Abstract

Introduction

Methods of decontamination or sanitization of toothbrushes have been questioned.

Objective

This study assessed the effectiveness of pomegranate peels infusion as a disinfectant of toothbrushes against Streptococcus mutans.

Material and method

A sample of 16 schoolchildren aged between 7 and 9 years performed brushing 5 days/week, with a careful brushing once a day. After each day of brushing, the toothbrushes were washed and sprayed with one disinfectant solution. This procedure was repeated for 4 weeks using one of the different solutions per week: distilled water (G1; negative control), pomegranate (Punica granatum Linn) peels infusion (G2), 1% sodium hypochlorite (G3) and 0.12% chlorhexidine digluconate (G4). After the fifth day, toothbrushes were collected for laboratory analysis. Toothbrushes heads were subjected to agitation in saline dilution of 10^–1, 10^–2,10^–3, and 25 μL of each dilution were seeded in mitis salivarius agar culture medium for S. mutans colony-forming unit (CFU) counting. One calibrated examiner (Kappa = 0.91) performed the CFU (mL^–1 × 10⁴) counts. Kruskal-Wallis and Dunn Multiple Comparison tests were used at a significance level of 5%.

Result

G1 presented the highest number of CFU (3.9 ± 8.4), followed by G2 (3.2 ± 4.0). No S. mutans growth was observed in G3 and G4. There was no statistically significant difference between G1 and G2 and between G3 and G4 (p>0.05).

Conclusion

Pomegranate infusion was completely ineffective for the disinfection of toothbrushes against S. mutans when compared with 1% sodium hypochlorite and 0.12% chlorhexidine digluconate solutions.

Descriptors:
Disinfection; toothbrushing; pomegranate; Streptococcus mutans

Resumo

Introdução

Os métodos de descontaminação ou desinfecção de escovas dentais têm sido questionados.

Objetivo

Este estudo avaliou a eficácia da infusão de cascas de romã como um desinfetante de escovas dentais contra Streptococcus mutans.

Material e método

Uma amostra de 16 escolares com idade entre 7 e 9 anos realizou escovação dentária cuidadosa, uma vez ao dia por 5 dias/semana durante 4 semanas. Após cada dia de escovação, as escovas foram lavadas e pulverizadas com uma solução desinfetante. Este procedimento foi repetido por 4 semanas utilizando uma das diferentes soluções por semana: água destilada (G1; grupo controle), infusão de casca de romã (Punica granatum Linn) (G2), hipoclorito de sódio a 1% (G3) e digluconato de clorexidina a 0,12% (G4). Após o quinto dia, as escovas foram coletadas para análise laboratorial. As cabeças das escovas foram agitadas em solução salina diluída em 10^–1, 10^–2,10^–3, e 25μL de cada diluição foi semeada em meio de cultura agar mitis salivarius para contagem de unidade formadora de colônias (UFC) de S. mutans. Um examinador calibrado (Kappa = 0,91) realizou a contagem de UFC mL^–1 × 10⁴. Os testes de Kruskal-Wallis e de Comparações Múltiplas de Dunn foram usados em um nível de significância de 5%.

Resultado

G1 apresentou o maior número de UFC (3,9 ± 8,4), seguido de G2 (3,2 ± 4,0). Não foi observado crescimento de S. mutans em G3 e G4. Não houve diferença estatisticamente significante entre G1 e G2 e entre G3 e G4 (p>0,05).

Conclusão

A infusão de romã foi completamente ineficaz para a desinfecção de escovas dentais contra S. mutans quando comparada às soluções de hipoclorito de sódio a 1% e digluconato de clorexidina a 0,12%.

Descritores:
Desinfecção; escovação dentária; Punica granatum; Streptococcus mutans

INTRODUCTION

The toothbrush is the most effective tool to remove dental biofilm. The mechanical action of its bristles on the tooth surfaces promotes breakdown and removal of microorganisms adhered to these surfaces, enabling the maintenance of a healthy oral microbiota. Although it is important to maintain control of the Streptococcus mutans levels, toothbrushes become sites of colonization by microorganisms because they offer ideal conditions for bacteria, viral, fungal and parasitic growth¹1 Sato S, Ito IY, Lara EH, Panzeri H, Albuquerque RF Jr, Pedrazzi V. Bacterial survival rate on toothbrushes and their decontamination with antimicrobial solutions. J Appl Oral Sci. 2004 Jun;12(2):99-103. http://dx.doi.org/10.1590/S1678-77572004000200003. PMid:21365129.
http://dx.doi.org/10.1590/S1678-77572004... . A single use is sufficient to contaminate the bristles, and microorganisms are capable of surviving from 24 hours up to 7 days²2 Nelson-Filho P, Faria G, Silva RA, Rossi MA, Ito IY. Evaluation of the contamination and disinfection methods of toothbrushes used by 24- to 48 month-old children. J Dent Child (Chic). 2006 Sep-Dec;73(3):152-8. PMid:17367032..

