Probiotic lactobacilli and bifidobacteria in the mouth – in vitro studies on saliva-mediated functions and acid production

Probiotic lactobacilli and bifidobacteria in the mouth – in vitro studies on saliva-mediated functions and acid production Probiotics are viable bacteria which, when used in adequate amounts, are beneficial to the health of the host. Although most often related to intestinal health, probiotic bacteria can be found also in the mouth after consumption of products that contain them. This study aimed at evaluating the oral effects of probiotic bacteria already in commercial use. In a series of in vitro studies, the oral colonisation potential of different probiotic bacteria, their acid production and potential saliva-mediated effects on oral microbial ecology were investigated. The latter included effects on the salivary pellicle, the adhesion of other bacteria, and the activation of the peroxidase system. Streptococcus mutans, Streptococcus gordonii, Aggregatibacter actinomycetemcomitans and Helicobacter pylori were used as bacterial indicators of the studied phenomena. There were significant differences between the probiotic strains in their colonisation potential. They all were acidogenic, although using different sugars and sugar alcohols. However, their acid production could be inhibited by the peroxidase system. Based on the results, it can be suggested that probiotic bacteria might influence the oral microbiota by different, partly species or strain-specific means. These include the inhibition of bacterial adhesion, modification of the enamel pellicle, antimicrobial activity, and activation of the peroxidase system. To conclude, probiotic strains differed from each other in their colonisation potential and other oral effects as evaluated in vitro. Both positive and potentially harmful effects were observed, but the significance of the perceived results needs to be further evaluated in vivo.

Innate and Adaptive Immunity in Orolabial Herpes Simplex

Oral mucosa is a frequent site of primary herpes simplex virus type 1 (HSV-1) infection, whereas intraoral recurrent disease is very rare. Instead, reactivation from latency predominantly results in asymptomatic HSV shedding to saliva or recurrent labial herpes (RLH) with highly individual frequency. The current study aimed to elucidate the role of human oral innate and acquired immune mechanisms in modulation of HSV infection in orolabial region. Saliva was found to neutralize HSV-1, and to protect cells from infection independently of salivary antibodies. Neutralization capacity was higher in saliva from asymptomatic HSV-seropositive individuals compared to subjects with history of RLH or seronegative controls. Neutralization was at least partially associated with salivary lactoferrin content. Further, lactoferrin and peroxidase-generated hypothiocyanite were found to either neutralize HSV-1 or interfere with HSV-1 replication, whereas lysozyme displayed no anti-HSV-1 activity. Lactoferrin was also shown to modulate HSV-1 infection by inhibiting keratinocyte proliferation. RLH susceptibility was further found to be associated with Th2 biased cytokine responses against HSV, and a higher level of anti- HSV-IgG with Th2 polarization, indicating lack of efficiency of humoral response in the control of HSV disease. In a three-dimensional cell culture, keratinocytes were found to support both lytic and nonproductive infection, suggesting HSV persistence in epithelial cells, and further emphasizing the importance of peripheral immune control of HSV. These results suggest that certain innate salivary antimicrobial compounds and Th1 type cellular responses are critically important in protecting the host against HSV disease, implying possible applications in drug, vaccine and gene therapy design.

A Comparative Study of the Effectiveness of a Prefabricated Appliance and a Stabilization Appliance in the Treatment of Myofascial pain and Headache

The aim of this thesis was to evaluate the short- and long-term effectiveness of a prefabricated occlusal appliance (R) on patients with myofascial pain and headache by comparing it with the treatment of the stabilization appliance (S). Another aim was to evaluate the effect of appliance treatment on stress-related salivary parameters like cortisol and IgA, as well as on flow rate values in these patients. Sixty-five patients diagnosed with myofascial temporomandibular disorder (TMD) pain, of whom 94% suffered concomitantly from headache, at two centres for Stomatognathic Physiology, one in Sweden and one in Finland, were included in this randomized controlled trial using Research Diagnostic Criteria for TMD (RDC/TMD), with history questionnaires and clinical examinations performed at baseline and at 6- and 10-weeks, and 6- and 12-month follow-ups. Patients were randomly assigned either to the R or the S group. Treatment outcome was measured according to IMMPACT (Initiative on Methods, Measurements, and Pain Assessment in Clinical Trials), i.e. four chronic pain outcome domains: pain intensity, overall improvement, physical and emotional functioning. Changes in frequency and intensity of headache were recorded. Thirty-nine patients participated in the saliva study. Salivary analyses were performed at 6 and 10 weeks. The results revealed no differences between groups at baseline. At all follow-ups, all four outcome measures, as well as frequency and intensity of headache, showed statistically significant within-group improvement compared to baseline, without significant differences between groups. No treatment-induced changes in saliva parameters could be registered. In conclusion, the effectiveness of the prefabricated appliance seemed to be similar to that of the stabilization appliance in alleviating myofascial pain, and frequency and intensity of headache, in the short as well as the long term. However, no changes in salivary parameters were observed during treatment.

