Effects of Chlorhexidine mouthwash on the oral microbiome

Raul Bescos, Ann Ashworth, Craig Cutler, Zoe L Brookes, Louise Belfield, Ana Rodiles, Patricia Casas-Agustench, Garry Farnham, Luke Liddle, Mia Burleigh, Desley White, Chris Easton, Mary Hickson, Raul Bescos, Ann Ashworth, Craig Cutler, Zoe L Brookes, Louise Belfield, Ana Rodiles, Patricia Casas-Agustench, Garry Farnham, Luke Liddle, Mia Burleigh, Desley White, Chris Easton, Mary Hickson

Abstract

Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Absolute abundance and Linear Discriminant Analysis (LDA) scores in the main bacterial phyla (A,B), genus (D,E) and family (F,G) after a 7-day treatment with placebo and chlorhexidine (CHX). Figure C shows the ratio between the two main phyla (Firmicutes:Bacteroidetes) following each treatment. Operational Taxonomic Units (OTUs) with an asterisk were statistically significant (False Discovery Rate > 0.05).
Figure 2
Figure 2
Shannon’s index representing alpha-diversity (A) and Bray-Curtis index representing beta-diversity (B) (each dot represents an individual sample and ellipsis represents the 95% confidence regions for group) after a 7-day treatment with placebo and chlorhexidine (CHX).
Figure 3
Figure 3
Saliva pH (A), saliva buffering capacity (B) and concentration of salivary lactate (C), glucose (D), nitrite (F), nitrate (G), as well as the oral nitrate-reducing capacity of bacteria (ONRC) (E) and concentration of plasma nitrite (H) and nitrate (I) after a 7-day treatment with placebo and chlorhexidine (CHX).
Figure 4
Figure 4
Moderate degree and significant Pearson correlations (r > 0.40; P < 0.05) found between abundance of oral bacteria (Operational Taxonomic Units [OTUs] %) at phylum level and salivary markers after the placebo and chlorhexidine treatment. In the placebo condition, abundance of Proteobacteria was negatively correlated to the oral nitrate-reducing capacity of bacteria (ONRC) (A), and with lower levels of diastolic blood pressure (D). Abundance of Bacteroidetes was negatively associated with plasma nitrite (B), while abundance of Actinobacteria was positively correlated (E). Abundance of the phylum SR1 was positively associated with greater salivary pH. Following 7-day use of chlorhexidine, the abundance of Fusobacteria was correlated with greater concentration of glucose in saliva (F). Abundance of Actinobacteria was negatively correlated with saliva lactate (G), and abundance of Proteobacteria was also negatively correlated with saliva nitrite concentration (H).
Figure 5
Figure 5
Changes in systolic (SBP) and diastolic (DBP) blood pressure after 7-day use of placebo and chlorhexidine (CHX).