In recent years, the issue of toothbrush disinfection has become progressively important³3 Peker I, Akarslan Z, Basman A, Haciosmanoglu N. Knowledge and behavior of dentists in a dental school regarding toothbrush disinfection. Braz Oral Res. 2015;29(1):48. http://dx.doi.org/10.1590/1807-3107BOR-2015.vol29.0048. PMid:25789506.
http://dx.doi.org/10.1590/1807-3107BOR-2... . Methods of decontamination or sanitization of toothbrushes have been questioned. The literature has demonstrated that the use of chemical agents, such as sodium hypochlorite, chlorhexidine gluconate, hydrogen peroxide, cetylpyridinium chloride, sodium perborate, essential oils, Brushtox^® (40% activated ethanol with biocide parabens), is considered an efficient and inexpensive method of decontamination²2 Nelson-Filho P, Faria G, Silva RA, Rossi MA, Ito IY. Evaluation of the contamination and disinfection methods of toothbrushes used by 24- to 48 month-old children. J Dent Child (Chic). 2006 Sep-Dec;73(3):152-8. PMid:17367032.^,44 Spolidorio DM, Tardivo TA, Derceli JR, Neppelenbroek KH, Duque C, Spolidorio LC, et al. Evaluation of two alternative methods for disinfection of toothbrushes and tongue scrapers. Int J Dent Hyg. 2011 Nov;9(4):279-83. http://dx.doi.org/10.1111/j.1601-5037.2011.00503.x. PMid:21356033.
http://dx.doi.org/10.1111/j.1601-5037.20...

5 Chandrdas D, Jayakumar HL, Katodia L, Sreedevi A, Chandra M. Evaluation of antimicrobial efficacy of garlic, tea tree oil, cetylpyridinium chloride, chlorhexidine, and ultraviolet sanitizing device in the decontamination of toothbrush. Indian J Dent. 2014 Oct;5(4):183-9. http://dx.doi.org/10.4103/0975-962X.144718. PMid:25565751.
http://dx.doi.org/10.4103/0975-962X.1447... ^-66 Nascimento C, Trinca NN, Pita MS, Pedrazzi V. Genomic identification and quantification of microbial species adhering to toothbrush bristles after disinfection: a cross-over study. Arch Oral Biol. 2015 Jul;60(7):1039-47. http://dx.doi.org/10.1016/j.archoralbio.2015.03.012. PMid:25912552.
http://dx.doi.org/10.1016/j.archoralbio.... . In this context, phytoplants have demonstrated to be a good alternative to synthetic chemical antimicrobial agents, because they do not produce side effects and acquire antimicrobial resistance⁷7 Nabar BM, Pawar PL. Effect of plant extracts formulated in different ointment bases on MDR strains. Indian J Pharm Sci. 2010 May;72(3):397-401. http://dx.doi.org/10.4103/0250-474X.70494. PMid:21188057.
http://dx.doi.org/10.4103/0250-474X.7049... ^,88 Devatkal SK, Jaiswal P, Jha SN, Bharadwaj R, Viswas KN. Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat. J Food Sci Technol. 2013 Jun;50(3):555-60. http://dx.doi.org/10.1007/s13197-011-0351-y. PMid:24425952.
http://dx.doi.org/10.1007/s13197-011-035... .

