness against the different microorganisms. It is believed that components such as sodium fluoride, zinc sulfate and sodium monoflurophosphate, eugenol, sorbitol, benzyl alcohol, sodium laurylsulfate, limonene, cocamidopropyl betaine and potassium nitrate may be related to antibacterial effects. All toothpastes investigated in the present study contain essentially the same active ingredients with little variation, but have demonstrated varying degrees of effectiveness against the tested microorganisms. Fluoride and sorbitol are present in all toothpastes in this study, according to the manufacturers. Sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphates and sodium fluoride-eugenol toothpastes showed greater antibacterial effects when compared to sodium fluoride-sodium laurylsulfate and sodium fluoride potassium nitrate toothpastes. This may have been due to the presence of active components with greater antimicrobial capacity. Sodium fluoridezinc sulphate toothpaste and sodium fluorideeugenol have in their composition the substance eugenol, whose strong antiseptic potential has been reported in the literature [27]. Only benzyl alcohol-sodium fluorophosphate toothpaste possesses benzyl alcohol as constituent. The alcohol has antibacterial potential and is also added to toothpastes for its fragrance [28].
All the toothpastes have limonene as a constituent except sodium fluoride-zinc sulphate toothpaste. Limonene is a chemical substance found in the peels of citrus fruits. Its major use in toothpaste is as solvent for cleansing purpose and as fragrance, but is known to have antimicrobial activities [30]. Sorbitol is a sugar-alcohol compound used as a sweetener in toothpaste, but it has a preventive effect against caries [29].
There are conflicting results on the antibacterial effects of different concentrations of sodium fluoride toothpastes against some oral microbiome [31]. This was re-affirmed by Imran et al. [32]. Toothpastes having higher concentrations of fluoride may record higher antibacterial potentials. The present study agreed with the report of Marinho et al. [4] that fluoride toothpaste is associated with inhibition of oral microbiome and capable of achieving reduction in tooth decay. The reported ability of benzyl alcohol-sodium fluorophosphates toothpaste (containing sodium monofluorophosphate and sodium laurylsulfate) in the reduction of dental decay [4] appears supported by the present study. When formulated correctly and used as directed, fluoride toothpastes will help to prevent tooth decay. It is well documented that fluoride has the ability to inhibit or even reverse the initiation and progression of dental caries [33].
In our study, sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphate, sodium fluoride-eugenol and sodium fluoride-sodium laurylsulfate toothpastes did not inhibit E. coli, only sodium fluoride-potassium nitrate toothpaste at 1000 mg / ml, 500 mg / ml, 250 mg / ml and 125 mg / ml inhibited the bacterium. Among the toothpastes used in this study, only sodium fluoride-potassium nitrate toothpaste contained potassium nitrate and cocamidopropyl betaine in its formulations. Potassium nitrate has been reported to have strong antimicrobial activities against E. coli [34] and cocamidopropyl betaine has antibacterial activity [35].
All the toothpastes at different dilutions showed no inhibitory effects on P. aeruginosa. This could be due to its intrinsic resistance to many antimicrobial agents, mainly due to the synergistic effect of multi-drug efflux system and the bacteria's low outer membrane permeability [36] The MIC and MBC results revealed that sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphates and sodium fluoride-eugenol toothpastes had bacteriostatic and bactericidal effects on S. aureus, S. mitis, S. salivarius and S. pyogenes. Of these three toothpastes, sodium fluorideeugenol was shown to be the most inhibitory for microorganisms, followed by benzyl alcohol-sodium fluorophosphates and sodium fluoride-zinc sulphate. Sodium fluoridesodium laurylsulfate and sodium fluoridepotassium nitrate toothpastes did not show inhibitory activity against S. aureus, S. mitis, S. salivarius, but both toothpastes inhibited S. pyogenes. The low inhibition potential of sodium fluoride-sodium laurylsulfate toothpaste compared to other toothpastes is supported by Bhattcharjee et al. [37]. All toothpastes contain sodium laurysulfate (SLS) in their constitution, except sodium fluoride-potassium nitrate toothpaste. SLS has been reported to have strong antimicrobial properties [38]. This may be the explanation for the lower inhibitory effects presented by sodium fluoride-potassium nitrate toothpaste. Although the substance is present in Sodium fluoride-sodium laurylsulfate toothpaste, there was no inhibition by it. It may be that the concentration of SLS present in this toothpaste is not satisfactory to cause any significant inhibitory effect on bacteria.
The sterility test carried out on the toothpaste showed that they were all sterile. This is in agreement with the findings of Okpalugo et al. [39]. The test was important to guarantee the quality of these products. No visible growth was observed in the present study in any of the toothpastes, indicating that manufacturers' claims about quality with respect to sterility can be trusted.
Since dilutions can affect the effectiveness of toothpastes on the bacteria tested in the in vitro study, toothpastes that showed higher rates of inhibition, being considered to have higher antibacterial properties, may not necessarily be higher than those that had lower rates of inhibition. In addition, it should be taken into account that toothpaste when used in vivo is likely to be diluted by saliva, reducing or losing such antimicrobial properties [40]. In addition, different toothpastes have different active ingredients and other substances, which can spread at different rates, resulting in different potential antimicrobials. Therefore, it cannot be fully assumed that the results of antimicrobial efficacy can be directly proportional or transferable to the clinical efficacy of toothpaste. The accumulation of oral microorganisms represents the main etiological factor for oral diseases, including cavities and periodontal diseases. Thus, the use of toothpastes with adequate formulations is essential, since oral hygiene is considered the main factor that influences the degree of dental caries and the control of periodontal disease. The manufacturers' claims about the potential antibacterial properties of toothpaste against microorganisms capable of causing infection in the oral cavity appeared to be supported by this research for sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphates and sodium fluoride-eugenol toothpastes. However, the same antibacterial potential against these microorganisms was not demonstrated by sodium fluoride-sodium laurylsulfate toothpaste, except for S. pyogenes and sodium fluoride-potassium nitrate toothpaste, which was effective against S. pyogenes and E. coli. Thus, sodium fluoridesodium laurylsulfate and sodium fluoridepotassium nitrate toothpastes may not satisfy the claim required by the manufacturers.
The limitation to this study is that antibacterial effects of the toothpastes on many other periodontal anaerobic pathogens such as P. gingivalis, T. forsythia, T. denticola and facultative anaerobic pathogen, A. actinomycetemcomitans, were not determined in this study. Also, tests for the presence of the constituents claimed by the manufacturers or the concentrations of the constituents were not included in this study.

CONCLUSION
Most of the toothpastes in Ado-Ekiti are essentially similar in composition, but differ in relation to the inhibition potentials on the microorganisms tested. While some toothpastes had greater antimicrobial effects, others had less potential against the tested microorganisms. This variation may be related to the concentration of active components used in the preparation of different toothpastes. In general, the manufacturers' claims about the potential antibacterials of toothpaste against microorganisms capable of causing infection in the oral cavity appear to be supported by this research for toothpastes sodium fluoride-zinc sulphate, benzyl alcohol-sodium fluorophosphate, sodium fluoride-eugenol.