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Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans
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Journal of Infection and Public Health (2015) xxx, xxx—xxx
Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans Cristiane Aparecida Pereira a,∗, Anna Carolina Borges Pereira Costa a,1, Priscila Christiane Suzy Liporoni b,2, Marcos Augusto Rego b,2, Antonio Olavo Cardoso Jorge a,1 a
Unesp — Univ Estadual Paulista, School of Dentistry, Institute of Science and Technology, Department of Biosciences and Oral Diagnosis, Francisco José Longo 777, São Dimas, São José dos Campos, CEP: 12245-000 SP, Brazil b Unitau — Univ de Taubaté, School of Dentistry, Department of Restorative Dentistry, Expedicionário Ernesto Pereira 110, Centro, Taubaté, CEP: 12020-330 SP, Brazil Received 2 May 2015 ; received in revised form 21 July 2015; accepted 11 October 2015
KEYWORDS Streptococcus mutans; Baccharis; Biofilms; Dental caries
Summary Streptococcus mutans is an important cariogenic microorganism, and alternative methods for its elimination are required. Different concentrations of Baccharis dracunculifolia essential oil (EO) were tested to determine its minimal inhibitory concentration (MIC) in planktonic cultures, and this concentration was used in S. mutans biofilms. Additionally, we assessed the effect of a 0.12% chlorhexidine (CHX) and saline solution in S. mutans biofilms. The biofilms were grown in discs of composite resin for 48 h and exposed to B. dracunculifolia, CHX or saline solution for 5 min. The viability of the biofilms was determined by counting the colony-forming units per milliliter (CFU/ml) in agar, which was statistically significant (P < 0.05). The MIC of the B. dracunculifolia EO to planktonic growth of S. mutans was 6%. In biofilms of S. mutans clinical isolates, B. dracunculifolia EO (6%) and CHX resulted in reductions of 53.3—91.1% and 79.1—96.6%, respectively. For the
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Corresponding author. Tel.: +55 12 39479033; fax: +55 12 39479010. E-mail addresses: [email protected] (C.A. Pereira), carol [email protected] (A.C.B.P. Costa), [email protected] (P.C.S. Liporoni), [email protected] (M.A. Rego), [email protected] (A.O.C. Jorge). 1 Tel.: +55 12 39479033; fax: +55 12 39479010. 2 Tel.: +55 12 3625 4149. http://dx.doi.org/10.1016/j.jiph.2015.10.012 1876-0341/© 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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C.A. Pereira et al. biofilm formed by the S. mutans reference strain, the reductions achieved with B. dracunculifolia EO and CHX were, respectively, 39.3% and 88.1%. It was concluded that B. dracunculifolia EO showed antibacterial activity and was able to control this oral microorganism, which otherwise causes dental caries. © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.
Introduction A biofilm is a naturally occurring accumulation of microorganisms of either a single or multiple species embedded in an extracellular polymeric matrix and adherent to a biologic or non-biologic surface. Bacteria in a biofilm differ profoundly from their planktonic counterparts by growing in an enclosed, nutrient-sufficient ecosystem as a community of sessile organisms that exhibit an altered phenotype with respect to the growth rate, gene transcription [1], and resistance to antimicrobial agents [2]. The resistance of biofilms to antibiotics is increased compared with planktonic cells. In fact, when cells exist in a biofilm, they can become 10—1000 times more resistant to the effects of antimicrobial agents [3—5]. The oral cavity is heavily colonized by a complex, relatively specific and highly interrelated range of microorganisms, which are organized in biofilms. There is substantial evidence linking oral disease and respiratory tract infections with the pathogens in oral biofilms [6]. Approximately 20% of oral bacteria are streptococci [7]. The oral streptococci pioneer early dental plaque formation and have a specific temporal and spatial distribution that is crucial for the development of oral biofilms. Streptococcus mutans is considered to be the most cariogenic of all oral streptococci [8], and the main factors associated with its cariogenicity include adhesion, acidogenicity and acid tolerance [9,10]. These bacteria produce glucosyltransferases and synthesize glucans from sucrose, which mediate the adherence of S. mutans and other oral bacterial microbiota on tooth surfaces, contributing to the formation of dental biofilm. Selection for a cariogenic microbiota in dental biofilm increases the magnitude of the decrease in pH following the fermentation of available carbohydrates as well as increases the probability of enamel demineralization, which leads to the formation of dental caries [9].