Punica granatum Linn (pomegranate) is native to the region extending from northern India to Iran and it is also widely cultivated in parts of America and Africa⁹9 Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008 Jun;13(2):128-44. PMid:18590349.^,1010 Viuda-Martos M, Fernández-López J, Pérez-Álvarez JA. Pomegranate and its many functional components as related to human health: a review. Compr Rev Food Sci Food Saf. 2010 Nov;9(6):635-54. http://dx.doi.org/10.1111/j.1541-4337.2010.00131.x.
http://dx.doi.org/10.1111/j.1541-4337.20... . It is composed of tannins and alkaloids that are antimicrobial substances¹¹11 Qabaha KI. Antimicrobial and free radical scavenging activities of five Palestinian medicinal plants. Afr J Tradit Complement Altern Med. 2013 May 16;10(4):101-8. eCollection 2013. PMid: 24146509. PMCID: PMC3794399.. Pomegranate has many potential effects, such as bactericidal, antifungal, antiviral, immune modulation, vermifuge, stimulant, refrigerant, astringent, stomachic, stypic, laxative, diuretic and anthelmintic⁹9 Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008 Jun;13(2):128-44. PMid:18590349.^,1212 Abdollahzadeh S, Mashouf R, Mortazavi H, Moghaddam M, Roozbahani N, Vahedi M. Antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens. J Dent (Tehran). 2011;8(1):1-6. PMid:21998800..

The capacity of the pomegranate extract to prevent dental biofilm adherence and dental bacterial growth may be due to the effect of its flavonoid components, which have antiglycosyltransferase activity, and polyphenols may affect the bacterial cell wall, inhibit enzymes by oxidized agents, interact with proteins and disturb co-aggregation of microorganisms¹³13 Vasconcelos LC, Sampaio FC, Sampaio MC, Pereira MS, Higino JS, Peixoto MH. Pereira M do S, Higino JS, Peixoto MH. Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S. mutans, S. mitis and C. albicans. Braz Dent J. 2006;17(3):223-7. http://dx.doi.org/10.1590/S0103-64402006000300009. PMid:17262129.
http://dx.doi.org/10.1590/S0103-64402006... ^,1414 Pereira JV, Pereira MSV, Sampaio FC, Sampaio MCC, Alves PM, Araújo CRF, et al. Efeito antibacteriano e antiaderente in vitro do extrato da Punica granatum Linn. sobre microrganismos do biofilme dental. Rev Bras Farmacogn. 2006 Mar;16(1):88-93. http://dx.doi.org/10.1590/S0102-695X2006000100016.
http://dx.doi.org/10.1590/S0102-695X2006... . Its methanolic extract has been shown to be effective against some common oral pathogens, such as Staphylococcus epidermidis, Staphylococcus aureus, Actynomyces viscosus, Candida spp, Lactobacillus acidophilus, S. mutans, Streptococcus sanguinis, Streptococcus salivarius and Streptococcus aureus⁷7 Nabar BM, Pawar PL. Effect of plant extracts formulated in different ointment bases on MDR strains. Indian J Pharm Sci. 2010 May;72(3):397-401. http://dx.doi.org/10.4103/0250-474X.70494. PMid:21188057.
http://dx.doi.org/10.4103/0250-474X.7049... ^,1212 Abdollahzadeh S, Mashouf R, Mortazavi H, Moghaddam M, Roozbahani N, Vahedi M. Antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens. J Dent (Tehran). 2011;8(1):1-6. PMid:21998800.

13 Vasconcelos LC, Sampaio FC, Sampaio MC, Pereira MS, Higino JS, Peixoto MH. Pereira M do S, Higino JS, Peixoto MH. Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S. mutans, S. mitis and C. albicans. Braz Dent J. 2006;17(3):223-7. http://dx.doi.org/10.1590/S0103-64402006000300009. PMid:17262129.
http://dx.doi.org/10.1590/S0103-64402006...

14 Pereira JV, Pereira MSV, Sampaio FC, Sampaio MCC, Alves PM, Araújo CRF, et al. Efeito antibacteriano e antiaderente in vitro do extrato da Punica granatum Linn. sobre microrganismos do biofilme dental. Rev Bras Farmacogn. 2006 Mar;16(1):88-93. http://dx.doi.org/10.1590/S0102-695X2006000100016.
http://dx.doi.org/10.1590/S0102-695X2006... ^-1515 Mehta VV, Rajesh G, Rao A, Shenoy R, Pai M. Antimicrobial efficacy of Punica granatum mesocarp, Nelumbo nucifera leaf, Psidium guajava leaf and Coffea Canephora extract on common oral pathogens: an in-vitro study. J Clin Diagn Res. 2014 Jul;8(7):ZC65-8. http://dx.doi.org/10.7860/JCDR/2014/9122.4629. PMid:25177642.
http://dx.doi.org/10.7860/JCDR/2014/9122... . Pomegranate peels infusion is widely used by the Brazilian population, especially in Northeastern Brazil, as an antiseptic in throat infections; it is easily accessible, low cost and its extract has proven potential antimicrobial effect¹⁶16 De Albuquerque UP, Monteiro JM, Ramos MA, Amorim EL. Medicinal and magic plants from a public Market in northeastern Brazil. J Ethnopharmacol. 2007 Mar;110(1):76-91. http://dx.doi.org/10.1016/j.jep.2006.09.010. PMid:17056216.
http://dx.doi.org/10.1016/j.jep.2006.09.... . However, to our knowledge, no study has evaluated its antimicrobial power to disinfect toothbrushes.

