Revista de Odontologia da UNESP
https://revodontolunesp.com.br/article/doi/10.1590/1807-2577.03323
Revista de Odontologia da UNESP
Original Article

Establishment of a saliva donor selection for in vitro biofilm growth

Seleção de doadores de saliva para crescimento de biofilme in vitro

Thalita MENDES; Luciana Solera SALES; Marcelle DANELON; Fernanda Lourenção BRIGHENTI

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Abstract

Introdução: O emprego de biofilmes polimicrobianos, utilizando a saliva como inóculo, é um modelo promissor para o estudo de biofilmes cariogênicos in vitro. Entretanto, ainda não existe uma padronização para seleção de doadores de saliva.

Objetivo: O objetivo deste estudo foi estabelecer uma metodologia para seleção de doadores de saliva utilizando fatores salivares microbianos e características in vitro do biofilme.

Material e método: Para doação de saliva foram selecionados vinte voluntários. Os voluntários permaneceram 24 horas sem escovar os dentes e ficaram em jejum por 2 horas antes da coleta da saliva. Foram avaliados os seguintes parâmetros: viabilidade das bactérias anaeróbias totais e mutans streptococci; concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) da clorexidina; capacidade de formação de biofilme por meio da biomassa; e a suscetibilidade dos biofilmes à clorexidina.

Resultado: A viabilidade bacteriana da saliva, a capacidade de formação de biofilme e a suscetibilidade do biofilme à clorexidina foram apresentadas como média e intervalo de confiança (95%). A diferença entre a viabilidade do biofilme (mutans streptococci e bactérias totais) após tratamento com NaCl 0,9% e diacetato de clorexidina 0,2% foi comparada pelo teste t de Student com nível de significância estabelecido em 5%. A viabilidade total de bactérias anaeróbias (mediana) foi de 7,28 log 1+UFC/mL (unidades formadoras de colônia/mL). A viabilidade dos mutans streptococci na saliva apresentou mediana de 5,47 log 1+UFC/mL. Para capacidade de formação de biofilme a mediana da biomassa foi de 0,1172 A570.

Conclusão: O tratamento com clorexidina reduziu significativamente os mutans streptococci e a viabilidade total das bactérias. A metodologia para seleção do doador de saliva foi estabelecida com sucesso.

Keywords

Biofilme, biomassa, clorexidina, viabilidade microbiana, doador de saliva

Resumo

Introduction: The utilization of polymicrobial biofilms, with saliva as an inoculum, represents a promising model for in vitro studies on cariogenic biofilms. However, there is still no standardization for selecting saliva donors.

Objective: The aim of this study is to establish a methodology for the selection of saliva donors using microbial salivary factors and in vitro biofilm characteristics.

Material and method: For saliva donation, twenty volunteers were selected. Volunteers remained 24 h without brushing their teeth and fasted for 2 h before saliva collection. The following parameters were evaluated: total anaerobic bacteria and mutans streptococci viability; minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) of chlorhexidine; biofilm forming capacity by biomass assessment; and the susceptibility of biofilms to chlorhexidine.

Result: Saliva bacterial viability, biofilm forming capacity and biofilm susceptibility to chlorhexidine were presented as mean and confidence interval (95%). The difference between biofilm (mutans streptococci and Total bacteria) viability after treatment with NaCl 0.9% and 0.2% chlorhexidine diacetate was compared using the Student t-test with a significance level established at 5%. Total anaerobic bacteria viability (median) was 7.28 log 1+CFU/mL (colony forming units/ mL). Mutans streptococci viability in the saliva showed a median of 5.47 log 1+CFU/mL. Biofilm forming capacity showed that biomass had a median of 0.1172 A570.

Conclusion: Treatment with chlorhexidine significantly reduced mutans streptococci and total bacteria viability. The methodology for the selection of the saliva donor was successfully established.
 

