This thesis investigates the acid tolerance (AT) of oral biofilms and how this property is affected by changes in the local environment, to further understand the phenotypical modifications of the bacterial communities that occur during caries development.

Paper I explored the effect of biofilm growth and presence of salivary proteins on the AT of five oral species, as well as their ability to induce an acid tolerance response (ATR). The results show that surface contact, presence of salivary proteins and sensing of environmental pH can contribute to high levels of AT.

In paper II, the AT, metabolic profile and microbial composition of plaque samples from children with oral health and children with severe caries were compared. Differences between the two groups were observed in organic acid production and species composition of the plaque samples, and significantly higher levels of AT was observed in plaque collected from caries active children.

In paper III, the effect of probiotic strains on the ATR of four oral species when grown in dual-species biofilms was investigated. The results showed that the presence of the probiotic strain L. reuteri PTA5289 was able to inhibit the ATR of oral species and led to a down-regulation of three key genes involved in acid stress tolerance.

The results of this thesis suggest that many species of the oral biofilm can obtain high levels of acid tolerance and that signals in the extracellular environment contribute to an increase and decrease of this property. Biofilm AT might therefore be a future focus in the development of new predictive biomarkers and therapeutic interventions for dental caries.

Avhandlingen undersöker orala biofilmers syratolerans (AT) och hur denna egenskap påverkas av ändringar i den omgivande miljön. Detta för att närmare första de fenotypiska förändringar som de orala bakterierna genomgår under kariesutveckling.

I delarbete I analyseras hur växt i biofilm och närvaro av salivproteiner påverkar 5 orala bakteriearters AT samt deras förmåga att utveckla en ATR. Resultaten av studien visar att adhesion till en yta, närvaro av salivprotein och pH-förändringar i den omgivande miljön kan bidra till höga nivåer av syratolerans hos orala bakterier.

I delarbete II undersöks kliniska plackprover från barn med hög kariesaktivitet med plackprover från barn utan tecken på kariesaktivitet, vad gäller bakteriernas AT, kolhydratmetabolism och inbördes komposition. Skillnader i produktionen av organiska syror och bakteriell komposition kunde ses mellan de två grupperna och signifikant högre nivåer av AT visades i plackprover från barnen med hög kariesaktivitet.

I delarbete III, undersöktes effekten av probiotiska bakterier på orala arters AT. Resultaten av studien visar att närvaro av den probiotiska arten L. reuteri PTA5289 inhiberade utvecklingen av en ATR hos de orala arterna och ledde till en nedreglering av tre gener involverade i ATR.

Resultaten i denna avhandling indikerar att många orala arter kan uppnå höga nivåer av syratolerans och att denna egenskap kan påverkas av signaler i den omgivande miljön. AT hos orala biofilmer kan därför vara ett framtida fokusområde vid utvecklingen av nya prediktiva biomarkörer och terapeutiska interventioner för karies.

Introduction: During development of dental caries, oral biofilms undergo changes in microbial composition and phenotypical traits. The aim of this study was to compare the acid tolerance (AT) of plaque from two groups of children: one with severe caries (CA) and one with no caries experience (CF) and to correlate this to the microbial composition and metabolic profile of the biofilms.

Methods: Dental plaque samples from 20 children (2–5 years) in each group were studied. The AT was analyzed by viability assessment after exposure to an acid challenge (pH 3.5), using LIVE/DEAD® BacLight™ stain and confocal microscopy. Levels of acid tolerance (AT) were evaluated using a scoring system ranging from 1 (no/low AT), to 5 (high/all AT). Metabolic profiles were investigated following a 20 mM glucose pulse for one hour through Nuclear Magnetic Resonance (NMR). Microbial composition was characterized by 16S rRNA Illumina sequencing.

Results: The mean AT score of the CA group (4.1) was significantly higher than that of the CF group (2.6, p < 0.05). When comparing the end-products of glucose metabolism detected after a glucose-pulse, the CA samples showed a significantly higher lactate to acetate, lactate to formate, lactate to succinate and lactate to ethanol ratio than the CF samples (p < 0.05). The bacterial characterization of the samples revealed 25 species significantly more abundant in the CA samples, including species of Streptococcus, Prevotella, Leptotrichia and Veillonella (p < 0.05).

Discussion: Our results show that AT in pooled plaque from the oral cavity of children with severe caries is increased compared to that in healthy subjects and that this can be related to differences in the metabolic activity and microbial composition of the biofilms. Thus, the overall phenotype of dental plaque appears to be a promising indicator of the caries status of individuals. However, longitudinal studies investigating how the AT changes over time in relation to caries development are needed before plaque AT could be considered as a prediction method for the development of dental caries.