Astringent Food Compounds and Their Interactions with Taste Properties

Astringency is traditionally thought to be induced by plant tannins in foods. Because of this current research concerning the mechanism of astringency is focused on tannin‐protein interactions and thus on precipitation, which may be perceived by mechanoreceptors. However, astringency is elicited by a wide range of different phenolic compounds, as well as, some non‐phenolic compounds in various foods. Many ellagitannins or smaller compounds that contribute to astringent properties do not interact with salivary proteins and may be directly perceived through some receptors. Generally, the higher degree of polymerization of proanthocyanidins can be associated with more intense astringency. However, the astringent properties of smaller phenolic compounds may not be directly predicted from the structure of a compound, although glycosylation has a significant role. The astringency of organic acids may be directly linked to the perception of sourness, and this increases along with decreasing pH. Astringency can be divided into different sub‐qualities, including even other qualities than traditional mouth‐drying, puckering or roughing sensations. Astringency is often accompanied by bitter or sour or both taste properties. The different sub‐qualities can be influenced by different astringent compounds. In general, the glycolysation of the phenolic compound results in more velvety and smooth mouthdrying astringency. Flavonol glycosides and other flavonoid compounds and ellagitannins contribute to this velvety mouthdrying astringency. Additionally, they often lack the bitter properties. Proanthocyanidins and phenolic acids elicit more puckering and roughing astringency with some additional bitter properties. Quercetin 3‐O‐rutinoside, along with other quercetin glycosides, is among the key astringent compounds in black tea and red currants. In foods, there are always various other additional attributes that are perceived at the same with astringency. Astringent compounds themselves may have other sensory characteristics, such as bitter or sour properties, or they may enhance or suppress other sensory properties. Components contributing to these other properties, such as sugars, may also have similar effects on astringent sensations. Food components eliciting sweetness or fattiness or some polymeric polysaccharides can be used to mask astringent subqualities. Astringency can generally be referred to as a negative contributor to the liking of various foods. On the other hand, perceptions of astringent properties can vary among individuals. Many genetic factors that influence perceptions of taste properties, such as variations in perceiving a bitter taste or variations in saliva, may also effect the perception of astringency. Individuals who are more sensitive to different sensations may notice the differences between astringent properties more clearly. This may not have effects on the overall perception of astringency. However, in many cases, the liking of astringent foods may need to be learned by repetitive exposure. Astringency is often among the key sensory properties forming the unique overall flavour of certain foods, and therefore it also influences whether or not a food is liked. In many cases, astringency may be an important sub‐property suppressed by other more abundant sensory properties, but it may still have a significant contribution to the overall flavour and thus consumer preferences. The results of the practical work of this thesis show that the astringent phenolic compounds are mostly located in the skin fractions of black currants, crowberries and bilberries (publications I–III). The skin fractions themselves are rather tasteless. However, the astringent phenolic compounds can be efficiently removed from these skin fractions by consecutive ethanol extractions. Berries contain a wide range of different flavonol glycosides, hydroxycinnamic acid derivatives and anthocyanins and some of them strongly contribute to the different astringent and bitterness properties. Sweetness and sourness are located in the juice fractions along with the majority of sugars and fruit acids. The sweet and sour properties of the juice may be used to mask the astringent and bitterness properties of the extracts. Enzymatic treatments increase the astringent properties and fermented flavour of the black currant juice and decrease sweetness and freshness due to the effects on chemical compositions (IV). Sourness and sweetness are positive contributors to the liking of crowberry and bilberry fractions, whereas bitterness is more negative (V). Some astringent properties in berries are clearly negative factors, whereas some may be more positive. The liking of berries is strongly influenced by various consumer background factors, such as motives and health concerns. The liking of berries and berry fractions may also be affected by genetic factors, such as variations in the gene hTAS2R38, which codes bitter taste receptors (V).