Biofilms must be removed mechanically, but antibacterial mouth rinses are effective at decreasing tooth surface biofilms. In general, mouth rinses contain fluorides, alcohols, and detergents or antibacterial substances [11]. Ideal antibacterial substances must be effective against many microorganisms, act rapidly, maintain activity at low concentrations, lack side effects, and not cause discomfort [12]. Frequently used antibacterial chemicals include povidone iodine products, chlorhexidine, and cetylpyridinium chloride [13]. However, natural products have significantly contributed to the discovery of chemical structures and the creation of new medicaments that can be used as innovative therapeutic agents against this prevalent disease [14,15] Baccharis dracunculifolia DC (Asteraceae) is a native plant from Brazil, which is commonly known as ‘‘alecrim-do-campo’’’ and ‘‘vassoura’’. B. dracunculifolia is the most important botanical source of South-eastern Brazilian propolis, known as green propolis because of its color [16]. Teas, decoctions and tinctures prepared from the flowering plant are widely used in alternative medicine to treat inflammation, hepatic disorders and stomach ulcers [17]. A variety of chemical compounds and pharmacological activities have been attributed to this plant, including antiulcerative [18], antibacterial [19] and antifungal [20] properties. The aim of this study was to determine the minimal inhibitory concentration (MIC) of B. dracunculifolia essential oil (EO) against planktonic cultures of S. mutans and its antibacterial activity in biofilms formed in the discs of composite resin.
Materials and methods Microorganisms and standard suspensions Seven S. mutans strains that had been previously isolated and identified from the oral cavities
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Antibacterial activity of Baccharis dracunculifolia of healthy individuals and one reference strain (American Type Culture Collection — ATCC® 35688, Manassas, USA) of S. mutans that was maintained in our laboratory stock collection were included in the study. Standard suspensions of each isolate, containing 106 cells/ml, were prepared. For this purpose, isolates were seeded onto brain heart infusion (BHI) agar (Difco, Detroit, USA) and incubated for 48 h. All incubations were performed 37 ◦ C and at a partial pressure of 5% CO2 . After incubation, the cells were suspended in sterile saline solution [0.9% sodium chloride (NaCl)], and the number of cells in suspension was counted with a spectrophotometer (B582, Micronal, São Paulo, Brazil). The optical density and wavelength were 0.620 and 398 nm, respectively.
Essential oil isolation Leaves of the B. dracunculifolia plant were collected from the region of Belo Horizonte, Minas Gerais, Brazil to extract EO. The plant sample was identified, and a voucher specimen was deposited in the herbarium of the Zoobotanic Foundation, which was localized in the same city. Air-dried plant leaves were placed in a round-bottom distillation flask to which distilled water was added. The EO was obtained by hydrodistillation for 3 h with Clevenger apparatus [20]. The EO was separated, dried over anhydrous sodium sulfate, and stored in an amber bottle at 4 ◦ C until used.
Determination of the minimal inhibitory concentrations (MIC) of essential oil The MIC of B. dracunculifolia EO was determined by the microdilution in broth method in sterile 96well flat-bottom microtiter plates with a lid (Costar Corning, New York, USA). Different concentrations of B. dracunculifolia EO were prepared by dissolving various amounts of EO in distilled water. Tween-80 (0.05%, v/v) was added to enhance oil the solubility. The dilutions were sterilized in disposable membrane filters with a 0.22-m pore size (Millipore, São Paulo, Brazil). A total of 0.1 ml of each dilution of B. dracunculifolia EO and 0.1 ml of bacterial standard suspensions, as described above, were used in each test, and the mixtures were incubated for 48 h at 37 ◦ C. Each sample was tested in duplicate. The lowest concentration of B. dracunculifolia EO required to completely inhibit the growth of S. mutans was designated as the MIC. The MIC was used in the experiments that evaluated the viability of S. mutans biofilms.