Palavras-chave

Biofilm, biomass, chlorhexidine, microbial viability, saliva donor

References

1 Arweiler NB, Netuschil L. The oral microbiota. Adv Exp Med Biol. 2016;902:45-60. http://dx.doi.org/10.1007/978-3-319-31248-4_4. PMid:27161350.

2 Signori C, van de Sande FH, Maske TT, de Oliveira EF, Cenci MS. Influence of the inoculum source on the cariogenicity of in vitro microcosm biofilms. Caries Res. 2016;50(2):97-103.; http://dx.doi.org/10.1159/000443537. PMid:26919718.

3 Edlund A, Yang Y, Hall AP, Guo L, Lux R, He X, et al. An in vitro biofilm model system maintaining a highly reproducible species and metabolic diversity approaching that of the human oral microbiome. Microbiome. 2013 Oct;1(1):25. http://dx.doi.org/10.1186/2049-2618-1-25. PMid:24451062.

4 Liljemark WF, Bloomquist CG, Reilly BE, Bernards CJ, Townsend DW, Pennock AT, et al. Growth dynamics in a natural biofilm and its impact on oral disease management. Adv Dent Res. 1997 Apr;11(1):14-23. http://dx.doi.org/10.1177/08959374970110010501. PMid:9524438.

5 Blanc V, Isabal S, Sánchez MC, Llama-Palacios A, Herrera D, Sanz M, et al. Characterization and application of a flow system for in vitro multispecies oral biofilm formation. J Periodontal Res. 2014 Jun;49(3):323-32. http://dx.doi.org/10.1111/jre.12110. PMid:23815431.

6 Kutsch VK. Dental caries: an updated medical model of risk assessment. J Prosthet Dent. 2014 Apr;111(4):280-5. http://dx.doi.org/10.1016/j.prosdent.2013.07.014. PMid:24331852.

7 Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res. 2008;42(6):409-18. http://dx.doi.org/10.1159/000159604. PMid:18832827.

8 Farias AL, Meneguin AB, Silva Barud H, Brighenti FL. The role of sodium alginate and gellan gum in the design of new drug delivery systems intended for antibiofilm activity of morin. Int J Biol Macromol. 2020 Nov;162:1944-58. http://dx.doi.org/10.1016/j.ijbiomac.2020.08.078. PMid:32791274.

9 Matos BM, Brighenti FL, Do T, Beighton D, Koga-Ito CY. Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans. Clin Oral Investig. 2017 Jun;21(5):1769-76. http://dx.doi.org/10.1007/s00784-016-1958-1. PMid:27660160.

10 Valdez RM, Dos Santos VR, Caiaffa KS, Danelon M, Arthur RA, Negrini TC, et al. Comparative in vitro investigation of the cariogenic potential of bifidobacteria. Arch Oral Biol. 2016 Nov;71:97-103. http://dx.doi.org/10.1016/j.archoralbio.2016.07.005. PMid:27475723.

11 Braga AS, Girotti LD, Melo Simas LL, Pires JG, Pelá VT, Buzalaf MAR, et al. Effect of commercial herbal toothpastes and mouth rinses on the prevention of enamel demineralization using a microcosm biofilm model. Biofouling. 2019 Aug;35(7):796-804. http://dx.doi.org/10.1080/08927014.2019.1662897. PMid:31514534.

12 Sissons CH. Artificial dental plaque biofilm model systems. Adv Dent Res. 1997 Apr;11(1):110-26. http://dx.doi.org/10.1177/08959374970110010201. PMid:9524448.

13 Maske TT, van de Sande FH, Arthur RA, Huysmans MCDNJM, Cenci MS. In vitro biofilm models to study dental caries: a systematic review. Biofouling. 2017 Sep;33(8):661-75. http://dx.doi.org/10.1080/08927014.2017.1354248. PMid:28792234.