Therefore, the aim of this comparative study was to assess the effectiveness of pomegranate peels infusion as a toothbrush disinfectant against S. mutans. The null hypothesis is that there is no difference among the pomegranate peels infusion comparing to different solutions (1% sodium hypochlorite and 0.12% chlorhexidine digluconate) for the disinfection of toothbrushes.

MATERIAL AND METHOD

This study was approved by the local ethical Review Board for Human Studies. All the responsibles for the children signed a term of free and informed consent allowing their participation.

Manufacture of Pomegranate Peel Infusion

Fresh pomegranates (500 g) were obtained from a public market in the city of João Pessoa, Paraíba, Brazil. The pomegranate peels were separated and oven dried at 33 °C for 7 days to eliminate the moisture and stabilize the enzyme content. Water was boiled to a temperature of 100 °C and then poured over the pomegranate peels (20 g pomegranate peels/200 mL water), which were allowed to steep in the liquid in a closed glass container for a period of 10 minutes. The liquid was strained and stored in a sterile spray bottle for 5 days, while it was being tested as disinfectant solution.

Sample

The sample comprised 16 schoolchildren, aged between 7 and 9 years, who met the following inclusion criteria: children whose parents consented to their participation in the study; children who had not been using antimicrobial substances such as mouthwashes and antibiotics or immunosuppressants for at least three months before the study began; children who attended classes during the week of collection, and those without systemic disorders.

Groups and Experimental Periods

A sample of 16 schoolchildren aged between 7 and 9 years performed brushing 5 days/week during 4 weeks. The children carefully brushed their teeth once a day for two minutes at school under supervision. After each day of brushing, the toothbrushes were washed and sprayed with one of the following solutions: distilled water (G1; negative control), pomegranate (Punica granatum Linn) peels infusion (G2), 1% sodium hypochlorite (G3) and 0.12% chlorhexidine digluconate (G4).

The researcher visited the school daily and supervised the toothbrushing performed by the schoolchildren in the classroom, in first period of the morning. Each child received in the first day of the experiment a new toothbrush (Condor Trip®, Condor, São Bento do Sul – Santa Catarina, Brazil) and the dentifrice (about 0.5 grams) was placed on the child’s toothbrush by the transverse technique (Colgate Tripla Ação^®,1450 ppm fluoride, São Bernardo do Campo – São Paulo, Brazil). The child was provided with a glass of water to perform a mouth rinse and wash the brush. After brushing, the toothbrushes were collected and washed in tap water for one minute. Then the disinfection procedure was performed using the disinfectant substance, which was sprayed six times on the bristles, maintaining a distance of approximately five centimeters between the spray nozzle and brush bristles. This procedure was performed 5 days a week, with the same toothbrushes and the same disinfectant substance.

After the fifth day, toothbrushes were collected for laboratory analysis. This procedure was repeated for 4 weeks using one of the different solutions per week. New toothbrushes were provided when a new disinfection solution had been used. There were 54 toothbrushes used in the entire experiment.

