In Vitro Screen of Lactobacilli Strains for Gastrointestinal and Vaginal Benefits.

Traditional probiotics comprise mainly lactic acid bacteria that are safe for human use, tolerate acid and bile, and adhere to the epithelial lining and mucosal surfaces. In this study, one hundred commercial and non-commercial strains that were isolated from human feces or vaginal samples were tested with regards to overall growth in culture media, tolerance to acid and bile, hydrogen peroxide (H2O2) production, and adhesion to vaginal epithelial cells (VECs) and to blood group antigens. As a result, various of the tested lactobacilli strains were determined to be suitable for gastrointestinal or vaginal applications. Commercial strains grew better than the newly isolated strains, but tolerance to acid was a common property among all tested strains. Tolerance to bile varied considerably between the strains. Resistance to bile and acid correlated well, as did VEC adhesion and H2O2 production, but H2O2 production was not associated with resistance to bile or acid. Except for L. iners strains, vaginal isolates had better overall VEC adhesion and higher H2O2 production. Species- and strain-specific differences were evident for all parameters. Rank-ordered clustering with nine clusters was used to identify strains that were suitable for gastrointestinal or vaginal health, demonstrating that the categorization of strains for targeted health indications is possible based on the parameters that were measured in this study.

Deep sequencing of blood and gut T-cell receptor ß-chains reveals gluten-induced immune signatures in celiac disease.

Celiac disease (CD) patients mount an abnormal immune response to gluten. T-cell receptor (TCR) repertoires directed to some immunodominant gluten peptides have previously been described, but the global immune response to in vivo gluten exposure in CD has not been systematically investigated yet. Here, we characterized signatures associated with gluten directed immune activity and identified gluten-induced T-cell clonotypes from total blood and gut TCR repertoires in an unbiased manner using immunosequencing. CD patient total TCR repertoires showed increased overlap and substantially altered TRBV-gene usage in both blood and gut samples, and increased diversity in the gut during gluten exposure. Using differential abundance analysis, we identified gluten-induced clonotypes in each patient that were composed of a large private and an important public component. Hierarchical clustering of public clonotypes associated with dietary gluten exposure identified subsets of highly similar clonotypes, the most proliferative of which showing significant enrichment for the motif ASS[LF]R[SW][TD][DT][TE][QA][YF] in PBMC repertoires. These results show that CD-associated clonotypes can be identified and that common gluten associated immune response features can be characterized in vivo from total repertoires, with potential use in disease stratification and monitoring.

Secretor Status Is Strongly Associated with Microbial Alterations Observed during Pregnancy.

During pregnancy there are significant changes in gut microbiota composition and activity. The impact of secretor status as determined by genotyping FUT2 (fucosyltransferase 2) gene was taken as one of the confounding factors associated with faecal microbiota changes during pregnancy. In this prospective study, we followed women during pregnancy (total = 123 of which secretors = 108, non-secretors = 15) and characterised their gut microbiota by quantitative polymerase chain reaction (qPCR), Fluorescence In situ Hybridisation (FISH), Denaturing Gradient Gel Electrophoresis (DGGE) and pyrosequencing. qPCR revealed that C. coccoides group counts decreased significantly in non-secretors in comparison to secretors (p = 0.02). Similar tendency was found by FISH analysis in Clostridium histolyticum and Lactobacillus-Enterococcus groups between the secretor and the non-secretor pregnant women. DGGE analysis showed significant decrease in richness of Clostridium sp. between secretor and non-secretor mothers during pregnancy. Pyrosequencing based analysis at phyla level showed that there is greater increase in Actinobacteria in secretors in comparison to non-secretors, whereas Proteobacteria showed more increase in non-secretors. Change in relative abundance of Clostridiaceae family from first to third trimester were significantly associated with secretor status of pregnant women (p = 0.05). Polyphasic approach for microbiota analysis points out that the host secretor status (FUT2 genotype) affects the gut microbiota during pregnancy. This may lead to altered infant gut microbiota colonization.