Probiotic bacteria show promising results in prevention of the biofilm-mediated disease caries, but the mechanisms are not fully understood. The acid tolerance response (ATR) allows biofilm bacteria to survive and metabolize at low pH resulting from microbial carbohydrate fermentation. We have studied the effect of probiotic strains: Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus on ATR induction in common oral bacteria. Communities of L. reuteri ATCC PTA5289 and Streptoccus gordonii, Streptococcus oralis, Streptococcus mutans or Actinomyces naeslundii in the initial stages of biofilm formation were exposed to pH 5.5 to allow ATR induction, followed by a low pH challenge. Acid tolerance was evaluated as viable cells after staining with LIVE/ DEAD & REG;BacLightTM. The presence of L. reuteri ATCC PTA5289 caused a significant reduction in acid tolerance in all strains except S. oralis. When S. mutans was used as a model organism to study the effects of additional probiotic strains (L. reuteri SD2112, L. reuteri DSM17938 or L. rhamnosus GG) as well as L. reuteri ATCC PTA5289 supernatant on ATR development, neither the other probiotic strains nor supernatants showed any effect. The presence of L. reuteri ATCC PTA5289 during ATR induction led to down-regulation of three key genes involved in tolerance of acid stress (luxS, brpA and ldh) in Streptococci. These data suggest that live cells of probiotic L. reuteri ATCC PTA5289 can interfere with ATR development in common oral bacteria and specific strains of L. reuteri may thus have a role in caries prevention by inhibiting development of an acid-tolerant biofilm microbiota.

BACKGROUND: In caries, low pH drives selection and enrichment of acidogenic and aciduric bacteria in oral biofilms, and development of acid tolerance in early colonizers is thought to play a key role in this shift. Since previous studies have focussed on planktonic cells, the effect of biofilm growth as well as the role of a salivary pellicle on this process is largely unknown. We explored acid tolerance and acid tolerance response (ATR) induction in biofilm cells of both clinical and laboratory strains of three oral streptococcal species (Streptococcus gordonii, Streptococcus oralis and Streptococcus mutans) as well as two oral species of Actinomyces (A. naeslundii and A. odontolyticus) and examined the role of salivary proteins in acid tolerance development.

METHODS: Biofilms were formed on surfaces in Ibidi® mini flow cells with or without a coating of salivary proteins and acid tolerance assessed by exposing them to a challenge known to kill non-acid tolerant cells (pH 3.5 for 30 min) followed by staining with LIVE/DEAD BacLight and confocal scanning laser microscopy. The ability to induce an ATR was assessed by exposing the biofilms to an adaptation pH (pH 5.5) for 2 hours prior to the low pH challenge.

RESULTS: Biofilm formation significantly increased acid tolerance in all the clinical streptococcal strains (P < 0.05) whereas the laboratory strains varied in their response. In biofilms, S. oralis was much more acid tolerant than S. gordonii or S. mutans. A. naeslundii showed a significant increase in acid tolerance in biofilms compared to planktonic cells (P < 0.001) which was not seen for A. odontolyticus. All strains except S. oralis induced an ATR after pre-exposure to pH 5.5 (P < 0.05). The presence of a salivary pellicle enhanced both acid tolerance development and ATR induction in S. gordonii biofilms (P < 0.05) but did not affect the other bacteria to the same extent.

CONCLUSIONS: These findings suggest that factors such as surface contact, the presence of a salivary pellicle and sensing of environmental pH can contribute to the development of high levels of acid tolerance amongst early colonizers in oral biofilms which may be important in the initiation of caries.

The aim of this study was to compare the concentration of salivary immunoglobulin A (IgA) and the selected interleukins (IL)-1β, IL-6, IL-8 and IL-10 in young individuals with presence and non-presence of Lactobacillus reuteri in saliva after a three-week intervention with probiotic lozenges. The study group consisted of 47 healthy individuals aged 18-32 years with no clinical signs of oral inflammation. In a randomised, double-blind, placebo-controlled, cross-over trial participants ingested two lozenges per day containing two strains of the probiotic bacterium L. reuteri or placebo lozenges. The intervention and wash-out periods were three weeks. Stimulated and unstimulated whole saliva was collected at baseline and immediately after termination of the intervention periods. The samples were analysed for total protein, salivary IgA and selected cytokines. In this extended analysis, data were collected by analysing baseline and follow-up saliva samples related to ingestion of the probiotic lozenges for the presence of L. reuteri through DNA-extraction, PCR-amplification and gel-electrophoresis. At baseline, 27% of the individuals displayed presence of L. reuteri and 42% were positive immediately after the three-week probiotic intervention. Individuals with presence of L. reuteri in saliva had significantly higher (P<0.05) concentrations of salivary IgA and %IgA/protein at the termination of the probiotic intake compared with non-presence. No differences in the cytokine levels were observed. In conclusion, detectable levels of L. reuteri in saliva coincided with higher concentrations of salivary IgA and %IgA/protein in stimulated whole saliva after the three-week daily intake of probiotic lozenges. Our findings suggest that monitoring the presence of probiotic candidates in the oral environment is important to interpret and understand their possible immune-modulating role in maintaining oral health.