Abstract
Objectives
This in situ study aims to evaluate the effects of chlorhexidine (CHX) mouth rinsing on biofilm formation and moreover on the disruption of existing mature dental biofilms.
Methods
Biofilms were formed in situ by five volunteers on bovine enamel specimens fixed to individual acrylic splints. For biofilm formation analysis, the volunteers intraorally exposed the splint for 48 h. Mouth rinsing using 10 ml of 0.2% CHX or water as control was performed for 30 s every 12 h. For analysis of biofilm disruption, the biofilm was formed on enamel specimens for 48 h. Then, the first CHX rinse was carried out. A second rinse followed after an additional 12 h, again for 30 s using 10 ml of 0.2% CHX. Biofilm vitality was imaged by fluorescence microscopy after vital fluorescence staining. Additionally, the ultrastructure of the biofilm was examined by transmission electron microscopy.
Results
Rinses with 0.2% CHX significantly reduced biofilm formation on enamel. Both biofilm colonization and vitality were dramatically impaired. Moreover, a considerable biofilm disruption induced by the CHX rinses was observed. Remarkably, a single application of CHX to a 48-h mature biofilm causes biofilm ultrastructure alterations and induces a substantial reduction in biofilm thickness and bacterial vitality.
Conclusions
CHX mouth rinses induced a significant inhibition of biofilm formation on native enamel. Furthermore, an important biofilm disrupting effect under in situ conditions was detected.
Clinical Relevance: CHX rinses could be used as a short-term treatment protocol for biofilm management focused on patients unable to reach adequate oral hygiene.
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References
Zaura E, Keijser BJ, Huse SM, Crielaard W (2009) Defining the healthy “core microbiome” of oral microbial communities. BMC Microbiol 9:259. https://doi.org/10.1186/1471-2180-9-259
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43(11):5721–5732. https://doi.org/10.1128/JCM.43.11.5721-5732.2005
Hannig M (1999) Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period. Clin Oral Investig 3(2):88–95. https://doi.org/10.1007/s007840050084
Socransky SS (2000) Haffajee AD (2002) Dental biofilms: difficult therapeutic targets. Periodontol 28:12–55. https://doi.org/10.1034/j.1600-0757.2002.280102.x
Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, Arweiler NB (2005) Effect of two antimicrobial agents on early in situ biofilm formation. J Clin Periodontol 32(2):147–152. https://doi.org/10.1111/j.1600-051X.2005.00650.x
Arweiler NB, Hellwig E, Sculean A, Hein N, Auschill TM (2004) Individual vitality pattern of in situ dental biofilms at different locations in the oral cavity. Caries Res 38(5):442–447. https://doi.org/10.1159/000079625
Takahashi N, Nyvad B (2008) Caries ecology revisited: microbial dynamics and the caries process. Caries Res 42(6):409–418. https://doi.org/10.1159/000159604
Brading MG, Marsh PD (2003) The oral environment: the challenge for antimicrobials in oral care products. Int Dent J 53:353–362. https://doi.org/10.1111/j.1875-595X.2003.tb00910.x
Loe H, Theilade E, Jensen SB (1965) Experimental gingivitis in man. J Periodontol 36:177–187. https://doi.org/10.1902/jop.1965.36.3.177
Listgarten MA (1988) The role of dental plaque in gingivitis and periodontitis. J Clin Periodontol 15(8):485–487. https://doi.org/10.1111/j.1600-051X.1988.tb01019.x
Lindhe J, Koch G (1967) The effect of supervised oral hygiene on the gingivae of children. J Periodontal Res 2(3):215–220. https://doi.org/10.1111/j.1600-0765.1967.tb01892.x
Swallow JN, Davies DE, Hawkins SD (1969) Gingival disease prevalence in mentally handicapped adults. The effects of an oral hygiene programme. Br Dent J 127(8):376–379
Addy M (2000) Moran JM (1997) Clinical indications for the use of chemical adjuncts to plaque control: chlorhexidine formulations. Periodontol 15:52–54. https://doi.org/10.1111/j.1600-0757.1997.tb00104.x
ten Cate JM, Marsh PD (1994) Procedures for establishing efficacy of antimicrobial agents for chemotherapeutic caries prevention. J Dent Res 73(3):695–703. https://doi.org/10.1177/00220345940730031601
Van Strydonck DAC, Slot DE, Velden U, Weijden F (2012) Effect of a chlorhexidine mouthrinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J Clin Periodontol 39:1042–1055. https://doi.org/10.1111/j.1600-051X.2012.01883.x
Addy M, Moran J (2008) Chemical supragingival plaque control. In: Lindhe J, Karring T, Lang NP (eds) Clinical periodontology and implant dentistry, 5rd edn. Blackwell Munksgaard, Oxford, pp 734–765
Lang N, Brecx MC (1986) Chlorhexidine digluconate-an agent for chemical plaque control and prevention of gingival inflammation. J Periodontal Res 21(s16):74–89. https://doi.org/10.1111/j.1600-0765.1986.tb01517.x
Gunsolley JC (2010) Clinical efficacy of antimicrobial mouthrinses. J Dent 38(Suppl 1):S6–S10. https://doi.org/10.1016/S0300-5712(10)70004-X