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Production of biofilms and antibacterial assays The biofilm formation was developed as a methodology proposed by Pereira et al. [21]. The biofilms were grown on 240 sterile discs (30 for each isolate) of composite resin, which were prepared in a cylindrical-shaped Teflon mold that was 6.0 mm in diameter and 3.00 mm in height. The mold was filled in a single increment with microhybrid (Vit-lescenceTM , Ultradent Products, South Jordan, USA) and covered with a mylar matrix strip. The top surface was cured for 40 s in a curing unit device (Optilux 401, Demetron/Kerr, Danbury, CT, USA) that was operating between 700 and 800 mW/cm2 . Samples were retrieved from the mold, and the excess resin was removed with surgical blades. The discs were immersed in distilled water at 37 ◦ C and sterilized by autoclaving at 121 ◦ C for 15 min. The discs were placed in 24-well plates (Costar Corning, New York, USA) with 2 ml of sterile Gibbons and Nygaard broth [22]. The wells were inoculated with 0.1 ml of standard suspensions, and the plates were incubated for 48 h. The broth was changed after 24 h of incubation. After 48 h of incubation, the discs containing the biofilms were washed twice with 0.9% NaCl to remove loosely bound material, and they were submitted to antibacterial assays. The antibacterial effects of B. dracunculifolia EO in the concentration of 6% (MIC obtained) were evaluated. The 0.9% NaCl was used as a negative control, and 0.12% chlorhexidine (CHX) solution (Byoformula, São Paulo, Brazil) was used as a positive control. The 240 discs (30 for each isolate) with biofilms established after 48 h were treated for 5 min with 2 ml of B. dracunculifolia EO (n = 10), 0.12% CHX (n = 10) and sterile 0.9% NaCl (n = 10). After the experimental periods, each biofilm was washed with 2 ml of 0.9% NaCl to remove the tested substances. The discs were placed in tubes containing 10 ml of 0.9% NaCl and they were sonicated (Sonoplus HD 2200, 50 W. Bandelin Eletronic, Berlin, Germany) for 30 s to disperse the biofilms. Biofilm suspensions were serially diluted in 0.9% NaCl to give dilutions of 10−1 to 10−5 times the original concentration. One hundred microliter aliquots of each dilution were seeded on BHI agar (in duplicate). After 48 h of incubation, the number of colony-forming units per milliliter (CFU/ml) was determined. The results were analyzed by analysis of variance (ANOVA) and the Tukey test. A P value <0.05 was considered to indicate a statistically significant difference. The percentage of reduction was calculated with 0.9% NaCl results as a reference.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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C.A. Pereira et al. Table 1 Means and standard deviation of reductions for biofilms formed by each strain of S. mutans, treated with B. dracunculifolia essential oil (6%) or chlorhexidine (0.12%), compared with the control group, that was treated only with physiological solution (NaCl 0.9%). Biofilms formed by
Figure 1 The mean and standard deviation of the CFU/ml (105 ) obtained in the three experimental conditions tested for biofilms formed by S. mutans clinical strains, B. dracunculifolia essential oil (6%), chlorhexidine (0.12%), and physiological solution (0.9% NaCl). Tukey test for each tested group. The values followed by different capital letters differed significantly between the tested experimental conditions.
Biofilms reductions with B. dracunculifolia essential oil (6%)
Clinical strain 15 Clinical strain 22 Clinical strain 24 Clinical strain 26 Clinical strain 28 Clinical strain 29 Clinical strain 31 Reference strain
53.3% 78.9% 90.9% 71.8% 91.1% 75% 61.7% 39.3%
± ± ± ± ± ± ± ±
21.4% 9% 5% 24.5% 4.9% 15.9% 20.6% 17.5%
Chlorhexidine (0.12%) 88.1% 79.1% 91.7% 80% 96.6% 90.6% 84.8% 88.1%
± ± ± ± ± ± ± ±
13.5% 11.9% 11.4% 21% 3.1% 3.2% 10.9% 13.4%
Results In this study, we initially determined the lowest concentration of B. dracunculifolia EO required to completely inhibit the growth of S. mutans in planktonic cultures, which was designated as the MIC. The MIC of the B. dracunculifolia EO to S. mutans, both standard and clinical strains, was 6%. This concentration was used in subsequent assays to evaluate the viability of the S. mutans biofilms. The mean and standard deviation values of the CFU/ml (105 ) obtained in the three experimental conditions for each biofilm group are shown in Figs. 1 and 2. Significant decreases in the viability of biofilms formed by S. mutans clinical isolates treated with B. dracunculifolia (P = 0.0254) and CHX (P = 0.0038) were observed compared to the
Figure 2 The mean and standard deviation of the CFU/ml (105 ) obtained in the three experimental conditions tested for biofilms formed by the S. mutans reference strain, B. dracunculifolia essential oil (6%), chlorhexidine (0.12%), and physiological solution (0.9% NaCl). The Tukey test for each tested group. Values followed by different capital letters differed significantly between the tested experimental conditions.
control group (treated with 0.9% NaCl only). In biofilms formed by the S. mutans reference strain, treatment with B. dracunculifolia (P = 0.0032) and CHX (P = 0.0001) also promoted significant reductions in the viability. The means of reduction for biofilms formed by each strain treated with B. dracunculifolia EO or CHX, compared to the untreated controls biofilms, are shown in Table 1.
