Effect of Fluoride on Probiotics Involved in Gut Health

Abstract

Introduction
Fluoride can prevent dental caries by inhibiting demineralisation and promoting remineralisation of teeth while affecting the physiology of oral microbiota, inhibiting cellular enzymes. However, the impact of fluoride on the gut microbiome is unclear. The gut microbiome is an ecosystem of probiotics and opportunistic microorganisms inhabiting the human body. Individual health is closely linked to the gut microbiome, which could be affected by fluoride exposure. Conversely, the responses of specific probiotics and non-probiotic gut strains towards various fluoridated environments remain unknown. Therefore, this research investigated the responses of probiotics and non-probiotic gut strains to fluoride.
Methods
A systematic review was carried out following the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) (Chapter 2). Bacteriostatic, bactericidal and growth parameters of probiotics and Escherichia coli strains in the presence of fluoride were determined following the turbidimetric method. Pharmacodynamics/pharmacokinetics were determined using the static model in a shake flask, and fluoride was measured using an ion-selective electrode. RNA extracted from the L. rhamnosus and E. coli strains was used for transcriptomic sequencing (Illumina sequencing) for gene expression analysis. Proteins extracted from L. rhamnosus and E. coli strains were used for Liquid Chromatography, Coupled Tandem Mass Spectrometry (LC-MS/MS) for protein expression analysis. Physiological response under fluoride was assessed using culturing techniques applied to probiotics and E. coli strains in Chapter 3. Gene and protein expressions of Lacticasiebacillus rhamnosus and E. coli strains were assessed using molecular techniques in Chapters 4 and 5.
Results
A systematic review of the available human and animal literature till 2024 suggested that in humans, high doses were potentially detrimental to the microbiome, whereas doses (≤ 2 mg/l NaF) had positive effects. However, in animals, the doses (≥50 mg/l NaF) were deemed to be risky in terms of gut microbial changes, whereas doses (≤25 mg/l NaF) had harmless effects. In vitro experiments completed within this thesis suggested that high fluoride concentrations of 2272 and 1136 mg/l inhibited probiotics' growth and, to some extent, E. coli as well. The strains grew well at low fluoride concentrations (36, 28, 18, 14, 9, 7, 4, 2,1, 0.4 and 0.2 mg/l F) where growth was comparable with the control. Moreover, a positive correlation was found between the bacterial growth rate and the percentage of fluoride reduction, which indicates that the resident gut flora can thrive in the fluoridated environment, but up to some limit. Molecular analysis revealed that the most significantly upregulated genes in E. coli after exposure to high fluoride were mainly enriched in pathways involved in energy metabolism, oxidative stress responses and chemotactic movements, whereas in Lacticasiebacillus rhamnosus, genes involved in ribosomal protein synthesis and biosynthesis of amino acids were upregulated, which are important for bacterial existence and growth. The significantly impacted proteins in E. coli were mainly involved in translation, carbohydrate transport and energy metabolism, transcription, defence mechanism, and amino acid transport. In Lacticasiebacillus rhamnosus, fluoride exposure significantly impacted ribosomal assembly, translation, transcription, DNA repair, amino acid transport and metabolism.
Discussion
The findings from the culturing experiments complement the previous observations of the dose and time response effects of fluoride on the growth of probiotics and non-probiotics gut strains, and also that fluoride has antimicrobial properties at high concentrations. This study also highlighted the importance of molecular mechanisms that enable gut bacteria to cope with stress. It revealed that exposure to high fluoride induced changes in genes and proteins. The observed changes affecting genes and proteins involved in energy metabolism and chemotactic movements in E. coli and translation and amino acid synthesis in LGG suggested the adaptive response enabling the bacteria to adapt and cope with fluoride stress. These findings also indicate cellular damage by fluoride, which delays cell division, the reason for reduced growth at high fluoride.

Date of Award	6 Dec 2025
Original language	English
Awarding Institution	Teesside University
Supervisor	Caroline Orr (Supervisor), Vida Zohoori (Supervisor) & Paul Dean (Supervisor)