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.

Microorganisms are often located within surface-associated multicellular aggregates known as biofilms. The human gastrointestinal tract (GI-tract) carries a microbiota that is constantly affected by extraneous influences, and hence upholding and improving the microbial balance has increased the request for probiotics. Several Lactobacillus reuteri strains have probiotic properties and are used in food technology. In this study the growth characteristics of L. reuteri was investigated using the morphology and stability of L. reuteri ATCC PTA 5289 after encapsulation through spray drying. The bacterial cells were assessed and visualized by Scanning Electron Microscopy (SEM) and Light Microscopy (LM) as well as spectrophotometry. L. reuteri ATCC PTA 5289 showed stable growth on polystyrene surfaces and adherence was also observed on aluminium surfaces. SEM images demonstrated morphological changes of the bacteria that occurred during the spray drying. After spray drying the preparation showed 15 % viable cells. The survival percentage of the spray-dried end product after 4 weeks of storage was approximately 33 % when stored at 5 °C, compared to 5 % at 25 °C.

We investigated whether ingestion of probiotic bacteria could influence salivary IgA levels, specific anti-mutans streptococci IgA levels and specific antibodies towards the ingested probiotic bacterium. The study was a randomised, double-blind, placebo-controlled trial, where the test group (n = 11) received twice daily chewing of gum containing Lactobacillus reuteri (2 9 108 CFU per dose) and the control group (n = 12) received placebo. Resting saliva was collected before and after 12 weeks of treatment and 4 weeks after end of treatment. Total salivary IgA concentrations were measured by ELISA. Specific IgA reactivity was determined using a whole-cell ELISA. Results were expressed as % IgA per protein in saliva. The level of total IgA% per protein increased significantly between pretreatment levels (13.5%) and follow-up treatment levels (14.4%) within the test group only (P < 0.05). No changes were seen in the control group during the trial. The level of probiotic-reactive antibodies decreased significantly between pre- and post-treatment samples (from 12.2% to 9.0%, P < 0.05) in the test group. Similarly, the level of specific mutans streptococci antibodies decreased significantly between pre- and post-treatment samples (P < 0.05) in the test group only (for Streptococcus mutans from 20.1% to 15.0%; for Streptococcus sobrinus from 7.4% to 5.3%). Ingestion of probiotic bacteria might influence the adaptive immune response of the host.

Avsikten med studien var att undersöka förekomsten av Lactobacillus reuteri i saliv ef- ter användandet av ett probiotiskt kosttillskott innehållande L. reuteri samt fastställa om L. reuteri har effekt på plackindex (PlI) samt på den supra- och subgingivala mikrofloran. Studien var dubbelblind, randomiserad, placebokontrollerad och löpte över 12 veckor. Totalt deltog 23 friska individer randomiserade i test- respektive kontrollgrupp. I försöket testades placebotuggummi mot tuggummi med L. reuteri (ATCC 55730 och ATCC PTA 5289). Tuggummi togs 2 gånger per dag, morgon och kväll efter tandborstning. Startda- gen och efter 12 veckor samlades vilosaliv samt plack supra- och subgingivalt. Dessutom bedömdes plackindex enligt Silness & Löe på 4 indextänder mesialt och distalt. Efter ytterligare 4 veckor gjordes en uppföljning med prov på saliv och bedömning av plack på indextänderna. Mikrobiologisk analys utfördes med hjälp av checkerboard DNA-DNA hybridiseringsteknik samt odling på selektiva medier för bestämning av laktobacill- populationen. Totala laktobacillhalten i saliv ökade signifikant i båda grupperna (p<0,05) med en signifikant ökning av L. reuteri i testgruppen (p=0,008). Efter 16 veckor påträffades ingen L. reuteri i saliv. Kontrollgruppen visade en signifikant ökning av PII efter 12 veckor (p=0,023), medan testgruppen inte visade någon förändring. I båda grupperna regist- rerades en signifikant ökning supra- och subgingivalt av de flesta bakteriearter, men det fanns ingen signifikant skillnad mellan grupperna för undersökta bakterier. Kvoten mellan ”onda/goda” supragingivala bakterier minskade i testgruppen men var inte sig- nifikant. Motsvarande kvot för subgingivala bakterier minskade signifikant i båda grup- perna. Slutsats: Intag av L. reuteri resulterade i förekomst av L. reuteri i saliv men bakterien måste tillföras kontinuerligt. Ingen signifikant förändring i den undersökta supra- och subgingivala mikrofloran kunde påvisas. Den signifikanta ökningen i PlI i kontrollgruppen utan motsvarande förändring i testgruppen kan tyda på en probiotisk effekt av L. reuteri i denna population