14 Souza BM, Fernandes Neto C, Salomão PMA, Vasconcelos LRSM, Andrade FB, Magalhães AC. Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel. J Appl Oral Sci. 2018;26(0):e20170304. http://dx.doi.org/10.1590/1678-7757-2017-0304. PMid:29489933.

15 Viana CS, Maske TT, Signori C, Van de Sande FH, Oliveira EF, Cenci MS. Influence of caries activity and number of saliva donors: mineral and microbiological responses in a microcosm biofilm model. J Appl Oral Sci. 2021 Sep;29:e20200778. http://dx.doi.org/10.1590/1678-7757-2020-0778. PMid:34495103.

16 Maske TT, Brauner KV, Nakanishi L, Arthur RA, van de Sande FH, Cenci MS. An in vitro dynamic microcosm biofilm model for caries lesion development and antimicrobial dose-response studies. Biofouling. 2016;32(3):339-48. http://dx.doi.org/10.1080/08927014.2015.1130824. PMid:26905384.

17 McBain AJ, Sissons C, Ledder RG, Sreenivasan PK, De Vizio W, Gilbert P. Development and characterization of a simple perfused oral microcosm. J Appl Microbiol. 2005;98(3):624-34. http://dx.doi.org/10.1111/j.1365-2672.2004.02483.x. PMid:15715865.

18 Feio M, Sapeta P. Xerostomia em cuidados paliativos. Acta Med Port. 2005;18(6):459-65. PMid:16684486.

19 van de Sande FH, Azevedo MS, Lund RG, Huysmans MC, Cenci MS. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies. Biofouling. 2011 Oct;27(9):1057-63. http://dx.doi.org/10.1080/08927014.2011.625473. PMid:22044385.

20 Exterkate RA, Crielaard W, Ten Cate JM. Different response to amine fluoride by Streptococcus mutans and polymicrobial biofilms in a novel high-throughput active attachment model. Caries Res. 2010;44(4):372-9. http://dx.doi.org/10.1159/000316541. PMid:20668379.

21 Engelkirk PG, Duben-Engelkirk JL, Dowell VR. Principles and practice of clinical anaerobic bacteriology. Belmont: Star Publishing Company; 1992.

22 Gold OG, Jordan HV, van Houte J. A selective medium for Streptococcus mutans. Arch Oral Biol. 1973 Nov;18(11):1357-64. http://dx.doi.org/10.1016/0003-9969(73)90109-X. PMid:4518755.

23 Eldeniz AU, Hadimli HH, Ataoglu H, Orstavik D. Antibacterial effect of selected root-end filling materials. J Endod. 2006 Apr;32(4):345-9. http://dx.doi.org/10.1016/j.joen.2005.09.009. PMid:16554209.

24 Albuquerque YE, Danelon M, Salvador MJ, Koga-Ito CY, Botazzo Delbem AC, Ramirez-Rueda RY, et al. Mouthwash containing Croton doctoris essential oil: in vitro study using a validated model of caries induction. Future Microbiol. 2018 May;13(6):631-43. http://dx.doi.org/10.2217/fmb-2017-0209. PMid:29771131.

25 Oliveira RVD, Bonafé FSS, Spolidorio DMP, Koga-Ito CY, Farias AL, Kirker KR, et al. Streptococcus mutans and Actinomyces naeslundii interaction in dual-species biofilm. Microorganisms. 2020 Jan;8(2):194. http://dx.doi.org/10.3390/microorganisms8020194. PMid:32023892.

26 Fernandez Y, Mostajo M, Exterkate RAM, Buijs MJ, Crielaard W, Zaura E. Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro. Clin Oral Investig. 2017 May;21(4):1221-30. http://dx.doi.org/10.1007/s00784-016-1876-2. PMid:27337976.

27 Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends Microbiol. 2005 Dec;13(12):589-95. http://dx.doi.org/10.1016/j.tim.2005.09.006. PMid:16214341.