Discussion S. mutans is an important microorganism for early colonization on tooth surfaces during the formation of a dental plaque [23]. The acidogenic properties of S. mutans, in addition to its ability to synthesize extracellular glucans, are the major factors involved in the development and establishment of cariogenic biofilms [24]. Various chemical plaque control approaches have been suggested to reduce cariogenic biofilms. Among them, CHX and triclosan, which are synthetic chemical antimicrobial agents, are common materials in oral care products [25,26]. Although these compounds have broad and strong antibacterial characteristics, they have long-term side effects, such as intra-oral staining and disturbance of the oral bacterial ecology [27]. Therefore, there has been increasing attention on the discovery of new compounds that lack side effects with long-term use. The aim of this study was to evaluate the activity of B. dracunculifolia EO against planktonic cultures, biofilms of reference (ATCC) and clinical strains of S. mutans. In S. mutans planktonic cultures, the effective concentration that could inhibit
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Antibacterial activity of Baccharis dracunculifolia the growth of the strains was 6%. This concentration was used in subsequent assays to evaluate the viability of S. mutans biofilms and resulted in significant reductions in the viability of these biofilms without total elimination. In biofilms formed by S. mutans clinical isolates, B. dracunculifolia EO produced reductions of 53.3 ± 21.4% to 91.1 ± 4.9%. For the biofilm formed by the S. mutans reference strain, the reduction achieved with B. dracunculifolia EO was of 39.3 ± 17.5%. The presence of microbial biofilms is the main cause of inefficient eradication with antibiotic therapy. Biofilm bacteria often tolerate antibiotic doses that are several 100-fold over the minimum lethal dose for bacteria living outside the biofilm [4]. The mechanism of biofilm resistance includes physical or chemical diffusion barriers to antimicrobial penetration into the biofilm, slow growth of the biofilm from nutrient limitation, activation of the general stress response and the emergence of a biofilm-specific phenotype [28]. These issues have made it necessary to develop alternative strategies to control biofilms, particularly in the context of treating oral disease. Selected natural products that originate in plants can influence microbial biofilm formation. Plantderived compounds inhibit peptidoglycan synthesis [29], damage microbial membrane structures [30], modify bacterial membrane surface hydrophobicity [31] and modulate quorum sensing [29], all of which could influence biofilm formation. The mechanisms by which EO can inhibit microorganisms involve different modes of action and may be due, in part, to their hydrophobicity. As a result, the oils are partitioned into the lipid bilayer of the cell membrane, affecting the respiratory chain and leading to the leakage of vital cell contents [32]. The impairment of bacterial enzyme systems may also be a potential mechanism of action. Various components of EO can permeabilize the cell membrane, increasing the penetration of antibiotics. Interference with the bacterial enzyme systems may be another potential mechanism of action [33]. The antimicrobial activity of B. dracunculifolia EO found in this study may be due to the presence of high prenylated p-coumaric acid levels, primarily of artepillin C and baccharin, flavonoids, diterpenes, and triterpenes isolated from the aerial parts of the plant [34,35]. Leitão et al. [36] verified that green propolis and B. dracunculifolia extracts have a bacteriostatic effect on the planktonic cultures of S. mutans as well as have inhibitory effects on the cariogenic traits (glucan synthesis and acidogenic potential) exhibited by these bacteria. The leaves
5 of B. dracunculifolia have also exhibited antifungal activity against Candida spp. [20]. A clinical study evaluated the efficacy of green propolis, collected by Apis mellifera bees from B. dracunculifolia, in treating patients diagnosed with gingivitis and chronic periodontitis, observing regression of these diseases [37]. Another relevant study conducted by Resende et al. [38] noted the absence of mutagenic activity of B. dracunculifolia extracts and Artepillin C using Salmonella typhimurium strains. In the present study, CHX was used as a negative control, and there were significant reductions in the viability of S. mutans biofilms. In biofilms formed by S. mutans clinical isolates, CHX had reductions of 79.1 ± 11.9% to 96.6 ± 3.1%. For the biofilm formed by the S. mutans reference strain, the reduction achieved with CHX was of 88.1 ± 13.4%. These findings agree with the literature on the action of CHX on aerobic and anaerobic Gram-positive and Gram-negative bacteria and yeast. This chemical substance has a high affinity for the cell wall of microorganisms, and it induces cell surface alterations that cause the loss of osmotic balance and cytoplasmic precipitation [39]. Currently, CHX is the safest and most efficient antimicrobial agent for reducing the microorganisms in the oral cavity. However, CHX is associated with a number of adverse effects, such as the formation of stains on teeth and dentures, dysgeusia, parotid enlargement, and desquamation of the oral mucosa [40]. These factors have encouraged the search for other antimicrobial agents. Based on the current results, it can be concluded that B. dracunculifolia EO was effective at reducing the viability of S. mutans biofilms. In addition, in planktonic cultures, B. dracunculifolia EO was able to inhibit the complete growth of S. mutans. These data indicate its usefulness for controlling oral microorganisms that cause of dental caries.
Funding No funding sources.
Conflict of interest None declared.
Ethical approval Not required.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Acknowledgments This work was supported by the Fundac ¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant 2009/52048-1, Scholarships 2011/21346-7 and 2013/07411-6). The authors are thankful to PharmaNéctar® (Belo Horizonte, Brazil) for donating the Baccharis dracunculifolia leaves.
