Probiotic Modulation of Oral Microbiota

OBJECTIVES

This is a monocentric, prospective, cross-over, randomized, double-blinded study in which all participants will receive placebo and active probiotic treatment, 15 of which will first be treated with placebo and then will be given probiotics. The other 15 participants will first get probiotics and then, after a 3-week wash out period, the placebo.

The main objective of the study is to assess changes in salivary microbiota profiles and inflammatory markers following S. salivarius probiotic treatment. Our secondary objective is to identify correlations between specific salivary microbial taxa (subspecies to phylum levels) and inflammatory markers. Clinical outcome is not the focus of this study, although basic information about the oral health will be measured.

METHODOLOGY

BIOTICS-O (Burgerstein) probiotic that will be used is a commercial food supplement available over the counter in the pharmacy as blister packs of 30 lozenges containing 10^9 CFU of S. salivarius K12. Participants will let melt the lozenge after tooth brushing in the evening.

Placebo lozenges (inactivated probiotic) will have the same look, taste and smell as the active treatment lozenges, and they will be administered in the same way as the active treatment lozenges.

Metagenomic analysis of the microbiota:

Indexed paired-end metagenomic libraries will be prepared using DNA extracted from saliva and sequenced for 2x150 cycles on an Illumina NovaSeq 6000 instrument to generate 5-10 million read pairs per sample. Our standard metagenomic analysis pipeline (HUGE-MAP) will be used; it includes: (i) read quality filtering; (ii) removal of replicate sequences; (iii) removal of read pairs that match human genome sequence and, (iv) classification of read pairs with CLARK against the collection of NCBI reference and representative bacterial, archaeal and fungal genomes, as well as Latest RefSeq NCBI genomes of prophages and DNA virus families whose members may infect humans. Functional assignments i.e. profiling the presence/absence and abundance of microbial gene families and pathways will be performed using MG-RAST server. Bacterial abundance will be measured by qPCR and/or relative to the number of reads obtained from the spiked calibrator.

Cytokine measurements:

Salivary cytokines (IL-1β, IL-6, IL-8, TNF-alpha) analysis will be performed using a Salimetrcs Core Cytokine Panel - 4-plex.

OBJECTIVES

This is a monocentric, prospective, cross-over, randomized, double-blinded study in which all participants will receive placebo and active probiotic treatment, 15 of which will first be treated with placebo and then will be given probiotics. The other 15 participants will first get probiotics and then, after a 3-week wash out period, the placebo.

The main objective of the study is to assess changes in salivary microbiota profiles and inflammatory markers following S. salivarius probiotic treatment. Our secondary objective is to identify correlations between specific salivary microbial taxa (subspecies to phylum levels) and inflammatory markers. Clinical outcome is not the focus of this study, although basic information about the oral health will be measured.

METHODOLOGY

BIOTICS-O (Burgerstein) probiotic that will be used is a commercial food supplement available over the counter in the pharmacy as blister packs of 30 lozenges containing 10^9 CFU of S. salivarius K12. Participants will let melt the lozenge after tooth brushing in the evening.

Placebo lozenges (inactivated probiotic) will have the same look, taste and smell as the active treatment lozenges, and they will be administered in the same way as the active treatment lozenges.

Metagenomic analysis of the microbiota:

Indexed paired-end metagenomic libraries will be prepared using DNA extracted from saliva and sequenced for 2x150 cycles on an Illumina NovaSeq 6000 instrument to generate 5-10 million read pairs per sample. Our standard metagenomic analysis pipeline (HUGE-MAP) will be used; it includes: (i) read quality filtering; (ii) removal of replicate sequences; (iii) removal of read pairs that match human genome sequence and, (iv) classification of read pairs with CLARK against the collection of NCBI reference and representative bacterial, archaeal and fungal genomes, as well as Latest RefSeq NCBI genomes of prophages and DNA virus families whose members may infect humans. Functional assignments i.e. profiling the presence/absence and abundance of microbial gene families and pathways will be performed using MG-RAST server. Bacterial abundance will be measured by qPCR and/or relative to the number of reads obtained from the spiked calibrator.

Cytokine measurements:

Salivary cytokines (IL-1β, IL-6, IL-8, TNF-alpha) analysis will be performed using a Salimetrix Core Cytokine Panel - 4-plex..

Samplings will be performed at weeks 1, 4, 7, 10, 13, 16 and 19.

Inclusion criteria

• Informed Consent as documented by signature
• Good oral and general health

Exclusion criteria

• Women who are pregnant or breast feeding
• Intention to become pregnant during the course of the study
• Systemic disease
• Previous enrolment into the current study
• Clinically diagnosed severe oral lesions
• Use of antibiotics and topical oral probiotics within the 3 months preceding the study or during the study
• Dental treatments and use of oral disinfectants within 30 days preceding the study or during the study