Arch Health Invest (2013) 2(1): 3-9 ISSN 2317-3009 Antimicrobial Activity of Plants Infusions on Oral Fusobacteria and their Adherence to Human Erythrocytes Atividade antimicrobiana de infusões de vegetais sobre fusobactérias bucais e sua capacidade de adesão a eritrócitos humanos Actividad antimicrobiana de infusiones vegetales en fusobacterias orales y su capacidad de adhesión a los eritrocitos humanos Elerson Gaetti Jardim Jr1 Luís Fernando Landucci2 Ana Cláudia Okamoto1 Haroldo Akeshigue3 1Universidade Estadual Paulista “Júlio de Mesquita Filho”, UNESP - Faculdade de Odontologia de Araçatuba – Departamento de Patologia e Propedêutica Clínica 2Fundação Educacional de Santa Fé do Sul, FUNEC; Centro Universitário de Rio Preto, UNIRP 3 Universidade Católica de Brasília, UCB Periodontal disease is the result of the interrelationship between microbiotic aggression and the host’s organic defence. Amongst the microorganisms involved in periodontopathies, Fusobacterium nucleatum is conspicuous by establishing a link between the initial and final colonizers, besides producing toxic compounds and adhering to the host’s cells. Control of bacterial biofilm can be achieved by use of chemical agents, many of which extracted from plants. Thus the object of this study was to evaluate the inhibitory activity in vitro of some teas, generally taken in a normal diet, on Fusobacterium nucleatum and your adherence to host’s cells. Minimum inhibitory and bactericidal concentrations were established and haemagglutinative test in microplaques was effected. It was ascertained that all plant extracts have inhibitory activity and that infusions of Camellia sinensis (black tea and green tea), Mentha piperita (mint) and Pimpinella anixem (aniseed) added to the bacteria/erythrocyte compound reduced significantly the adherence of microorganisms. Keywords: Dental Plaque; Periodontitis; Disease Prevention; Plant Extracts; Hemagglutination; Cell Adhesion. INTRODUCTION their ability to co-aggregate with other oral anaerobes 4 seems to be involved in mixed infections . The oral fusobacteria are one of the most relevant Periodontitis represent serious health problems as microorganism in the biofilm, representing a bridge they involve, to a greater or lesser extension, all ethnic between the early and late colonizers of the biofilm1. groups, regardless gender or socio-economic levels2,3. The association of oral Fusobacterium species with However, inflammation of periodontal tissues might be gingivitis and periodontitis has received studies2,3 and minimized or even blocked, in a significant part of the Arch Health Invest 2(1) 2013 Arch Health Invest (2013) 2(1): 3-9 ISSN 2317-3009 population, by establishing means for bacterial biofilm chamomile (Matricaria chamomilla ), Japanese green control. tea (Camellia sinensis, Ban-cha variety), aniseed Fusobacterium nucleatum is able to adhere and (Pimpinella anixem), balm mint (Melissa officinalis) colonize a large variety of human cells, particularly and boldo (Peumus boldus) were prepared. The different erythrocytes, lymphocytes, neutrophils, fibroblasts, plants were acquired at the shops and represent the main epithelial cells, and HeLa cells5. Studies of microbial trademarks available. adherence to the host‘s cells are important instruments In preparation of the infusions, 10 g of leaves for understanding the colonization of the periodontal were added to 100ml of 0,1M buffered phosphate saline environment by resident microorganisms. In addition, solution (PBS) and left for 5 minutes at 100OC, for one the adherence of oral fusobacteria to erythrocytes was hour at 55ºC and overnight at 25oC. The aqueous closely related to their adhesion to epithelial cells and extracts were then submitted to fractioned filtration in other human cell lines6. cellulose membrane with 0.65 µm and sterilized through There is a cultural tendency in the population, in filtration in membranes of 0.22 µm (Millipore®). The considering the use of medicinal plants in infusions as infusions were prepared immediately before use the adequate pharmaceutical resources in the treatment of tests. oral problems, and there is the false belief that these 3. EVALUATION OF THE ANTIMICROBIAL compounds have no collateral effects7. However, some ACTIVITY OF PLANT INFUSIONS infusions, as tea, the beverage most largely consumed in In order to determine the higher inhibitory the world8, are valuable sources of fluoride and tannin dilution of the infusions, the agar dilution method was (polyphenols) with antibiofilm and anticariogenic employed as previously described10. It was used activity9. However, it is opportune to check whether Wilkins-Chalgren agar supplemented with yeast extract compounds of the human diet have an antimicrobial (0.5%). In the preparation of different dilutions of the effect on oral fusobacteria or if they might inhibit the infusions in agar, water originally used in preparation of microbial capacity of hemagglutination. Thus, this study the culture medium was partially substituted by plant aimed to evaluate the inhibitory activity of several plant infusions in order