A possible link between early probiotic intervention and the risk of neuropsychiatric disorders later in childhood: a randomized trial.

BACKGROUND: Recent experimental evidence suggests that gut microbiota may alter function within the nervous system providing new insight on the mechanism of neuropsychiatric disorders. METHODS: Seventy-five infants who were randomized to receive Lactobacillus rhamnosus GG (ATCC 53103) or placebo during the first 6 mo of life were followed-up for 13 y. Gut microbiota was assessed at the age of 3 wk, 3, 6, 12, 18, 24 mo, and 13 y using fluorescein in situ hybridization (FISH) and qPCR, and indirectly by determining the blood group secretor type at the age of 13 y. The diagnoses of attention deficit hyperactivity disorder (ADHD) and Asperger syndrome (AS) by a child neurologist or psychiatrist were based on ICD-10 diagnostic criteria. RESULTS: At the age of 13 y, ADHD or AS was diagnosed in 6/35 (17.1%) children in the placebo and none in the probiotic group (P = 0.008). The mean (SD) numbers of Bifidobacterium species bacteria in feces during the first 6 mo of life was lower in affected children 8.26 (1.24) log cells/g than in healthy children 9.12 (0.64) log cells/g; P = 0.03. CONCLUSION: Probiotic supplementation early in life may reduce the risk of neuropsychiatric disorder development later in childhood possible by mechanisms not limited to gut microbiota composition.

Altered duodenal microbiota composition in celiac disease patients suffering from persistent symptoms on a long-term gluten-free diet.

OBJECTIVES: A significant fraction of celiac disease patients suffer from persistent symptoms despite a long-term gluten-free diet (GFD) and normalized small bowel mucosa. The commonly suggested reasons, such as inadvertent gluten-intake or presence of other gastrointestinal disease, do not explain the symptoms in all these patients. Recently, alterations in intestinal microbiota have been associated with autoimmune disorders, including celiac disease. This led us to test a hypothesis that abnormal intestinal microbiota may be associated with persisting gastrointestinal symptoms in treated celiac disease patients. METHODS: Duodenal microbiota was analyzed in 18 GFD-treated patients suffering from persistent symptoms and 18 treated patients without symptoms by 16S rRNA gene pyrosequencing. The celiac disease patients had been following a strict GFD for several years and had restored small bowel mucosa and negative celiac autoantibodies. Their symptoms on GFD were assessed with Gastrointestinal Symptom Rating Scale. RESULTS: The results of several clustering methods showed that the treated celiac disease patients with persistent symptoms were colonized by different duodenal microbiota in comparison with patients without symptoms. The treated patients with persistent symptoms had a higher relative abundance of Proteobacteria (P=0.04) and a lower abundance of Bacteroidetes (P=0.01) and Firmicutes (P=0.05). Moreover, their microbial richness was reduced. The results indicated intestinal dysbiosis in patients with persistent symptoms even while adhering to a strict GFD. CONCLUSIONS: Our findings indicate that dysbiosis of microbiota is associated with persistent gastrointestinal symptoms in treated celiac disease patients and open new possibilities to treat this subgroup of patients.

Faecal microbiota composition in adults is associated with the FUT2 gene determining the secretor status.

The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota.

The duodenal microbiota composition of adult celiac disease patients is associated with the clinical manifestation of the disease.