Lima KC, Fava LR, Siqueira JF (2001) Susceptibilities of Enterococcus faecalis biofilms to some antimicrobial medications. J Endod 27(10):616–619. https://doi.org/10.1097/00004770-200110000-00004
Brecx M, Theilade J (1984) Effect of chlorhexidine rinses on the morphology of early dental plaque formed on plastic film. J Clin Periodontol 11(9):553–564. https://doi.org/10.1111/j.1600-051X.1984.tb00908.x
Hannig M, Khanafer AK, Hoth-Hannig W, Al-Marrawi F, Acil Y (2005) Transmission electron microscopy comparison of methods for collecting in situ formed enamel pellicle. Clin Oral Investig 9(1):30–37. https://doi.org/10.1007/s00784-004-0284-1
Feng J, Cheng L, Zhou X, Xu HH, Weir MD, Meyer M, Maurer H, Li Q, Hannig M, Rupf S (2015) In situ antibiofilm effect of glass-ionomer cement containing dimethylaminododecyl methacrylate. Dent Mater 31(8):992–1002. https://doi.org/10.1016/j.dental.2015.05.005
Rupf S, Balkenhol M, Sahrhage TO, Baum A, Chromik JN, Ruppert K, Wissenbach DK, Maurer HH, Hannig M (2012) Biofilm inhibition by an experimental dental resin composite containing octenidine dihydrochloride. Dent Mater 28(9):974–984. https://doi.org/10.1016/j.dental.2012.04.034
García-Caballero L, Quintas V, Prada-López I, Seoane J, Donos N, Tomas I (2013) Chlorhexidine substantivity on salivary flora and plaque-like biofilm: an in situ model. PLoS One 8(12):e83522. https://doi.org/10.1371/journal.pone.0083522
Tómas I, García-Caballero L, Cousido MC, Limeres J, Alvarez M, Diz P (2009) Evaluation of chlorhexidine substantivity on salivary flora by epifluorescence microscopy. Oral Dis 15(6):428–433. https://doi.org/10.1111/j.1601-0825.2009.01570.x
Cousido MC, Tomas Carmona I, Garcia-Caballero L, Limeres J, Alvarez M, Diz P (2010) In vivo substantivity of 0.12% and 0.2% chlorhexidine mouthrinses on salivary bacteria. Clin Oral Investig 14(4):397–402. https://doi.org/10.1007/s00784-009-0320-2
Mathur S, Mathur T, Srivastava R, Khatri R (2011) Chlorhexidine: The gold standard in chemical plaque control. Natl J Physiol Pharm Pharmacol 1(2):45–50
Hugo WB, Longworth AR (1965) Cytological aspects of the mode of action of chlorhexidine diacetate. J Pharm Pharmacol 17:28–32. https://doi.org/10.1111/j.2042-7158.1965.tb07562.x
Hugo WB, Longworth AR (1966) The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic constituents, dehydrogenase activity and cell walls of Escherichia coli and Staphylococcus aureus. J Pharm Pharmacol 18(9):569–578. https://doi.org/10.1111/j.2042-7158.1966.tb07935.x
Gilbert P, Moore LE (2005) Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol 99(4):703–715. https://doi.org/10.1111/j.1365-2672.2005.02664.x
Chawner JA, Gilbert P (1989) A comparative study of the bactericidal and growth inhibitory activities of the bisbiguanides alexidine and chlorhexidine. J Appl Bacteriol 66(3):243–252. https://doi.org/10.1111/j.1365-2672.1989.tb02475.x
Vitkov L, Hermann A, Krautgartner WD, Herrmann M, Fuchs K, Klappacher M, Hannig M (2005) Chlorhexidine-induced ultrastructural alterations in oral biofilm. Microsc Res Tech 68(2):85–89. https://doi.org/10.1002/jemt.20238
de Souza-E-Silva CM, da Silva Ventura TM, de Pau L, la Silva C, de Lima LA, Buzalaf MAR (2017) Effect of gels containing chlorhexidine or epigallocatechin-3-gallate on the protein composition of the acquired enamel pellicle. Arch Oral Biol 82:92–98. https://doi.org/10.1016/j.archoralbio.2017.05.024
von Ohle C, Gieseke A, Nistico L, Decker EM, DeBeer D, Stoodley P (2010) Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine. Appl Environ Microbiol 76(7):2326–2334. https://doi.org/10.1128/AEM.02090-09
Zaura-Arite E, van Marle J, ten Cate JM (2001) Confocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. J Dent Res 80:1436–1440. https://doi.org/10.1177/00220345010800051001
Pratten J, Barnett P, Wilson M (1998) Composition and susceptibility to chlorhexidine of multispecies biofilms of oral bacteria. Appl Environ Microbiol 64(9):3515–3519
Acknowledgements
The authors thank Birgit Leis and Belinda König for technical assistance with the samples processing for TEM analysis.
Funding
This study has been funded by the German Research Foundation (DFG, SFB 1027).
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All procedures performed in studies involving human participants were approved by the Ethical Committee of Saarland Medical Association, Germany (238/03, 2016). The study was also in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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Martínez-Hernández, M., Reda, B. & Hannig, M. Chlorhexidine rinsing inhibits biofilm formation and causes biofilm disruption on dental enamel in situ. Clin Oral Invest 24, 3843–3853 (2020). https://doi.org/10.1007/s00784-020-03250-3
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DOI: https://doi.org/10.1007/s00784-020-03250-3