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Journal of Infection and Public Health (2015) xxx, xxx—xxx
Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans Cristiane Aparecida Pereira a,∗, Anna Carolina Borges Pereira Costa a,1, Priscila Christiane Suzy Liporoni b,2, Marcos Augusto Rego b,2, Antonio Olavo Cardoso Jorge a,1 a
Unesp — Univ Estadual Paulista, School of Dentistry, Institute of Science and Technology, Department of Biosciences and Oral Diagnosis, Francisco José Longo 777, São Dimas, São José dos Campos, CEP: 12245-000 SP, Brazil b Unitau — Univ de Taubaté, School of Dentistry, Department of Restorative Dentistry, Expedicionário Ernesto Pereira 110, Centro, Taubaté, CEP: 12020-330 SP, Brazil Received 2 May 2015 ; received in revised form 21 July 2015; accepted 11 October 2015
KEYWORDS Streptococcus mutans; Baccharis; Biofilms; Dental caries
Summary Streptococcus mutans is an important cariogenic microorganism, and alternative methods for its elimination are required. Different concentrations of Baccharis dracunculifolia essential oil (EO) were tested to determine its minimal inhibitory concentration (MIC) in planktonic cultures, and this concentration was used in S. mutans biofilms. Additionally, we assessed the effect of a 0.12% chlorhexidine (CHX) and saline solution in S. mutans biofilms. The biofilms were grown in discs of composite resin for 48 h and exposed to B. dracunculifolia, CHX or saline solution for 5 min. The viability of the biofilms was determined by counting the colony-forming units per milliliter (CFU/ml) in agar, which was statistically significant (P < 0.05). The MIC of the B. dracunculifolia EO to planktonic growth of S. mutans was 6%. In biofilms of S. mutans clinical isolates, B. dracunculifolia EO (6%) and CHX resulted in reductions of 53.3—91.1% and 79.1—96.6%, respectively. For the
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Corresponding author. Tel.: +55 12 39479033; fax: +55 12 39479010. E-mail addresses: [email protected] (C.A. Pereira), carol [email protected] (A.C.B.P. Costa), [email protected] (P.C.S. Liporoni), [email protected] (M.A. Rego), [email protected] (A.O.C. Jorge). 1 Tel.: +55 12 39479033; fax: +55 12 39479010. 2 Tel.: +55 12 3625 4149. http://dx.doi.org/10.1016/j.jiph.2015.10.012 1876-0341/© 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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C.A. Pereira et al. biofilm formed by the S. mutans reference strain, the reductions achieved with B. dracunculifolia EO and CHX were, respectively, 39.3% and 88.1%. It was concluded that B. dracunculifolia EO showed antibacterial activity and was able to control this oral microorganism, which otherwise causes dental caries. © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.
Introduction A biofilm is a naturally occurring accumulation of microorganisms of either a single or multiple species embedded in an extracellular polymeric matrix and adherent to a biologic or non-biologic surface. Bacteria in a biofilm differ profoundly from their planktonic counterparts by growing in an enclosed, nutrient-sufficient ecosystem as a community of sessile organisms that exhibit an altered phenotype with respect to the growth rate, gene transcription [1], and resistance to antimicrobial agents [2]. The resistance of biofilms to antibiotics is increased compared with planktonic cells. In fact, when cells exist in a biofilm, they can become 10—1000 times more resistant to the effects of antimicrobial agents [3—5]. The oral cavity is heavily colonized by a complex, relatively specific and highly interrelated range of microorganisms, which are organized in biofilms. There is substantial evidence linking oral disease and respiratory tract infections with the pathogens in oral biofilms [6]. Approximately 20% of oral bacteria are streptococci [7]. The oral streptococci pioneer early dental plaque formation and have a specific temporal and spatial distribution that is crucial for the development of oral biofilms. Streptococcus mutans is considered to be the most cariogenic of all oral streptococci [8], and the main factors associated with its cariogenicity include adhesion, acidogenicity and acid tolerance [9,10]. These bacteria produce glucosyltransferases and synthesize glucans from sucrose, which mediate the adherence of S. mutans and other oral bacterial microbiota on tooth surfaces, contributing to the formation of dental biofilm. Selection for a cariogenic microbiota in dental biofilm increases the magnitude of the decrease in pH following the fermentation of available carbohydrates as well as increases the probability of enamel demineralization, which leads to the formation of dental caries [9].
