Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis

Danuta Mazurel; Miguel Carda-Diéguez; Thomas Langenburg; Miglė Žiemytė; William Johnston; Carlos Palazón Martínez; Fernando Albalat; Carmen Llena; Nezar Al-Hebshi; Shauna Culshaw; Alex Mira; Bob T. Rosier

doi:10.1038/s41522-023-00406-3

Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis

Danuta Mazurel, Miguel Carda-Diéguez, Thomas Langenburg, Miglė Žiemytė, William Johnston, Carlos Palazón Martínez, Fernando Albalat, Carmen Llena, Nezar Al-Hebshi, Shauna Culshaw, Alex Mira^*, Bob T. Rosier^*

^*Corresponding author for this work

Preventive Dentistry

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.

Original language	English
Article number	40
Pages (from-to)	1-12
Number of pages	12
Journal	npj Biofilms and Microbiomes
Volume	9
DOIs	https://doi.org/10.1038/s41522-023-00406-3
Publication status	Published - 17 Jun 2023

Bibliographical note

Funding Information:
We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R.

Funding Information:
We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R.

Publisher Copyright:
© 2023, The Author(s).

Funding

We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R. We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R.

Funders	Funder number
Centro Periodontal de Valencia
Department of Microbiology
Forsyth Institute
Lluis Alcanyis Foundation
Family Process Institute
Ministerio de Ciencia, Innovación y Universidades	RTI2018-102032-B-I00
University of Glasgow
Universitat de València
European Regional Development Fund
Agència Valenciana de la Innovació	INNVAL20/19/006

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Mazurel, D., Carda-Diéguez, M., Langenburg, T., Žiemytė, M., Johnston, W., Martínez, C. P., Albalat, F., Llena, C., Al-Hebshi, N., Culshaw, S., Mira, A., & Rosier, B. T. (2023). Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis. npj Biofilms and Microbiomes, 9, 1-12. Article 40. https://doi.org/10.1038/s41522-023-00406-3

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title = "Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis",

abstract = "A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.",

author = "Danuta Mazurel and Miguel Carda-Di{\'e}guez and Thomas Langenburg and Miglė {\v Z}iemytė and William Johnston and Mart{\'i}nez, {Carlos Palaz{\'o}n} and Fernando Albalat and Carmen Llena and Nezar Al-Hebshi and Shauna Culshaw and Alex Mira and Rosier, {Bob T.}",

note = "Funding Information: We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R. Funding Information: We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R. Publisher Copyright: {\textcopyright} 2023, The Author(s).",

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doi = "10.1038/s41522-023-00406-3",

language = "English",

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Mazurel, D, Carda-Diéguez, M, Langenburg, T, Žiemytė, M, Johnston, W, Martínez, CP, Albalat, F, Llena, C, Al-Hebshi, N, Culshaw, S, Mira, A & Rosier, BT 2023, 'Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis', npj Biofilms and Microbiomes, vol. 9, 40, pp. 1-12. https://doi.org/10.1038/s41522-023-00406-3

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T1 - Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis

AU - Mazurel, Danuta

AU - Carda-Diéguez, Miguel

AU - Langenburg, Thomas

AU - Žiemytė, Miglė

AU - Johnston, William

AU - Martínez, Carlos Palazón

AU - Albalat, Fernando

AU - Llena, Carmen

AU - Al-Hebshi, Nezar

AU - Culshaw, Shauna

AU - Mira, Alex

AU - Rosier, Bob T.

N1 - Funding Information: We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R. Funding Information: We would like to thank the staff and patients of the Lluis Alcanyis Foundation dental clinic of the University of Valencia (Valencia, Spain) and the private clinic Centro Periodontal de Valencia (Valencia, Spain) for their help and participation in this study. We are also very grateful to Dr. Tsute Chen (Department of Microbiology, Forsyth Institute, Cambridge, United States of America) for his help with the SMDI score analysis and to Prof. Gordon Ramage (Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland) for his help with setting up the 10-species biofilm model. A.M. was supported by a grant from the European Regional Development Fund and Spanish Ministry of Science, Innovation and Universities with the reference RTI2018-102032-B-I00, as well as a grant from the Valencian Innovation Agency with the reference INNVAL20/19/006. BR was supported by a FPI fellowship from the Spanish Ministry of Science, Innovation and Universities with the reference Bio2015-68711-R. Publisher Copyright: © 2023, The Author(s).

PY - 2023/6/17

Y1 - 2023/6/17

N2 - A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.

AB - A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.

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Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis

Abstract

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