Pregnancy and periodontium. A clinical, microbiological, and enzymological approach via a longitudinal study

The mechanisms leading to an enhanced susceptibility to gingivitis in pregnant women have not yet been completely described. Therefore, the current study series were performed to investigate longitudinally the influence of pregnancy on periodontal tissues, and to evaluate microbial and host response factors related to pregnancy gingivitis formation. Pregnancy-related periodontal changes were analysed in 30 generally healthy women (24- 35 years old) once per trimester, till the end of lactation. Matched non-pregnant women (n=24) served as the controls, and were examined three times, once per following month. Pregnancy-related gingival inflammation was observed as enhanced tendency towards gingival bleeding and pseudopocket formation with a concomitant decrease in plaque levels. Gingivitis reached its peak during mid-pregnancy and then decreased transiently visit by visit. After lactation, no differences in periodontal status were seen between the study and control populations. In contrast to previous studies reporting increased levels of Prevotella intermedia, a specific aim was to analyse phenotypically two identical species, P. intermedia and Prevotella nigrescens, separately using a 16S ribosomal DNA-based PCR. As a result, the increased levels of P. nigrescens were related to pregnancy gingivitis. Matrix metalloproteinases (MMPs) are involved in periodontal destruction. However, their role in pregnancy gingivitis is not well studied. Therefore, neutrophilic enzymes and proteinases, such as MMP and myeloperoxidase (MPO) levels were analysed from saliva and gingival crevicular fluid (GCF) samples during the follow-up. Despite increased inflammation and microbial shift towards anaerobes, the host response did not activate the MMP, elastase and MPO secretion during pregnancy. These results demonstrate that during pregnancy gingival inflammation is enhanced especially during the second trimester, when P. nigrescens levels in subgingival plaque were increased, whereas the neutrophilic enzymes and proteinase levels in both saliva and GCF remained low. These findings could explain, at least in part, why pregnancy gingivitis itself does not predispose or proceed to periodontitis.

Detection of Illicit Drugs and Drug Precursors with Cantilever-Enhanced Photoacoustic Spectroscopy

In this study, cantilever-enhanced photoacoustic spectroscopy (CEPAS) was applied in different drug detection schemes. The study was divided into two different applications: trace detection of vaporized drugs and drug precursors in the gas-phase, and detection of cocaine abuse in hair. The main focus, however, was the study of hair samples. In the gas-phase, methyl benzoate, a hydrolysis product of cocaine hydrochloride, and benzyl methyl ketone (BMK), a precursor of amphetamine and methamphetamine were investigated. In the solid-phase, hair samples from cocaine overdose patients were measured and compared to a drug-free reference group. As hair consists mostly of long fibrous proteins generally called keratin, proteins from fingernails and saliva were also studied for comparison. Different measurement setups were applied in this study. Gas measurements were carried out using quantum cascade lasers (QLC) as a source in the photoacoustic detection. Also, an external cavity (EC) design was used for a broader tuning range. Detection limits of 3.4 particles per billion (ppb) for methyl benzoate and 26 ppb for BMK in 0.9 s were achieved with the EC-QCL PAS setup. The achieved detection limits are sufficient for realistic drug detection applications. The measurements from drug overdose patients were carried out using Fourier transform infrared (FTIR) PAS. The drug-containing hair samples and drug-free samples were both measured with the FTIR-PAS setup, and the measured spectra were analyzed statistically with principal component analysis (PCA). The two groups were separated by their spectra with PCA and proper spectral pre-processing. To improve the method, ECQCL measurements of the hair samples, and studies using photoacoustic microsampling techniques, were performed. High quality, high-resolution spectra with a broad tuning range were recorded from a single hair fiber. This broad tuning range of an EC-QCL has not previously been used in the photoacoustic spectroscopy of solids. However, no drug detection studies were performed with the EC-QCL solid-phase setup.