Biofilms must be removed mechanically, but antibacterial mouth rinses are effective at decreasing tooth surface biofilms. In general, mouth rinses contain fluorides, alcohols, and detergents or antibacterial substances [11]. Ideal antibacterial substances must be effective against many microorganisms, act rapidly, maintain activity at low concentrations, lack side effects, and not cause discomfort [12]. Frequently used antibacterial chemicals include povidone iodine products, chlorhexidine, and cetylpyridinium chloride [13]. However, natural products have significantly contributed to the discovery of chemical structures and the creation of new medicaments that can be used as innovative therapeutic agents against this prevalent disease [14,15] Baccharis dracunculifolia DC (Asteraceae) is a native plant from Brazil, which is commonly known as ‘‘alecrim-do-campo’’’ and ‘‘vassoura’’. B. dracunculifolia is the most important botanical source of South-eastern Brazilian propolis, known as green propolis because of its color [16]. Teas, decoctions and tinctures prepared from the flowering plant are widely used in alternative medicine to treat inflammation, hepatic disorders and stomach ulcers [17]. A variety of chemical compounds and pharmacological activities have been attributed to this plant, including antiulcerative [18], antibacterial [19] and antifungal [20] properties. The aim of this study was to determine the minimal inhibitory concentration (MIC) of B. dracunculifolia essential oil (EO) against planktonic cultures of S. mutans and its antibacterial activity in biofilms formed in the discs of composite resin.
Materials and methods Microorganisms and standard suspensions Seven S. mutans strains that had been previously isolated and identified from the oral cavities
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Antibacterial activity of Baccharis dracunculifolia of healthy individuals and one reference strain (American Type Culture Collection — ATCC® 35688, Manassas, USA) of S. mutans that was maintained in our laboratory stock collection were included in the study. Standard suspensions of each isolate, containing 106 cells/ml, were prepared. For this purpose, isolates were seeded onto brain heart infusion (BHI) agar (Difco, Detroit, USA) and incubated for 48 h. All incubations were performed 37 ◦ C and at a partial pressure of 5% CO2 . After incubation, the cells were suspended in sterile saline solution [0.9% sodium chloride (NaCl)], and the number of cells in suspension was counted with a spectrophotometer (B582, Micronal, São Paulo, Brazil). The optical density and wavelength were 0.620 and 398 nm, respectively.
Essential oil isolation Leaves of the B. dracunculifolia plant were collected from the region of Belo Horizonte, Minas Gerais, Brazil to extract EO. The plant sample was identified, and a voucher specimen was deposited in the herbarium of the Zoobotanic Foundation, which was localized in the same city. Air-dried plant leaves were placed in a round-bottom distillation flask to which distilled water was added. The EO was obtained by hydrodistillation for 3 h with Clevenger apparatus [20]. The EO was separated, dried over anhydrous sodium sulfate, and stored in an amber bottle at 4 ◦ C until used.
Determination of the minimal inhibitory concentrations (MIC) of essential oil The MIC of B. dracunculifolia EO was determined by the microdilution in broth method in sterile 96well flat-bottom microtiter plates with a lid (Costar Corning, New York, USA). Different concentrations of B. dracunculifolia EO were prepared by dissolving various amounts of EO in distilled water. Tween-80 (0.05%, v/v) was added to enhance oil the solubility. The dilutions were sterilized in disposable membrane filters with a 0.22-m pore size (Millipore, São Paulo, Brazil). A total of 0.1 ml of each dilution of B. dracunculifolia EO and 0.1 ml of bacterial standard suspensions, as described above, were used in each test, and the mixtures were incubated for 48 h at 37 ◦ C. Each sample was tested in duplicate. The lowest concentration of B. dracunculifolia EO required to completely inhibit the growth of S. mutans was designated as the MIC. The MIC was used in the experiments that evaluated the viability of S. mutans biofilms.