After collecting all toothbrushes, microbiological analysis were performed. Each toothbrush was then decapitated and the head transferred to a tube containing 7.5 mL of sterile phosphate-buffered saline (PBS). The tube containing the toothbrush head was refrigerated (cooler with ice) and immediately taken to the laboratory. The tubes were submitted to sonication in an ultrasonic apparatus (Ultrasonic Cleaner^®, Odontobrás, Ribeirão Preto – São Paulo, Brazil) for five minutes to release the bacteria adhered to the bristles. The brush head was removed and the resulting suspension was serially diluted (10^–1, 10 ^–2, 10^–3; and 25 µl of each dilution were seeded in Mitis salivarius agar (Difco Laboratories, Detroit, MI, USA) with 30U/mL bacitracin¹⁷17 Tenuta LMA, Lima JEO, Cardoso CL, Tabchoury CPM, Cury JA. Effect of plaque accumulation and salivary factors on enamel demineralization and plaque composition in situ. Pesqui Odontol Bras. 2003 Dec;17(4):326-31. http://dx.doi.org/10.1590/S1517-74912003000400006. PMid:15107914.
http://dx.doi.org/10.1590/S1517-74912003... for S. mutans CFU (Colony Forming Units) counting. The plates were incubated at 37 °C, for 48 h under microaerophilic conditions (jar with candle system). A single trained investigator performed the CFU counts, manually in a counter (Phoenix CP 608^®, Phoenix Industry and Trade of Scientific Equipment Ltd., Araraquara – São Paulo, Brazil). For this step, calibration had previously been performed, and an excellent intra-examiner agreement (Kappa = 0.91) had been observed.

Statistical Analysis

The results of CFU counts (mL^–1 × 10⁴) for each disinfectant solution were analyzed by GraphPad Prism 3.4 for Windows (GraphPad Software®, San Diego, California, USA) software program. The Kolmogorov-Smirnov test was used to test the normal distribution of data. The nonparametric Kruskal-Wallis test was used to compare the number of S. mutans CFU among the four disinfectant solutions. The Dunn Multiple Comparison was applied to verify whether there was significant difference between groups tested. The significance level was set at 5%.

Statistical test power for the sample size was calculated online using the website OpenEpi¹⁸18 Dean AG, Sullivan KM, Soe MM. OpenEpi: open source epidemiologic statistics for Public Health [Internet]. 2016 [cited 2016 Feb 1]. Availble from: www.openepi.com.

RESULT

The negative control G1 (distilled water) and G2 (pomegranate peels infusion) groups presented the highest number of CFU of S. mutans. No bacteria growth was observed in the samples treated with 1% sodium hypochlorite (G3) and 0.12% chlorhexidine digluconate (G4) solutions. There was no statistically significant difference between G1 and G2 and between G3 and G4. Statistically significant difference (p<0.001) was found between the groups: G1 and G3, G1 and G4, G2 and G3, and G2 and G4 (Table 1).

Considering the difference of 3.2 ± 4.0 for the pomegranate peels infusion, a sample size of sixteen schoolchildren provided a statistical power of 92.1%.

DISCUSSION

Currently, there are no protocols on toothbrush disinfection for healthy individuals, and the contamination with microorganisms increases with repeated use¹⁹19 Efstratiou M, Papaioannou W, Nakou M, Ktenas E, Vrotsos IA, Panis V. Contamination of a toothbrush with antibacterial properties by oral microorganisms. J Dent. 2007 Apr;35(4):331-7. http://dx.doi.org/10.1016/j.jdent.2006.10.007. PMid:17118507.
http://dx.doi.org/10.1016/j.jdent.2006.1... . In this context, it is important to mention the antimicrobial properties of the pomegranate⁹9 Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008 Jun;13(2):128-44. PMid:18590349.. To our knowledge, this is the first study evaluating the effectiveness of pomegranate peels infusion on the disinfection of toothbrush bristles.

Pomegranate peels are constituted by phenolic punicalagins; gallic acid and other fatty acids; catechin; EGCG (Epigallocatechin gallate); quercetin, rutin, and other flavonols; flavones, flavonones; anthocyanidins⁹9 Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008 Jun;13(2):128-44. PMid:18590349.. The pomegranate tannins are capable of crossing the cell wall composed of several polysaccharides and proteins, and bind to its surface. Polyphenols may affect the bacterial cell wall, inhibit enzymes by oxidized agents, interact with proteins and disturb co-aggregation of microorganisms¹¹11 Qabaha KI. Antimicrobial and free radical scavenging activities of five Palestinian medicinal plants. Afr J Tradit Complement Altern Med. 2013 May 16;10(4):101-8. eCollection 2013. PMid: 24146509. PMCID: PMC3794399.

12 Abdollahzadeh S, Mashouf R, Mortazavi H, Moghaddam M, Roozbahani N, Vahedi M. Antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens. J Dent (Tehran). 2011;8(1):1-6. PMid:21998800.^-1313 Vasconcelos LC, Sampaio FC, Sampaio MC, Pereira MS, Higino JS, Peixoto MH. Pereira M do S, Higino JS, Peixoto MH. Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S. mutans, S. mitis and C. albicans. Braz Dent J. 2006;17(3):223-7. http://dx.doi.org/10.1590/S0103-64402006000300009. PMid:17262129.
http://dx.doi.org/10.1590/S0103-64402006... .

