Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Early childhood caries (ECC) is considered a serious public health issue among children all over the world. However, many aspects should be explored about the oral microbiota related to the ECC progression and how the bacterial community modifies according to the dentine lesion activity. This thesis, composed by two chapters, aimed to investigate and quantify with quantitative polymerase chain reaction (qPCR) the following bacteria Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, as well as members of the groups Lactobacillus casei and Mitis on biofilm from pre-school children with different stages of early childhood caries progression (Chapter 1) and on active and inactive dentine carious lesions (Chapter 2), and also to verify the association of these microorganisms on the process of health or disease. The sample consisted on preschool children aged between 2 and 5 years from nurseries and public preeschools in Fortaleza-CE. The children were examined for caries diagnosis with ICDAS II index (International Caries Detection Assessment System), and the Nyvad criteria, in order to evaluate prevalence and caries activity. The supragingival biofilm collection was taken from 75 children, who were divided in three groups according to the ICDAS II: CF (caries free) (n=20), ECL (presence of enamel caries lesion) (n=17) and DCL (presence of dentine caries lesion) (n=38). Samples of carious dentine were collected under rubber dam isolation of 56 lesions of dentine affected by caries, being 17 inactive and 39 active. The DNA of all the collected samples was extracted and purified, then tested for the presence of the formerly mentioned bacterial species/groups through qPCR. The quantity of bacteria was compared through the Kruskal-Wallis and Mann-Whitney tests. Besides, the association between the presence of bacteria and ECC was analyzed through the Chi-square test, with a 5% significance level and the multiple logistic regression was applied. Bacteria from the group L. casei and L. acidophilus presented low detection on biofilm of all evaluated groups. The presence of S. mutans and Bifidobacterium spp. showed a strong association with dental caries progression on the biofilm from children with dentine lesions with odds ratio of 21,5 and 5,9; respectively. On active dentine lesions, concentrations of Bifidobacterium spp. and species from the Lactobacillus casei group were significantly higher when compared to the inactive lesions (p<0.05). The levels of Actinomyces naeslundii, Streptococcus gordonni and species from Mitis group were not significantly different among biofilm groups as well as comparing dentine lesions. In conclusion, the microbial profile from biofilm samples presented differences on the proportion of acidogenic and aciduric bacteria with dental caries progression. The presence of Bifidobacterium spp. and S. mutans presented a strong association with the development of the more advanced stages of ECC. Regarding the activity of dentine lesions, higher detection levels of the group L. casei and Bifidobacterium spp. showed an important role of these bacteria in the