to obtain final dilutions representing infusions used in Brazilian diet on fusobacteria and the 1/2, 1/4, 1/8, 1/16, and 1/32 of the original preparation. effects of such infusions on capacity of adherence of In the test, microbial inoculum (105 colony these anaerobes on human erythrocytes. forming units-CFU) was transferred using a Steers’ replicator onto Wilkins-Chalgren agar plates in MATERIAL AND METHODS triplicate. The screening and control plates (without 1. MICROORGANISMS plant infusions) were incubated in anaerobiosis, at 37oC All tested isolates of Fusobacterium nucleatum for 48 hours. The higher antimicrobial dilution was were maintained at -80oC and were recovered from 1993 defined as the higher dilution of plant infusion that to 1997 from lesions of chronic periodontitis. inhibited completely microbial growth on agar. Reactivation of selected strains was performed in The contact inhibitory activity was evaluated as peptone yeast extract broth incubated at 37oC, under described hereinafter. Bacterial cells were cultivated in anaerobiosis (90% N2 + 10% CO2) for 48 hours. F. peptone yeast extract broth (Difco, Rochester, NY, nucleatum ATCC 10953 was used as control. USA) with glucose (1%) in anaerobiosis, at 370C for 48 2. PLANT INFUSIONS hours. Aliquots of 0,1 ml were transferred to Eppendorf Aqueous extracts of mint (Mentha piperita), mate tubes and rinsed three times in PBS, pH 7,2, by (Ilex paraguayensis) , black tea (Camelia sinensis), centrifuging at 3000xg per 8 minutes. Then PBS was Arch Health Invest 2(1) 2013 Arch Health Invest (2013) 2(1): 3-9 ISSN 2317-3009 removed and 1,5ml of plant infusion was added to the It was considered that inhibition of the inoculum (107 CFU) and incubated at 370C in hemagglutination occurred when the hemagglutination anaerobiosis. In the tests, phosphate buffered saline title of the microorganism was reduced to at least 50% solution was used as control. in the in the presence of plant infusion, in comparison to After 30 min. aliquots of 0,1ml of these mixtures control (PBS). were removed and submitted to serial ten-fold dilutions RESULTS in PBS and inoculated onto brain heart infusion (BHI) The values of higher inhibitory dilutions of plant agar plates supplemented with yeast extract (0.5%) and infusions on oral F. nucleatum are presented in Table 1, defibrinated horse blood (5%). The plates were whereas Table 2 showed the contact inhibitory activity o incubated at 37 C in anaerobiosis (90% N2 + 10% CO2), of these infusions on targeted anaerobes. at 37ºC, for 72 hours. The results were expressed as the plant infusion that reduced 90% of the microbial Table 1- Susceptibility of 32 isolates of Fusobacterium nucleatum to different plant infusions. inoculum, in relation of control (PBS). Higher dilution with antimicrobial activity 4. INHIBITION OF HEMAGGLUTINATION Plant extract 1RANGE 2MDI 3MDI This test was performed through methodology 50 90 Boldo (P. boldus) 1/4 - > 1a 1/2 1 6 described by Falker Jr., Hawley . The tests was carried Camomile (M. chamomilla) 1/4 - > 1d 1/2 1 out on microtitration plates, using human blood (type A, Black tea (C. sinensis) 1/4 -1 1/4 1 B, O and AB with the corresponding RH-positive and Green tea (Variety Banchá) 1/8 -1 1/8 1 RH-negative) collected in Alsever solution at 10%24. Balm mint (M. officinalis) 1/4 - > 1 1/2 1 The bacterial cells were cultivated in BHI broth Aniseed (P. anixem) 1/4 - > 1 1/2 1 Mint (M. piperita) 1/4 - > 1 1/4 1 supplemented with yeast extract (0.5%) for 48 hours. Mate (I. paraguayensis) 1/8 -1 1/8 1 The cells were then submitted to centrifugation at 3000xg per 8 minutes, in PBS, and rinsed three times to These data evidenced a significant heterogeneity eliminated residues of the culture medium and among clinical isolates of fusobacteria. The aqueous resuspended using plant infusions until reaching extracts of black tea, green tea and mate were able to 8 concentration of 10 CFU/ml. The erythrocytes were inhibit all tested strains. Only the infusions of black tea rinsed twice by centrifugation at 600xg per 5 minutes in and green tea (C. sinensis) and mate (I. paraguayensis) PBS and resuspended in plant infusions until reaching presented bactericide effect after 2 hours in contact with the concentration of 1%. the fusobacteria. Initially 50µl of the bacterial suspension was Table 2- Contact inhibitory activity of plant infusions on added to the microplates followed by serial dilutions on oral Fusobacterium nucleatum base 2, using plant infusion in the preparation of serial Isolates ( % ) Plant extract dilutions. This was followed by the addition of 50µl of Susceptible Resistant the erythrocyte suspension at each dilution. The Boldo (P. boldus) 25 (78.1) 7 (21.9) mixtures on the microplates were homogenized slightly Camomile (M. chamomilla) 27 (84.4) 5 (15.6) Black tea (C. sinensis) 32 (100.0) 0 (0.0) for 1 minute and maintained at 37ºC for 30 min., and at Green tea (variety Banchá) 32 (100.0) 0 (0.0) room temperature for 2 hours, the title of Balm mint (M. officinalis) 27 (84.4) 5 (15.6) hemagglutination being then defined and evaluated as Aniseed (P.