According to the results of the present study, the pomegranate peels infusion showed no antimicrobial activity on S. mutans, being similar to the negative control group (distilled water). It can be suggested that the inefficacy of the pomegranate to disinfect toothbrushes bristles might be due to the formulation of Punica granatun Linn prepared as peels infusion. Previous studies have shown effectiveness of pomegranate hydroalcoholic extract⁸8 Devatkal SK, Jaiswal P, Jha SN, Bharadwaj R, Viswas KN. Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat. J Food Sci Technol. 2013 Jun;50(3):555-60. http://dx.doi.org/10.1007/s13197-011-0351-y. PMid:24425952.
http://dx.doi.org/10.1007/s13197-011-035... ^,99 Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008 Jun;13(2):128-44. PMid:18590349.^,1212 Abdollahzadeh S, Mashouf R, Mortazavi H, Moghaddam M, Roozbahani N, Vahedi M. Antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens. J Dent (Tehran). 2011;8(1):1-6. PMid:21998800.^,2020 Fawole OA, Makunga NP, Opara UL. Antibacterial, antioxidant and tyrosinase-inhibition activities of pomegranate fruit peel methanolic extract. BMC Complement Altern Med. 2012 Oct;12(1):200. http://dx.doi.org/10.1186/1472-6882-12-200. PMid:23110485.
http://dx.doi.org/10.1186/1472-6882-12-2... or in the form of phytotherapeutic gel¹³13 Vasconcelos LC, Sampaio FC, Sampaio MC, Pereira MS, Higino JS, Peixoto MH. Pereira M do S, Higino JS, Peixoto MH. Minimum inhibitory concentration of adherence of Punica granatum Linn (pomegranate) gel against S. mutans, S. mitis and C. albicans. Braz Dent J. 2006;17(3):223-7. http://dx.doi.org/10.1590/S0103-64402006000300009. PMid:17262129.
http://dx.doi.org/10.1590/S0103-64402006... on the growth of dental biofilm microorganisms. When compared with methanol extracts, none of the aqueous pomegranate peel extracts exhibited good antibacterial activity at the highest screening concentration against Gram-positive (Bacillus subtitlis and S. aureus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae)²¹21 Su X, Howell AB, D’Souza DH. Antibacterial effects of plant-derived extracts on methicillin-resistant Staphylococcus aureus. Foodborne Pathog Dis. 2012 Jun;9(6):573-8. http://dx.doi.org/10.1089/fpd.2011.1046. PMid:22663188.
http://dx.doi.org/10.1089/fpd.2011.1046... .

It should be emphasized that there are different results regarding the use of the pomegranate against dental biofilm, depending on its preparation, such as dry powders, leaves, peels, fruits or seeds. A recent study tested the dry powders of pomegranate and extracts in propylene glycol (200 mg/mL). The authors observed that the Punica granatum Linn extract completely inhibited the growth of S. mutans, Staphylococcus spp. and Candida spp. Thus, S. mutans showed high sensitivity to the pomegranate extract⁶6 Nascimento C, Trinca NN, Pita MS, Pedrazzi V. Genomic identification and quantification of microbial species adhering to toothbrush bristles after disinfection: a cross-over study. Arch Oral Biol. 2015 Jul;60(7):1039-47. http://dx.doi.org/10.1016/j.archoralbio.2015.03.012. PMid:25912552.
http://dx.doi.org/10.1016/j.archoralbio.... . Another study tested the Punica granatum Linn mesocarp (middle layer of the pericarp of a fruit) aqueous extract and observed its effectiveness against Candida spp. and the cariogenic bacteria (S. mitis and S. mutans)¹⁵15 Mehta VV, Rajesh G, Rao A, Shenoy R, Pai M. Antimicrobial efficacy of Punica granatum mesocarp, Nelumbo nucifera leaf, Psidium guajava leaf and Coffea Canephora extract on common oral pathogens: an in-vitro study. J Clin Diagn Res. 2014 Jul;8(7):ZC65-8. http://dx.doi.org/10.7860/JCDR/2014/9122.4629. PMid:25177642.
http://dx.doi.org/10.7860/JCDR/2014/9122... .