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Production of biofilms and antibacterial assays The biofilm formation was developed as a methodology proposed by Pereira et al. [21]. The biofilms were grown on 240 sterile discs (30 for each isolate) of composite resin, which were prepared in a cylindrical-shaped Teflon mold that was 6.0 mm in diameter and 3.00 mm in height. The mold was filled in a single increment with microhybrid (Vit-lescenceTM , Ultradent Products, South Jordan, USA) and covered with a mylar matrix strip. The top surface was cured for 40 s in a curing unit device (Optilux 401, Demetron/Kerr, Danbury, CT, USA) that was operating between 700 and 800 mW/cm2 . Samples were retrieved from the mold, and the excess resin was removed with surgical blades. The discs were immersed in distilled water at 37 ◦ C and sterilized by autoclaving at 121 ◦ C for 15 min. The discs were placed in 24-well plates (Costar Corning, New York, USA) with 2 ml of sterile Gibbons and Nygaard broth [22]. The wells were inoculated with 0.1 ml of standard suspensions, and the plates were incubated for 48 h. The broth was changed after 24 h of incubation. After 48 h of incubation, the discs containing the biofilms were washed twice with 0.9% NaCl to remove loosely bound material, and they were submitted to antibacterial assays. The antibacterial effects of B. dracunculifolia EO in the concentration of 6% (MIC obtained) were evaluated. The 0.9% NaCl was used as a negative control, and 0.12% chlorhexidine (CHX) solution (Byoformula, São Paulo, Brazil) was used as a positive control. The 240 discs (30 for each isolate) with biofilms established after 48 h were treated for 5 min with 2 ml of B. dracunculifolia EO (n = 10), 0.12% CHX (n = 10) and sterile 0.9% NaCl (n = 10). After the experimental periods, each biofilm was washed with 2 ml of 0.9% NaCl to remove the tested substances. The discs were placed in tubes containing 10 ml of 0.9% NaCl and they were sonicated (Sonoplus HD 2200, 50 W. Bandelin Eletronic, Berlin, Germany) for 30 s to disperse the biofilms. Biofilm suspensions were serially diluted in 0.9% NaCl to give dilutions of 10−1 to 10−5 times the original concentration. One hundred microliter aliquots of each dilution were seeded on BHI agar (in duplicate). After 48 h of incubation, the number of colony-forming units per milliliter (CFU/ml) was determined. The results were analyzed by analysis of variance (ANOVA) and the Tukey test. A P value <0.05 was considered to indicate a statistically significant difference. The percentage of reduction was calculated with 0.9% NaCl results as a reference.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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C.A. Pereira et al. Table 1 Means and standard deviation of reductions for biofilms formed by each strain of S. mutans, treated with B. dracunculifolia essential oil (6%) or chlorhexidine (0.12%), compared with the control group, that was treated only with physiological solution (NaCl 0.9%). Biofilms formed by
Figure 1 The mean and standard deviation of the CFU/ml (105 ) obtained in the three experimental conditions tested for biofilms formed by S. mutans clinical strains, B. dracunculifolia essential oil (6%), chlorhexidine (0.12%), and physiological solution (0.9% NaCl). Tukey test for each tested group. The values followed by different capital letters differed significantly between the tested experimental conditions.
Biofilms reductions with B. dracunculifolia essential oil (6%)
Clinical strain 15 Clinical strain 22 Clinical strain 24 Clinical strain 26 Clinical strain 28 Clinical strain 29 Clinical strain 31 Reference strain
53.3% 78.9% 90.9% 71.8% 91.1% 75% 61.7% 39.3%
± ± ± ± ± ± ± ±
21.4% 9% 5% 24.5% 4.9% 15.9% 20.6% 17.5%
Chlorhexidine (0.12%) 88.1% 79.1% 91.7% 80% 96.6% 90.6% 84.8% 88.1%
± ± ± ± ± ± ± ±
13.5% 11.9% 11.4% 21% 3.1% 3.2% 10.9% 13.4%
Results In this study, we initially determined the lowest concentration of B. dracunculifolia EO required to completely inhibit the growth of S. mutans in planktonic cultures, which was designated as the MIC. The MIC of the B. dracunculifolia EO to S. mutans, both standard and clinical strains, was 6%. This concentration was used in subsequent assays to evaluate the viability of the S. mutans biofilms. The mean and standard deviation values of the CFU/ml (105 ) obtained in the three experimental conditions for each biofilm group are shown in Figs. 1 and 2. Significant decreases in the viability of biofilms formed by S. mutans clinical isolates treated with B. dracunculifolia (P = 0.0254) and CHX (P = 0.0038) were observed compared to the
Figure 2 The mean and standard deviation of the CFU/ml (105 ) obtained in the three experimental conditions tested for biofilms formed by the S. mutans reference strain, B. dracunculifolia essential oil (6%), chlorhexidine (0.12%), and physiological solution (0.9% NaCl). The Tukey test for each tested group. Values followed by different capital letters differed significantly between the tested experimental conditions.
control group (treated with 0.9% NaCl only). In biofilms formed by the S. mutans reference strain, treatment with B. dracunculifolia (P = 0.0032) and CHX (P = 0.0001) also promoted significant reductions in the viability. The means of reduction for biofilms formed by each strain treated with B. dracunculifolia EO or CHX, compared to the untreated controls biofilms, are shown in Table 1.