Investigate the presence of Lactobacillus reuteri in saliva after supplementation with L. reuteri and the probiotic effect of L. reuteri on plaque index and supra- and subgingival microbiota. Material and Methods: The study included 23 healthy individuals, randomised into test or control subjects. At baseline and after 12 weeks saliva samples, plaque index and supra- and subgingival plaque samples were obtained. The test subjects were given the study product (containing L. reuteri, ATCC 55730 and ATCC PTA 5289) and the control subjects placebo for 12 weeks. Microbiological analyses were done by checkerboard DNA-DNA hybridization technique and selective culturing for lactobacilli determination. Results: A significant increase in total Lactobacillus counts in saliva occurred in both groups (p<0.05) with a significant increase of L. reuteri (p=0.008) in the test group. Ter- mination of intervention resulted in a wash out of L. reuteri. The control group demon- strated a statistically significant increase in PlI after 12 weeks (p=0.023) whilst there was no significant change in the test group. A significant increase was found for most bacterial species in both groups in supra- and subgingival plaque with no significant difference for any of the species between the groups. The ratio between ”bad/good” supragingival bacteria decreased for the test group but this decrease did not reach significance. The corresponding ratio for subgingival bacteria decreased significantly in both groups. Supplementation of L. reuteri resulted in presence of L. reuteri in saliva but L. reuteri was washed out after termination of intervention. No significant effect on supra- or subgingival microbiota was observed. The significant increase in PlI in the control group with no significant change in the test group may, however, indicate a probiotic effect of L. reuteri in this study population.

People have exploited fermentation by lactobacilli for centuries as a means of preparing and preserving foods. Several different bacterial species are today used as probiotic bacteria in different products both for human and animal applications. By definition probiotic bacteria are “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The most commonly used bacteria for the probiotic concept are found within the lactic acid bacteria (LAB) group. One of several genera included in the LAB group is Lactobacillus. One species of Lactobacillus, Lactobacillus reuteri has been studied extensively and certain strains have been shown to be probiotic i.e. health promoting with diverse beneficial effects, and thus it was interesting to further investigate the properties of these bacteria. To put this thesis work into context, the field of probiotic research is described and examples of proven probiotic effects are discussed. The overall aim was to investigate L. reuteri and its microbial action in the microbiota of humans and its relationship to health and disease. L. reuteri was shown to be a natural component of human milk. It was found in approximately one in seven nursing mothers living in geographically widely separated countries. Breast milk may be considered as a natural synbiotic and evidence from these results suggest that L. reuteri is one of the beneficial components in this regard. L. reuteri was also proven to be effective in reducing both gingivitis and dental plaque in patients with moderate to severe gingivitis, suggesting an improvement in periodontal health. Bacterial antagonism through the probiotic administration in the oral cavity might have contributed to the observed alleviation of symptoms and clinical manifestations of periodontal disease. It was shown that L. reuteri supplementation of pregnant mothers and their offspring during the first year of life resulted in detection of L. reuteri in breast milk and infant stool. Administration of L. reuteri resulted in the presence of L. reuteri in saliva, but no significant effect on supra- or subgingival microbiota was observed. The significant increase in plaque index in the control group with no significant change in the test group may however indicate a probiotic effect of L. reuteri in this study population of healthy individuals.

Aim: The aim of this stydy was to assess whether supplementation of Lactobacillus reuteri could have an impact on the oral microbiota. Material and Methods: Twent-three healthy people aged 29 to 63 years were included. A randomised double-blind placebo-controlled study was executed consisting of 12 persons in the test group which was given the study product twice a day for twelve weeks containing L. reuteri (an equal mix of ATCC 55730 and PTA 5289 at a total of 2x108 CFU/dose) and the control group of 11 persons having corresponding placebo. Pre and post of study period plaque index and oral health status (gingivitis, probing pocket depth, bleeding on probing) were measured together with sampling of supra-, subgingival plaque and saliva collection. Microbiological analysis was done by using checkerboard DNA-DNA hybridization technique and selective culturing for lactobacilli determination. Four weeks after the last intake of the product reassessments of plaque and saliva was performed. Results: No difference in general oral health could be observed between the groups after L. reuteri supplementation. Plaque index increased significantly in the control group after twelve weeks (p= 0.023). A significant increase in total Lactobacillus counts in saliva occurred in both groups (p<0.05). The probiotic intervention resulted in a significant increase of L. reuteri (p=0.008) corresponding to 13.8% of the total lactobacilli couont. A distribution ratio of 1:4 (ATCC 55730/ATCC PTA 5289) between the two installed L. reuteri strains in saliva was noticed. Termination of intervention resulted in a wash out of L. reuteri. A significant increase was found for most bacterial species in both groups and both in supra- and subgingival plaque during the test period. There was no significant difference detected for any of the bacterial species between the groups neither in plaque location. The ratio between "bad/good" supragingival bacteria decreased for the test group, however, not significant. The corresponding ratio for subgingival bacteria decreased significantly for both groups (p= 0.05)with no significant difference between the groups. Conclusions: The supplementation of L. reuteri did not affect general oral health. Presence of L. reuteri in saliva is only temporary and washed out after termination of intervention. Microbiologically no significant effect of the probiosis was observed. It can not be concluded whether L. reuteri was established in the plaque of the test group or not.