Another possible explanation for the ineffectiveness of pomegranate infusion might be the time of exposure to the solution and its concentration, because the bactericidal action is dose and time dependent²¹21 Su X, Howell AB, D’Souza DH. Antibacterial effects of plant-derived extracts on methicillin-resistant Staphylococcus aureus. Foodborne Pathog Dis. 2012 Jun;9(6):573-8. http://dx.doi.org/10.1089/fpd.2011.1046. PMid:22663188.
http://dx.doi.org/10.1089/fpd.2011.1046... . It should be mentioned that the period between the last disinfection and laboratory examination did not exceed 4 hours. Devatkal et al.⁸8 Devatkal SK, Jaiswal P, Jha SN, Bharadwaj R, Viswas KN. Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat. J Food Sci Technol. 2013 Jun;50(3):555-60. http://dx.doi.org/10.1007/s13197-011-0351-y. PMid:24425952.
http://dx.doi.org/10.1007/s13197-011-035... verified that pomegranate peel extracts (1%, 5% and 10%) significantly reduced the growth of bacterial cell right from the fourth hour of incubation, and the concentration of 1 g/10 mL of pomegranate peel to water may not be enough to release a sufficient amount of antibacterial compounds.

It was also found that all the samples sprayed with distilled water were infected by S. mutans. Contamination by S. mutans after use in samples sprayed with distilled water has also been observed in previous in vivo studies²2 Nelson-Filho P, Faria G, Silva RA, Rossi MA, Ito IY. Evaluation of the contamination and disinfection methods of toothbrushes used by 24- to 48 month-old children. J Dent Child (Chic). 2006 Sep-Dec;73(3):152-8. PMid:17367032.^,66 Nascimento C, Trinca NN, Pita MS, Pedrazzi V. Genomic identification and quantification of microbial species adhering to toothbrush bristles after disinfection: a cross-over study. Arch Oral Biol. 2015 Jul;60(7):1039-47. http://dx.doi.org/10.1016/j.archoralbio.2015.03.012. PMid:25912552.
http://dx.doi.org/10.1016/j.archoralbio.... ^,2222 Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent. 2000 Sep-Oct;22(5):381-4. PMid:11048305.^,2323 Nelson-Filho P, Pereira MS, De Rossi A, Silva RA, Mesquita KS, Queiroz AM, et al. Children’s toothbrush contamination in day-care centers: how to solve this problem? Clin Oral Investig. 2014 Nov;18(8):1969-74. http://dx.doi.org/10.1007/s00784-013-1169-y. PMid:24366670.
http://dx.doi.org/10.1007/s00784-013-116... , which justifies the use of this solution as negative control in this study.

On the other hand, the toothbrushes sprayed with 1% sodium hypochlorite presented no S. mutans growth. This result is in agreement with the findings of previous studies²²22 Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent. 2000 Sep-Oct;22(5):381-4. PMid:11048305.^,2424 Peker I, Akca G, Sarikir C, Alkurt MT, Celik I. Effectiveness of alternative methods for toothbrush disinfection: an in vitro study. Scientific World Journal. 2014;2014:726190. http://dx.doi.org/10.1155/2014/726190. PMid: 24971388. PMCID: PMC4058182.
http://dx.doi.org/10.1155/2014/726190... , in which there was absence or low percentage of S. mutans growth on toothbrushes after using this solution to disinfect the bristles. Sodium hypochlorite has bactericidal and fungicidal effect on the surface and depth of biofilm. The chlorine released by hypochlorous acid in contact with the tissue proteins, produces nitrogen, formaldehyde and acetaldehyde, which break the sequence of peptides and dissolve proteins. The hydrogen of the amino groups (–NH) is replaced by chlorine (–NCL) forming chloramines, highly toxic compounds, which interfere in cellular metabolism²³23 Nelson-Filho P, Pereira MS, De Rossi A, Silva RA, Mesquita KS, Queiroz AM, et al. Children’s toothbrush contamination in day-care centers: how to solve this problem? Clin Oral Investig. 2014 Nov;18(8):1969-74. http://dx.doi.org/10.1007/s00784-013-1169-y. PMid:24366670.
http://dx.doi.org/10.1007/s00784-013-116... . In spite of the excellent results of disinfection with sodium hypochlorite, its daily use without rinsing the brush properly, could lead to irritation of the oral mucosa, and development of stomach problems, therefore, it is not indicated for use by unsupervised children²⁵25 Sánchez-Ramírez CA, Larrosa-Haro A, Vásquez Garibay EM, Larios-Arceo F. Caustic ingestion and oesophageal damage in children: clinical spectrum and feeding practices. J Paediatr Child Health. 2011 Jun;47(6):378-80. http://dx.doi.org/10.1111/j.1440-1754.2010.01984.x. PMid:21309879.
http://dx.doi.org/10.1111/j.1440-1754.20... .