dentine caries activity. / A cÃrie precoce da infÃncia (CPI) Ã considerada um grave problema de saÃde pÃblica em crianÃas prÃ-escolares em todo mundo. No entanto, muitos aspectos ainda devem ser explorados acerca da microbiota oral relacionada com a progressÃo da CPI e como a comunidade bacteriana se modifica de acordo com a atividade da lesÃo dentinÃria. Esta tese, constituÃda de dois capÃtulos, teve como objetivo identificar e quantificar atravÃs da tÃcnica de reaÃÃo em cadeia da polimerase quantitativa (qPCR) as bactÃrias Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, bem como espÃcies dos grupos Lactobacillus casei e Mitis em biofilme de crianÃas prÃ-escolares com diferentes estÃgios de progressÃo da cÃrie precoce da infÃncia (CapÃtulo 1) e em lesÃes cariosas dentinÃrias ativas e inativas (CapÃtulo 2) e ainda verificar a associaÃÃo destes microrganismos ao processo de saÃde ou de doenÃa. A amostra consistiu em prÃ-escolares com idade entre 2 e 5 anos de idade que frequentavam creches e escolas pÃblicas de Fortaleza-CE. As crianÃas foram examinadas com o uso de Ãndices visuais ICDAS II (International Caries Detection Assessment System) e Nyvad, a fim de avaliar a prevalÃncia e atividade de cÃrie. A coleta de biofilme supragengival foi realizada em 75 crianÃas, as quais foram agrupadas de acordo com Ãndice ICDAS II em trÃs grupos: CF (livres de cÃrie) (n=20), ECL (presenÃa de lesÃes de cÃrie em esmalte) (n=17) e DCL (presenÃa de lesÃes de cÃrie em dentina) (n=38). Amostras de dentina cariada foram coletadas sob isolamento absoluto de 56 lesÃes cariosas dentinÃrias, sendo 17 inativas e 39 ativas. O DNA de todas as amostras coletadas foi extraÃdo e purificado e, em seguida, testado para a presenÃa das espÃcies/grupos bacterianos acima citados atravÃs de qPCR. A quantidade das bactÃrias foi comparada pelos testes Kruskal-Wallis e Mann-Whitney. AlÃm disso, a associaÃÃo da presenÃa de bactÃrias e CPI foi analisada atravÃs do teste Qui-quadrado, com nÃvel de significÃncia de 5% e aplicado a regressÃo logÃstica mÃltipla. BactÃrias L. acidophilus e do grupo L. casei apresentaram baixa detecÃÃo no biofilme de todos os grupos avaliados. A presenÃa de S. mutans e Bifidobacterium spp. mostrou forte associaÃÃo com a progressÃo da doenÃa no biofilme de crianÃas com lesÃes dentinÃrias com âodds ratioâ de 21,5 e 5,9, respectivamente. Em lesÃes dentinÃrias ativas, concentraÃÃes de Bifidobacterium spp. e bactÃrias do grupo L. casei foram significativamente maiores quando comparadas Ãs lesÃes inativas (p<0.05). Os nÃveis de A. naeslundii, bactÃrias do grupo Mitis e S. gordonni nÃo apresentaram diferenÃa significativa entre os grupos de biofilme, assim como nas lesÃes dentinÃrias. Conclui-se que as amostras de biofilme apresentaram alteraÃÃo na proporÃÃo de bactÃrias acidogÃnicas e acidÃricas com a progressÃo da doenÃa cÃrie. A presenÃa de Bifidobacterium spp. e S. mutans apresentou forte associaÃÃo com os estÃgios mais avanÃados da CPI. Em relaÃÃo Ãs lesÃes dentinÃrias, o aumento da concentraÃÃo de bactÃrias Bifidobacterium spp. e do grupo L. casei evidenciou um papel importante destas bactÃrias na atividade de lesÃes dentinÃrias.