References

    1. Löe H, Rindom Schiøtt C. The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Period Res. 1970;5:79–83. doi: 10.1111/j.1600-0765.1970.tb00696.x.
    1. Afennich, F., Slot, D., Hossainian, N. & Van der Weijden, G. The effect of hexetidine mouthwash on the prevention of plaque and gingival inflammation: a systematic review. Int J Dent Hyg. 9 (2011).
    1. Gunsolley JC. Clinical efficacy of antimicrobial mouthrinses. J Dent. 2010;38:S6–S10. doi: 10.1016/S0300-5712(10)70004-X.
    1. James, P. et al. Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. Cochrane Database Syst Rev. (2017).
    1. Eick, S., Goltz, S., Nietzsche, S., Jentsch, H. & Pfister, W. Efficacy of chlorhexidine digluconate–containing formulations and other mouthrinses against periodontopathogenic microorganisms. Quintessence Int. 42 (2011).
    1. Fedorowicz, Z., Aljufairi, H., Nasser, M., Outhouse, T. L. & Pedrazzi, V. Mouthrinses for the treatment of halitosis. Cochrane Database Syst Rev. (2008).
    1. Tribble GD, et al. Frequency of Tongue Cleaning Impacts the Human Tongue Microbiome Composition and Enterosalivary Circulation of Nitrate. Front Cell Infect Microbiol. 2019;9:39–39. doi: 10.3389/fcimb.2019.00039.
    1. Kapil V, et al. Physiological role for nitrate-reducing oral bacteria in blood pressure control. Free Rad. Biol Med. 2013;55:93–100.
    1. Ashworth A, et al. Dietary intake of inorganic nitrate in vegetarians and omnivores and its impact on blood pressure, resting metabolic rate and the oral microbiome. Free Rad Biol Med. 2019;138:63–72. doi: 10.1016/j.freeradbiomed.2019.05.010.
    1. Govoni M, Jansson EA, Weitzberg E, Lundberg JO. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide. 2008;19:333–337. doi: 10.1016/j.niox.2008.08.003.
    1. Bondonno CP, et al. Antibacterial Mouthwash Blunts Oral Nitrate Reduction and Increases Blood Pressure in Treated Hypertensive Men and Women. Am J Hypert. 2015;28:572–575. doi: 10.1093/ajh/hpu192.
    1. Rosier BT, Marsh PD, Mira A. Resilience of the Oral Microbiota in Health: Mechanisms That Prevent Dysbiosis. J Dent Res. 2018;97:371–380. doi: 10.1177/0022034517742139.
    1. Sanz Mariano, Beighton David, Curtis Michael A., Cury Jaime A., Dige Irene, Dommisch Henrik, Ellwood Roger, Giacaman Rodrigo A., Herrera David, Herzberg Mark C., Könönen Eija, Marsh Philip D., Meyle Joerg, Mira Alex, Molina Ana, Mombelli Andrea, Quirynen Marc, Reynolds Eric C., Shapira Lior, Zaura Egija. Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease. Journal of Clinical Periodontology. 2017;44:S5–S11. doi: 10.1111/jcpe.12682.
    1. Chen H, Jiang W. Application of high-throughput sequencing in understanding human oral microbiome related with health and disease. Front Microbiol. 2014;5:508.
    1. Belardinelli PA, Morelatto RA, Benavidez TE, Baruzzi AM, Lopez de Blanc SA. Effect of two mouthwashes on salivary pH. Acta Odontol Latinoam. 2014;27:66–71.
    1. Andreadis G, Topitsoglou V, Kalfas S. Acidogenicity and acidurance of dental plaque and saliva sediment from adults in relation to caries activity and chlorhexidine exposure. J Oral Microbiol. 2015;7:26197. doi: 10.3402/jom.v7.26197.
    1. Lenander-Lumikari M, Loimaranta V. Saliva and dental caries. Adv Dent Res. 2000;14:40–47. doi: 10.1177/08959374000140010601.
    1. Baliga S, Muglikar S, Kale R. Salivary pH: A diagnostic biomarker. J Ind Soc Period. 2013;17:461–465. doi: 10.4103/0972-124X.118317.
    1. Portenier I, Waltimo T, Orstavik D, Haapasalo M. Killing of Enterococcus faecalis by MTAD and chlorhexidine digluconate with or without cetrimide in the presence or absence of dentine powder or BSA. J Endod. 2006;32:138–141. doi: 10.1016/j.joen.2005.10.027.
    1. Spijkervet FK, et al. Chlorhexidine inactivation by saliva. Oral Surg Oral Med Oral Pathol. 1990;69:444–449. doi: 10.1016/0030-4220(90)90377-5.
    1. Abouassi T, et al. Does human saliva decrease the antimicrobial activity of chlorhexidine against oral bacteria? BMC Res Notes. 2014;7:711. doi: 10.1186/1756-0500-7-711.
    1. Seethalakshmi C, Reddy RCJ, Asifa N, Prabhu S. Correlation of Salivary pH, Incidence of Dental Caries and Periodontal Status in Diabetes Mellitus Patients: A Cross-sectional Study. J Clin Diagn Res. 2016;10:ZC12–ZC14.
    1. Gomes KM, Duarte RS, de Freire Bastos MDC. Lantibiotics produced by Actinobacteria and their potential applications (a review) Microbiology. 2017;163:109–121. doi: 10.1099/mic.0.000397.
    1. Ouwehand, A. C. & Vesterlund, S. In Lactic Acid Bacteria: Microbiological and Functional Aspects Vol. 139 (eds Salminen, S., von Wright, A., & Ouwehand, A.) 375–396 (Marcel Dekker, Inc, 2004).
    1. De Soet J, Nyvad B, Kilian M. Strain–Related Acid Production by Oral Streptococci. Caries Res. 2000;34:486–490. doi: 10.1159/000016628.
    1. Brooks GA. The science and translation of lactate shuttle theory. Cell Metab. 2018;27:757–785. doi: 10.1016/j.cmet.2018.03.008.
    1. Kilian M, et al. The oral microbiome–an update for oral healthcare professionals. Br Dent J. 2016;221:657–666. doi: 10.1038/sj.bdj.2016.865.
    1. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–167. doi: 10.1038/nrd2466.
    1. Hyde ER, et al. Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis. PLoS One. 2014;9:e88645. doi: 10.1371/journal.pone.0088645.
    1. Allaker RP, Silva Mendez LS, Hardie JM, Benjamin N. Antimicrobial effect of acidified nitrite on periodontal bacteria. Oral Microbiol Immunol. 2001;16:253–256. doi: 10.1034/j.1399-302X.2001.160410.x.
    1. Dejam A, et al. Nitrite Infusion in Humans and Nonhuman Primates. Circulation. 2007;116:1821–1831. doi: 10.1161/CIRCULATIONAHA.107.712133.
    1. Montenegro MF, et al. Blood Pressure–Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor. Hypertension. 2017;69:23–31. doi: 10.1161/HYPERTENSIONAHA.116.08081.
    1. Sundqvist ML, Lundberg JO, Weitzberg E. Effects of antiseptic mouthwash on resting metabolic rate: A randomized, double-blind, crossover study. Nitric Oxide. 2016;61:38–44. doi: 10.1016/j.niox.2016.10.003.
    1. Kapil V, et al. Sex differences in the nitrate-nitrite-NO• pathway: Role of oral nitrate-reducing bacteria. Free Rad Biol Med. 2018;126:113–121. doi: 10.1016/j.freeradbiomed.2018.07.010.
    1. Deschepper, M., Waegeman, W., Eeckloo, K., Vogelaers, D. & Blot, S. Effects of chlorhexidine gluconate oral care on hospital mortality: a hospital-wide, observational cohort study. Int Care Med. 44 (2018).
    1. Liddle L, et al. Changes in body posture alter plasma nitrite but not nitrate concentration in humans. Nitric Oxide. 2018;72:59–65. doi: 10.1016/j.niox.2017.11.008.
    1. Segata N, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12:R60. doi: 10.1186/gb-2011-12-6-r60.
    1. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J Royal Statistical Soc. Series B (Methodological) 1995;57:289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x.
    1. Dhariwal A, et al. MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 2017;45:W180–W188. doi: 10.1093/nar/gkx295.

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