In the present study excellent results were found in samples sprayed with 0.12% chlorhexidine digluconate. No bacterial growth was observed and this result corroborates the results shown in the literature²2 Nelson-Filho P, Faria G, Silva RA, Rossi MA, Ito IY. Evaluation of the contamination and disinfection methods of toothbrushes used by 24- to 48 month-old children. J Dent Child (Chic). 2006 Sep-Dec;73(3):152-8. PMid:17367032.^,2222 Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent. 2000 Sep-Oct;22(5):381-4. PMid:11048305.. Chlorhexidine is a broad-spectrum antimicrobial agent, whose action is related to its cationic bisbiguanide molecular structure that has substantivity and low-grade toxicity. At low concentration it is bacteriostatic, while at higher concentration it is bactericidal as it brings about coagulation and precipitation of cytoplasm²⁶26 Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod. 1994 Jun;20(6):276-8. http://dx.doi.org/10.1016/S0099-2399(06)80815-0. PMid:7931023.
http://dx.doi.org/10.1016/S0099-2399(06)... . Chlorhexidine digluconate inhibited biofilm formation of S. mutans on the toothbrush bristles²²22 Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent. 2000 Sep-Oct;22(5):381-4. PMid:11048305.^,2525 Sánchez-Ramírez CA, Larrosa-Haro A, Vásquez Garibay EM, Larios-Arceo F. Caustic ingestion and oesophageal damage in children: clinical spectrum and feeding practices. J Paediatr Child Health. 2011 Jun;47(6):378-80. http://dx.doi.org/10.1111/j.1440-1754.2010.01984.x. PMid:21309879.
http://dx.doi.org/10.1111/j.1440-1754.20... ^,2727 Nascimento AP, Faria G, Watanabe E, Ito IY. Efficacy of mouthrinse spray in inhibiting cariogenic biofilm formation on toothbrush bristles. Braz J Oral Sci. 2008 Jan-Mar;7(24):1489-92.. The 0.12% chlorhexidine has been widely used as a mouthwash and is the most effective solution to disinfect toothbrushes contaminated with S. mutans, Streptococcus pyogenes, S. aureus, Candida albicans²⁸28 Komiyama EY, Back-Brito GN, Balducci I, Koga-Ito CY. Evaluation of alternative methods for the disinfection of toothbrushes. Braz Oral Res. 2010 Jan-Mar;24(1):28-33. http://dx.doi.org/10.1590/S1806-83242010000100005. PMid:20339710.
http://dx.doi.org/10.1590/S1806-83242010... .

The limitations of the present study were not considering other specific microorganisms responsible of biofilm-related oral diseases and only six times of solution were sprayed on the bristles. Thus, other oral microorganisms should also be evaluated by counting of total microorganisms. Additionally, the higher standard deviation of CFU counts could be explained by a heterogeneous sample. No initial S. mutans screening was performed in schoolchildren in order to select them to participate in this study according to their dental caries risk. Further studies should include children with high count of S. mutans in their saliva with the presumption the brushes used by them predominately contain S. mutans.

Herbal medicines in particular in emerging countries are commendable due the availability of the flora, the low income population and the possibility of using products with low environmental risk. Punica granatum Linn can be cultivated locally and its extract can be obtained locally by public for therapeutic use. Further studies on this product and its extracts are recommended, perhaps testing different periods of exposure (immersion period or solution sprayed), the type of compounds responsible for its antibacterial effect and the development of its formulation, such as ethanol extract, different concentrations, infusion of the leaves, extract of fruit mesocarp, pulp extract gel and dry powders.

CONCLUSION

In conclusion, the pomegranate peels infusion was completely ineffective for the disinfection of toothbrushes against S. mutans when compared with 1% sodium hypochlorite and 0.12% chlorhexidine digluconate solutions, which were capable of inhibiting bacterial growth. The null hypothesis was accepted.

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