Objectives: This is to identify factors affecting the prevalence of Lactobacillus reuteri in maternal faeces and breast milk and infant faeces after oral supplementation with L. reuteri and to assess the influence on microbial ecology, particularly Clostridium difficile and Bifidobacterium colonization. Materials and Methods: In this double-blind trial, 232 mothers with a family history of atopic disease were randomized to a daily intake of either L. reuteri American-type culture collection (ATCC) 55730 (1 x 108 colony forming units (CFU)) or placebo for the last 4 weeks of pregnancy. Their babies then continued with tha same study product daily from birth until 12 months of age. Bacterial counts and prevalence were assessed in maternal breast milk and faeces and infant faeces, using conventional cultivation methods. Results: The prevalence of L. reuteri was higher during the first year of life in the stool samples from infants in the active as compared with the placebo-treated group. The highest prevalence was recorded at 5 to 6 days of age (82% in the treated vs 20% in the placebo group, P < 0.001). Lactobacillus reuteri was isolated from 12% and 2%, respectively, in the colostrum samples (P < 0.05). Breast-feeding seemed to reduce faecal L. reuteri counts, although antibiotics did not influence the levels of L. reuteri. The administration of L. reuteri did not affect bifidobacteria or C. difficile colonization. Conclusion: Lactobacillus reuteri may be detected in breats milk after oral supplementation to the mother and in almost all infants after oral supplementation during the first year of life, as well as occasionally in many untreated infants.

The nature and role of human milk microbiota in the early colonization and protection of infants from infection is the subject of increasing research. This study investigated the occurrence of Lactobacillus reuteri in milk of nursing mothers living in rural or urban areas in different geografic locations. Breat milk samples were collected from 220 mothers, 6-32 days after delivery, and analysed for the presence of total lactobacilli and L. reuteri. In all, 50% of mothers from rural areas in Japan and Sweden were L. reuteri-positive, whereas mothers from urban areas in South Africa, Israel and Denmark had very low and non-detectable levels. Overall, 15% of mothers had detectable L. reuteri in their milk. There were no significant differences in the prevalence of total Lactobacillus or L. reuteri in women from rural and urban habitats in the participating countries.

Avsikten med studien var att undersöka om dagligt användande av ett probiotiskt kosttillskott innehållande Lactobacillus reuteri kunde ge positiva effekter på patienter med måttlig eller svår gingivit. I försöket testades placebotuggummi mot 2 olika L. reuteri formuleringar (LR-1 och LR-2) innehållande 100 miljoner laktobaciller per tuggummi. Tuggummi togs 2 gånger per gång, på morgonen och på kvällen efter tandborstning. Studien var dubbelblind, randomiserad och löpte över 2 veckor. Totalt deltog 59 patienter. Två tandytor på varje patient utvaldes och gingival index och plack index mättes för varje yta, både startdagen och efter 2 veckor. För att minimera risken för observatörsberoende bedömning av index utfördes alla bedömningar på en och samma dag av en enda observatör, både vid start och avslut. Tandytorna fotograferades också för att ge ytterligare konsistens i bedömningen. Efter bedömning av index, rengjordes samtliga tandytor och patienterna instruerades av tandhygienist om korrekt tandborstning och behovet av dagligt användande av tandtråd. Salivprover togs både före och efter test behandling för att avgöra skillnader i laktobacillpopulationen över tiden. 20 patienter rendomiserades till LR-1, 21 till LR-2 och 18 till placebo. Gingival index förbättrades signifikant i samtliga grupper över de 14 dagarna. LR-1 formuleringen var dessutom signifikant överlägsen placebo. Plackindex var också signifikant förbättrat över tiden i de båda reuteri-grupperna- LR-1 koloniserades i 65% av patienterna och LR-2 i 95%.

The primary aim of this study was to assess if the probiotic Lactobacillus reuteri could be effective in the treatment of gingivitis and further to evaluate the influence of the probiotic on plaque and the lactobacilli population in the saliva. A randomised, placebo-controlled, double blind study was performed over 2 weeks. Fifty-nine petients with moderate to severe gingivitis were included and given one of two different Lactobacillus reuteri formulations (LR-1 or LR-2) at a dose of 2E+08 CFU per day, or a corresponding placebo. At baseline (day 0) gingival index and plaque index were measured on two surfaces and saliva for lactobacilli determination was collected. The patients were instructed how to brush and floss efficiently and study treatment was started. The patients returned on day 14 for final assessment of gingivitis and plaque and saliva was collected. 20 patients were randomised to LR-1, 21 to LR-2 and 18 to placebo. Gingival index fell significantly in all 3 groups (p<0.0001). LR-1, but not LR-2 improved more than placebo (p<0.0001). Plaque index fell significantly in LR-1 (p<0.05) and in LR-2 (p<0.01) between day 0 and day 14 but there was no significant change in the placebo group. At day 14, 65% of the patients in LR-1 were colonised with Lactobacillus reuteri and 95% in the LR-2 group. Lactobacillus reuteri was efficacious in reducing both gingivitis and plaque in patients with moderate to severe gingivitis.