The Diploma thesis deals with designing of probiotic dietary supplement for children with strains Lactobacillus acidophilus and Bifidobacterium breve and with prebiotics. Used prebiotics were Inulin, Chia fiber, Bamboo fiber, Chlorella + Spirulina and Yakon syrup. The theoretical part is focused on probiotics, prebiotics and their biological influence. In experimental part the possibilities of encapsulation into alginate particle and lyophilisation of probiotic cells were observed to find their good form to final nutritional product for children. Several types of probiotic with addition of prebiotics were tested in model conditions of gastrointestinal tract. It was found that addition of prebiotic highly increases viability of probiotic cells and their resistance to model conditions of gastrointestinal tract. In this case, the best prebiotic was found in Yakon syrup. The prebiotics were also characterised in terms of nutritional composition, amount of total and reducing sugars, oligosaccharides, proteins, lipids, polyphenols and chlorophyll were obtained. Finally, Chia fiber, Chlorella + Spirulina and Yakon syrup were chosen as prebiotics with best characterisation/properties. In conclusion, a dietary supplement with lyophilized alginate particles containing probiotic cells and with the most appropriate prebiotics were designed. Forms of the product were powder and gummy-bear.

Different species of genus Bifidobacterium are part of human and animal intestinal flora. These bacteria have benefit effects and therefore they are used in foods and pharmaceutical products as probiotics. Cheese is now suitable as a probiotic matrix except yoghurts and fermentated milks. This diploma thesis was focused on optimalization of DNA isolation from bacteria of genus Bifidobacterium. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for DNA isolation in presence of 8% polyethyleneglycol PEG 6000 and 5 M sodium chloride. Phenol extraction weas also used as an isolation method. Isolated DNA was used for amplification in domain, genus and species specific PCRs. Optimized method was tested for detection of bacteria of genus Bifidobacterium in experimentaly prepared probiotic cheeses. These cheeses contained potential probiotic bacteria from Laktoflóra collection. Bacteria were identified into species using species specific PCR. Species Bifidobacterium animalis was identified in all samples of probiotic cheeses.

The work was focused on isolation of PCR-ready DNA and the identification of probiotic lactic acid bacteria that were isolated from five milk product for infant nutrition. DNA was isolated from crude cell-lysates of the products by magnetic P(HEMA-co-GMA) microspheres. DNAs isolated from crude cell lysates of control strains using phenol extraction method were used as positive controls. Using PCRs DNA of genera Bifidobacterium and species B. animalis, B. bifidum, B. breve, B. infantis, B. longum and Streptococcus thermophilus species were identified in products. The results obtained are consistent with the data declared by the manufacturers.

The aim of thesis is focused on isolation of DNA in quality for polymerase chain reaction (PCR) and the identification of probiotic bacteria. From six probiotic supplements for children were isolated PCR-ready DNAs using magnetic carriers P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. DNAs of Lactobacillus, Bifidobacterium and Streptococcus genera were identified as: L. acidophilus, L. rhamnosus, L. casei, B. bifidum, B. longum ssp. longum, B. breve, B. longum ssp infantis, B. animalis and S. thermophilus. The identification corresponded with the data declared by the producers.

This diploma thesis is focused on encapsulation probiotics and co-encapsulation with some types of prebiotics. In theoretical part is aimed to probiotics, their general characteristics and application of probiotics in food industry. There are described prebiotics and their classification, there is described principles of encapsulation and encapsulation techniques. Methods, which are used for analysis of particles and encapsulation components were introduced too. The experimental part describes methods of prebiotics characterization by high performance liquid chromatography, thin layer chromatography and spectrophotometric methods. Cultivation of probiotics with prebiotics - hydrolyzed and non-hydrolyzed wad tested. Using flow cytometry cell viability was measured too. Finally probiotics and prebiotics were encapsulated, mainly by encapsulator machine. Long-term stability of particles during 6 week storage was observed. The particles were exposed to effect of artificial intestinal, gastric and bile juices.

The Diploma thesis deals with use of selected probiotic strains Lactobacillus acidophilus and Bifidobacterium breve in different forms in food for infants. The theoretical part is focused on describing probiotics, encapsulation methods and intestinal gut microbiota of infants. Further, characterization of individual periods of infant feeding and food for infants were introduced. In experimental part the possibilities of encapsulation and lyophilisation of probiotic cells were observed. Probiotic cells were encapsulated into alginate particles. The encapsulator was used for preparation of particles and the most appropriate particles were prepared by encapsulation nozzle with size of 300 µm. Moreover, probiotics viability was monitored by Flow Cytometry, Fluorescence Microscopy and by cultivation (CFU method). Viability of probiotics was monitored during long-term storage in selected food for infants. The appropriate shelf life of non-lyophilized alginate particles in real food have been set at 1 to 2 months. Lyophilized alginate particles could be stored for more than 3 months. Finally, the stability of the particles and viability of encapsulated and non-encapsulated cells in the gastrointestinal tract conditions were also examined. The viabilities of lyophilized cells and cells encapsulated in lyophilized particles were also compared. From the results obtained, non-encapsulated probiotic bacteria cells are more susceptible to negative effects of digestive juices, the percentage of dead probiotic cells after digestion was approximately 80 %. On the other hand, alginate particles showed cell protection from digestive juices, after incomplete cell releasing from particles the percentage of dead probiotic cells did not exceed 20 %. After adequate rehydration, similar results were gained with lyophilized alginate particles. Lyophilized alginate particles have been determined to be the most suitable application form for infants’ food.