Relationship between Daily Habits, Oral Microbiota and Caries with Special Reference to Xylitol

Caries is a plaque-associated multifactorial chronic disease. Oral hygiene habits, sugar, and oral micobiota interactions are important for caries to occur. Xylitol has been shown to reduce caries mainly due to its effects on mutans streptococci (MS). The purpose of this study was to evaluate the relationship of daily oral health habits and bacterial level on the caries occurrence and to study the effect of xylitol on the composition of oral microflora. A total of 192, 10-12 years old, male school children had been screened for salivary MS. Healthy subjects with high MS counts participated in two parallel double-blinded, randomised, controlled trials. In the first 5-week trial, subjects were assigned into xylitol (n=35) and sorbitol gum (n=38) groups. At baseline, children were examined using International Caries Detection and Assessment System (ICDAS) criteria and interviewed for oral health habits. In the second 4-week trial, subjects were assigned into xylitol (n=25) and saccharine mouthrinse (n=25) groups. In the end of both interventions, saliva samples were collected. The samples were analysed for changes in MS counts and changes in the composition of the oral microbiota assessed by the Human Oral Microbe Identification Microarray (HOMIM). Relationships between daily habits, bacterial levels and caries were evaluated. Daily use of sweets and soft drinks were the habits significantly associated with caries severity measured by ICDAS Caries Index (CI), while toothbrushing was the only habit associated with the low caries severity. Abiotrophia defectiva and Actinomyces meyeri/ A. odontolyticus were significantly higher in caries-affected children while Shuttleworthia satelles was significantly higher in caries-free children. Xylitol showed significant reduction in salivary levels of MS in both trials. No significant effects on other members of the microbiota were found when evaluated by HOMIM. In conclusion, other members of oral microbiota than MS may be associated with caries occurrence or absence. The use of xylitol had significant effect on MS with no effects on the other members of the salivary microbiota.

Probiotics and oral health: In vitro and clinical studies

Aino Toiviainen Probiotics and oral health: in vitro and clinical studies University of Turku, Faculty of Medicine, Institute of Dentistry, Periodontology, Finnish Doctoral Program in Oral Sciences (FINDOS-Turku), Turku, Finland Annales Universitatis Turkuensis, Sarja – Ser. D, Medica-Odontologica. Painosalama Oy, Turku, Finland, 2015 Probiotics are used, for example, to prevent and treat diarrhea, allergies and respiratory infections, and there is an increasing interest to use probiotics also for oral health purposes. The most commonly used probiotic bacteria are lactobacilli and bifidobacteria, which are acidogenic and aciduric. From the oral point of view, use of these probiotics may, at least in theory, mean an increased risk of caries. In this thesis, the effects of probiotics on oral microbial composition, acid production of dental plaque and gingival health were studied through in vitro studies and two clinical studies. In a randomized, double-blind and crossover study, 13 healthy adults were allocated into two groups. Half of the subjects first consumed Lactobacillus rhamnosus GG tablets twice a day for two weeks, and after the washout period, L. reuteri tablets twice a day for two weeks. The other half of the subjects used the tablets in reverse order. In another controlled, randomized and double-blind study, 62 healthy adults were allocated into two groups. One group used the test tablets containing L. rhamnosus GG and B. lactis BB-12 and the other group used control tablets without probiotics. The recommendation for the use of the tablets was 4 per day for 4 weeks. Probiotic lactobacilli interfered with S. mutans biofilm formation and the adhesion of S. mutans to saliva-coated hydroxyapatite in vitro. No effect was found in S. mutans levels in the three-species biofilms. In clinical studies, the studied probiotics had no effect on the acid production of plaque. The counts of mutans streptococci and the oral microbial composition remained the same. Tablets containing L. rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 did decrease the amount of plaque and gingival bleeding. According to our results, it seems that probiotics have beneficial effects on gingival health. The present results confirmed that probiotics are safe and have beneficial effects on oral health. Since the consumption of probiotics by the general population is steadily increasing, an understanding of the functions of probiotics in the oral cavity has become more important. Keywords: lactobacilli, bifidobacteria, caries, periodontal disease, mutans streptococci, probiotics