Discussion S. mutans is an important microorganism for early colonization on tooth surfaces during the formation of a dental plaque [23]. The acidogenic properties of S. mutans, in addition to its ability to synthesize extracellular glucans, are the major factors involved in the development and establishment of cariogenic biofilms [24]. Various chemical plaque control approaches have been suggested to reduce cariogenic biofilms. Among them, CHX and triclosan, which are synthetic chemical antimicrobial agents, are common materials in oral care products [25,26]. Although these compounds have broad and strong antibacterial characteristics, they have long-term side effects, such as intra-oral staining and disturbance of the oral bacterial ecology [27]. Therefore, there has been increasing attention on the discovery of new compounds that lack side effects with long-term use. The aim of this study was to evaluate the activity of B. dracunculifolia EO against planktonic cultures, biofilms of reference (ATCC) and clinical strains of S. mutans. In S. mutans planktonic cultures, the effective concentration that could inhibit
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Antibacterial activity of Baccharis dracunculifolia the growth of the strains was 6%. This concentration was used in subsequent assays to evaluate the viability of S. mutans biofilms and resulted in significant reductions in the viability of these biofilms without total elimination. In biofilms formed by S. mutans clinical isolates, B. dracunculifolia EO produced reductions of 53.3 ± 21.4% to 91.1 ± 4.9%. For the biofilm formed by the S. mutans reference strain, the reduction achieved with B. dracunculifolia EO was of 39.3 ± 17.5%. The presence of microbial biofilms is the main cause of inefficient eradication with antibiotic therapy. Biofilm bacteria often tolerate antibiotic doses that are several 100-fold over the minimum lethal dose for bacteria living outside the biofilm [4]. The mechanism of biofilm resistance includes physical or chemical diffusion barriers to antimicrobial penetration into the biofilm, slow growth of the biofilm from nutrient limitation, activation of the general stress response and the emergence of a biofilm-specific phenotype [28]. These issues have made it necessary to develop alternative strategies to control biofilms, particularly in the context of treating oral disease. Selected natural products that originate in plants can influence microbial biofilm formation. Plantderived compounds inhibit peptidoglycan synthesis [29], damage microbial membrane structures [30], modify bacterial membrane surface hydrophobicity [31] and modulate quorum sensing [29], all of which could influence biofilm formation. The mechanisms by which EO can inhibit microorganisms involve different modes of action and may be due, in part, to their hydrophobicity. As a result, the oils are partitioned into the lipid bilayer of the cell membrane, affecting the respiratory chain and leading to the leakage of vital cell contents [32]. The impairment of bacterial enzyme systems may also be a potential mechanism of action. Various components of EO can permeabilize the cell membrane, increasing the penetration of antibiotics. Interference with the bacterial enzyme systems may be another potential mechanism of action [33]. The antimicrobial activity of B. dracunculifolia EO found in this study may be due to the presence of high prenylated p-coumaric acid levels, primarily of artepillin C and baccharin, flavonoids, diterpenes, and triterpenes isolated from the aerial parts of the plant [34,35]. Leitão et al. [36] verified that green propolis and B. dracunculifolia extracts have a bacteriostatic effect on the planktonic cultures of S. mutans as well as have inhibitory effects on the cariogenic traits (glucan synthesis and acidogenic potential) exhibited by these bacteria. The leaves
5 of B. dracunculifolia have also exhibited antifungal activity against Candida spp. [20]. A clinical study evaluated the efficacy of green propolis, collected by Apis mellifera bees from B. dracunculifolia, in treating patients diagnosed with gingivitis and chronic periodontitis, observing regression of these diseases [37]. Another relevant study conducted by Resende et al. [38] noted the absence of mutagenic activity of B. dracunculifolia extracts and Artepillin C using Salmonella typhimurium strains. In the present study, CHX was used as a negative control, and there were significant reductions in the viability of S. mutans biofilms. In biofilms formed by S. mutans clinical isolates, CHX had reductions of 79.1 ± 11.9% to 96.6 ± 3.1%. For the biofilm formed by the S. mutans reference strain, the reduction achieved with CHX was of 88.1 ± 13.4%. These findings agree with the literature on the action of CHX on aerobic and anaerobic Gram-positive and Gram-negative bacteria and yeast. This chemical substance has a high affinity for the cell wall of microorganisms, and it induces cell surface alterations that cause the loss of osmotic balance and cytoplasmic precipitation [39]. Currently, CHX is the safest and most efficient antimicrobial agent for reducing the microorganisms in the oral cavity. However, CHX is associated with a number of adverse effects, such as the formation of stains on teeth and dentures, dysgeusia, parotid enlargement, and desquamation of the oral mucosa [40]. These factors have encouraged the search for other antimicrobial agents. Based on the current results, it can be concluded that B. dracunculifolia EO was effective at reducing the viability of S. mutans biofilms. In addition, in planktonic cultures, B. dracunculifolia EO was able to inhibit the complete growth of S. mutans. These data indicate its usefulness for controlling oral microorganisms that cause of dental caries.
Funding No funding sources.
Conflict of interest None declared.
Ethical approval Not required.
Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012
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Acknowledgments This work was supported by the Fundac ¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant 2009/52048-1, Scholarships 2011/21346-7 and 2013/07411-6). The authors are thankful to PharmaNéctar® (Belo Horizonte, Brazil) for donating the Baccharis dracunculifolia leaves.
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Please cite this article in press as: Pereira CA, et al. Antibacterial activity of Baccharis dracunculifolia in planktonic cultures and biofilms of Streptococcus mutans. J Infect Public Health (2015), http://dx.doi.org/10.1016/j.jiph.2015.10.012