Background: Mild Cognitive Impairment (MCI) is seen as a state between normal aging and dementia, with patients having an increased risk of developing Alzheimer’s disease (AD) and other sorts of dementia. MCI has been linked to a change in gut microbiota which impacts the microbiota-gut-brain axis (MGBA), consequently affecting neurological functions. A way of altering microbiota and thereby promoting cognitive health is through the administration of prebiotics, probiotics, and synbiotics. Aim: This systematic literature review aims to assess the impact of prebiotics, probiotics, and synbiotics on MCI by compiling existing data on the matter. Methods: Three databases - Web of Science, Cochrane, and PubMed - were searched and articles were included based on the following inclusion criteria: (1) randomized clinical trials (RCTs), (2) conducted on adults evaluated with MCI during the study, (3) including a prebiotic, probiotic, or synbiotic intervention of any kind, (4) comparing the intervention with a placebo or control group, (5) written in English, (6) reporting the main outcome of cognitive function using any neuropsychological evaluation test. Results: Five studies were included in the final selection. These studies showed that cognitive function improved after probiotic intervention, significantly affecting several cognitive domains: attention, calculation, orientation in time, and delayed memory. Two studies showed that subjects with low cognitive scores at baseline benefited more from probiotic supplementation compared to high-scoring subjects. Conclusions: Probiotics appear to improve cognition in MCI subjects; however, further research is needed to conclude the effects of prebiotics and synbiotics.

Afin de stabiliser le microbiote intestinal et prévenir ou traiter les infections entériques, il est suggéré depuis des décennies d’utiliser certaines bactéries lactiques dites ‘probiotiques’. La consommation de ces bactéries, qui sont des composants normaux du microbiote intestinal, aurait des effets bénéfiques sur la santé. Cependant, leur éventuel rôle prophylactique ou thérapeutique n’a été que très peu étudié. Dans une première partie, cinq bifidobactéries probiotiques isolées de fèces de nouveaux-nés ont été sélectionnées et caractérisées. Parmi celles-ci, une souche de Bifidobacterium thermacidophilum (RBL71) démontrant une forte résistance aux conditions retrouvées dans le tractus intestinal, une forte adhésion aux cellules intestinales Caco-2 et une inhibition de l’adhésion d’Escherichia coli O157:H7 (50%) aux cellules Caco-2 a été administrée par voie orale à des souris BALB/c. Ce gavage probiotique avant infection à E. coli O157:H7 a diminué les comptes fécaux de ce pathogène ainsi que les dommages histologiques intestinaux comparativement au groupe témoin. Une plus forte production d’anticorps spécifiques anti-E. coli O157:H7 a également été détectée chez les souris recevant la souche probiotique. Dans une seconde partie, l’efficacité de trois autres souches de bifidobactéries a été vérifiée contre des entéropathogènes viraux. Une souche de B. thermophilum (RBL67) démontrant la plus forte inhibition de l’attachement du rotavirus (98%) aux cellules intestinales Caco-2 et HT-29 a été administrée par voie orale à des souris néonatales CD-1 infectées par un rotavirus. La concentration de virus dans le contenu intestinal était significativement moins élevée à 48 heures post-infection chez le groupe recevant des probiotiques avant infection par rapport au groupe témoin. De plus, la durée de la diarrhée a été plus courte et une production d’anticorps spécifiques anti-rotavirus a été détectée chez les souris recevant des probiotiques avant l’infection. Ces résultats suggèrent que la souche RBL67 pourrait avoir un impact positif sur l’évolution des infections causées par des pathogènes viraux invasifs tel que rotavirus et que la souche RBL71 pourrait ainsi jouer un rôle dans la prévention ou le traitement d’infections entériques aux pathogènes bactériens non invasifs tel que E. coli O157:H7. Dans ces deux cas, l’inhibition de l’adhésion du pathogène serait le mécanisme d’action le plus probable. Cette démonstration de l’activité de ces nouvelles souches de bifidobactéries d’origine humaine contre E. coli O157:H7 et rotavirus, en interférant sur le mécanisme d’infection de ces entéropathogènes, soutient leur utilisation potentielle chez l’humain pour prévenir les infections entériques transmises par voie orale. / For decades, the use of certain lactic acid bacteria as so-called probiotics has been suggested in order to stabilize the intestinal microbiota and thus prevent or treat enteric infections. Consumption of these bacteria, which are normal components of human intestinal microbiota, is reputed to be beneficial to health. However, their possible role as therapeutic or prophylactic agents has been studied very little. Five probiotic bifidobacteria isolated from the feces of newborn infants were first selected and characterized. Among these, a strain of Bifidobacterium thermacidophilum (called RBL71) demonstrating strong resistance to the conditions prevailing in the digestive tract, strong adhesion to Caco-2 intestinal cells and inhibition of the adhesion of Escherichia coli O157:H7 (50%) to Caco-2 cells was administered via the oral route to BALB/c mice. Mice thus treated before challenge had reduced fecal counts of E. coli O157:H7 and less intestinal histological damage than the control group. Greater production of O157:H7-specific antibody was detected in mice receiving the probiotic. In a second study, the effectiveness of three other strains of bifidobacteria against viral enteropathogens was examined. A strain of B. thermophilum (called RBL67) demonstrating the strongest inhibition (98%) of rotavirus attachment to Caco-2 and HT-29 intestinal cells was administered via the oral route to neonatal CD-1 mice infected with rotavirus. The viral concentration of the intestinal contents 48 hours after infection was significantly lower in the probiotic-treated group than in the control group. In addition, the diarrhea was of shorter duration and rotavirus-specific antibody production was detected in the mice receiving the probiotic before infection. These results suggest that strain RBL67 has a positive impact on the evolution of infections by invasive viral pathogens such as rotavirus and that strain RBL71 could thus have a role to play in the prevention or treatment of enteric infections by non-invasive bacterial pathogens such as E. coli O157:H7. In both cases, inhibition of adhesion of the pathogen seems to be a plausible mechanism of action. This demonstration of the activities of these new bifidobacterial strains of human origin against E. coli O157:H7 and rotavirus suggests their potential for interfering with the mechanism of infection of enteropathogens and supports their use in humans as possible agents for preventing enteric infections transmitted by the oral route.