28 Filoche SK, Soma D, van Bekkum M, Sissons CH. Plaques from different individuals yield different microbiota responses to oral-antiseptic treatment. FEMS Immunol Med Microbiol. 2008 Oct;54(1):27-36. http://dx.doi.org/10.1111/j.1574-695X.2008.00443.x. PMid:18647353.

29 Filoche SK, Soma KJ, Sissons CH. Caries-related plaque microcosm biofilms developed in microplates. Oral Microbiol Immunol. 2007 Apr;22(2):73-9. http://dx.doi.org/10.1111/j.1399-302X.2007.00323.x. PMid:17311629.

30 Levine M, Ensom MH. Post hoc power analysis: an idea whose time has passed? Pharmacotherapy. 2001 Apr;21(4):405-9. http://dx.doi.org/10.1592/phco.21.5.405.34503. PMid:11310512.

31 di Stefano J. A confidence interval approach to data analysis. For Ecol Manage. 2004 Jan;187(2–3):173-83. http://dx.doi.org/10.1016/S0378-1127(03)00331-1.

32 Lim BS, Kim BH, Shon WJ, Ahn SJ. Effects of caries activity on compositions of mutans streptococci in saliva-induced biofilm formed on bracket materials. Materials (Basel). 2020 Oct;13(21):4764. http://dx.doi.org/10.3390/ma13214764. PMid:33114489.

33 Price RR, Viscount HB, Stanley MC, Leung KP. Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR. Biofouling. 2007;23(3-4):203-13. http://dx.doi.org/10.1080/08927010701251169. PMid:17653931.

34 Azevedo MS, van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res. 2011;45(6):510-7. http://dx.doi.org/10.1159/000331210. PMid:21967836.

35 Nasidze I, Li J, Quinque D, Tang K, Stoneking M. Global diversity in the human salivary microbiome. Genome Res. 2009 Apr;19(4):636-43. http://dx.doi.org/10.1101/gr.084616.108. PMid:19251737.

36 Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. 2012 Jun;486(7402):207-14. http://dx.doi.org/10.1038/nature11234. PMid:22699609.

37 Kistler JO, Pesaro M, Wade WG. Development and pyrosequencing analysis of an in-vitro oral biofilm model. BMC Microbiol. 2015 Feb;15(1):24. http://dx.doi.org/10.1186/s12866-015-0364-1. PMid:25880819.

38 Lamarque GCC, Méndez DAC, Gutierrez E, Dionisio EJ, Machado MAAM, Oliveira TM, et al. Could chlorhexidine be an adequate positive control for antimicrobial photodynamic therapy in- in vitro studies? Photodiagn Photodyn Ther. 2019 Mar;25:58-62. http://dx.doi.org/10.1016/j.pdpdt.2018.11.004. PMid:30399454.

39 Yousefi M, Parvaie P, Riahi SM. Salivary factors related to caries in pregnancy: a systematic review and meta-analysis. J Am Dent Assoc. 2020 Aug;151(8):576-588.e4. http://dx.doi.org/10.1016/j.aime.2020.04.021. PMid:32718487.

40 Mummolo S, Nota A, Albani F, Marchetti E, Gatto R, Marzo G, et al. Salivary levels of Streptococcus mutans and Lactobacilli and other salivary indices in patients wearing clear aligners versus fixed orthodontic appliances: An observational study. PLoS One. 2020 Apr;15(4):e0228798. http://dx.doi.org/10.1371/journal.pone.0228798. PMid:32330172.

41 Ryu M, Ueda T, Saito T, Yasui M, Ishihara K, Sakurai K. Oral environmental factors affecting number of microbes in saliva of complete denture wearers. J Oral Rehabil. 2010 Mar;37(3):194-201. http://dx.doi.org/10.1111/j.1365-2842.2009.02042.x. PMid:20050985.
 


Submitted date:
11/01/2023

Accepted date:
11/01/2023

6569f66fa953950440347382 rou Articles
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