This thesis focuses on the genomic study of symbionts of two different groups of hymenopterans: bees and ants. Both groups of insects have major ecological impact, and investigating their microbiomes increases our understanding of their health, diversity and evolution. The study of the bee gut microbiome, including members of Lactobacillus and Bifidobacterium, revealed genomic processes related to the adaptation to the gut environment, such as the expansion of genes for carbohydrate metabolism and the acquisition of genes for interaction with the host. A broader genomic study of these genera demonstrated that some lineages evolve under strong and opposite substitution biases, leading to extreme GC content values. A comparison of codon usage patterns in these groups revealed ongoing shifts of optimal codons. In a separate study we analysed the genomes of several strains of Lactobacillus kunkeei, which inhabits the honey stomach of bees but is not found in their gut. We observed signatures of genome reduction and suggested candidate genes for host-interaction processes. We discovered a novel type of genome architecture where genes for metabolic functions are located in one half of the genome, whereas genes for information processes are located in the other half. This genome organization was also found in other Lactobacillus species, indicating that it was an ancestral feature that has since been retained. We suggest mechanisms and selective forces that may cause the observed organization, and describe processes leading to its loss in several lineages independently. We also studied the genome of a species of Rhizobiales bacteria found in ants. We discuss its metabolic capabilities and suggest scenarios for how it may affect the ants’ lifestyle. This genome contained a region with homology to the Bartonella gene transfer agent (GTA), which is a domesticated bacteriophage used to transfer bacterial DNA between cells. We propose that its unique behaviour as a specialist GTA, preferentially transferring host-interaction factors, originated from a generalist GTA that transferred random segments of chromosomal DNA. These bioinformatic analyses of previously uncharacterized bacterial lineages have increased our understanding of their physiology and evolution and provided answers to old and new questions in fundamental microbiology.