BACKGROUND: Celiac disease is classically manifested in the gastrointestinal (GI) tract but extraintestinal symptoms, such as dermatitis herpetiformis (DH), are also common. Besides several well-known shared genetic risk factors and an environmental trigger, gliadin, factors determining the clinical outcome of the disease are not known. In this study, the role of duodenal microbiota in the celiac disease outcome was studied by analyzing mucosa-associated microbiota in celiac disease patients with a variety of intestinal and extraintestinal symptoms. METHODS: Microbiota in duodenal biopsy samples obtained from 33 patients with celiac disease with GI, DH, anemia, or mixed symptoms, as well as screen-detected asymptomatic celiac disease and 18 control subjects were analyzed using PCR denaturing gradient gel electrophoresis and a subset of samples additionally by the 16S ribosomal RNA gene sequencing. RESULTS: The composition and diversity of mucosal microbiota was associated with the manifestation of celiac disease when analyzed using PCR denaturing gradient gel electrophoresis and the 16S ribosomal RNA gene sequencing. The patients with celiac disease with GI symptoms or anemia had lower microbial diversity than those with DH. Moreover, the patients with GI symptoms had different intestinal microbiota composition and structure, dominated by Proteobacteria, in comparison to those with DH or control subjects (patients with dyspepsia). The relatively similar intestinal microbiota composition in the control subjects and those with DH was characterized by the high abundance of Firmicutes. CONCLUSIONS: The two common outcomes of celiac disease, classical GI and extraintestinal manifestations, had marked differences on the diversity and composition of intestinal microbiota. This association suggested that intestinal microbiota may have a role in the manifestation of the disease.

Association between the ABO blood group and the human intestinal microbiota composition.

BACKGROUND: The mucus layer covering the human intestinal epithelium forms a dynamic surface for host-microbial interactions. In addition to the environmental factors affecting the intestinal equilibrium, such as diet, it is well established that the microbiota composition is individually driven, but the host factors determining the composition have remained unresolved. RESULTS: In this study, we show that ABO blood group is involved in differences in relative proportion and overall profiles of intestinal microbiota. Specifically, the microbiota from the individuals harbouring the B antigen (secretor B and AB) differed from the non-B antigen groups and also showed higher diversity of the Eubacterium rectale-Clostridium coccoides (EREC) and Clostridium leptum (CLEPT) -groups in comparison with other blood groups. CONCLUSIONS: Our novel finding indicates that the ABO blood group is one of the genetically determined host factors modulating the composition of the human intestinal microbiota, thus enabling new applications in the field of personalized nutrition and medicine.

Isolation of bifidobacteria for blood group secretor status targeted personalised nutrition.

BACKGROUND: Currently, there is a constant need to find microbial products for maintaining or even improving host microbiota balance that could be targeted to a selected consumer group. Blood group secretor status, determining the ABO status, could be used to stratify the consumer group. OBJECTIVE: We have applied a validated upper intestinal tract model (TIM-1) and culturing methods to screen potential probiotic bacteria from faeces of blood secretor and non-secretor individuals. DESIGN: Faecal samples from healthy volunteers were pooled to age- and sex-matched secretor and non-secretor pools. Faecal pools were run through separate TIM-1 simulations, and bacteria were cultivated from samples taken at different stages of simulations for characterisation. RESULTS: Microbes in secretor pool survived the transit through TIM-1 system better than microbes of non-secretor pool, especially bifidobacteria and anaerobes were highly affected. The differences in numbers of bifidobacteria and lactobacilli isolates after plate cultivations and further the number of distinct RAPD-genotypes was clearly lower in non-secretor pool than in secretor pool. CONCLUSIONS: In the present study, we showed that microbiota of secretor and non-secretor individuals tolerate gastrointestinal conditions differently and that a combination of gastrointestinal simulations and cultivation methods proved to be a promising tool for isolating potentially probiotic bacteria.

Secretor genotype (FUT2 gene) is strongly associated with the composition of Bifidobacteria in the human intestine.

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.

Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

Strains of the cyanobacterial genera Calothrix and Rivularia isolated from the Baltic Sea display cryptic diversity and are distantly related to Gloeotrichia and Tolypothrix.

Strains of the cyanobacterial genus Calothrix display pronounced tapering filaments. These cyanobacteria are benthic, have a worldwide distribution and are among the most easily recognizable cyanobacterial genera. However, it is not clear whether cyanobacterial strains assigned to the genus Calothrix constitute a natural monophyletic group. We sequenced 16S rRNA genes from 42 cyanobacterial cultures and environmental samples belonging to the genus Calothrix, and the morphologically similar genera Rivularia, Gloeotrichia and Tolypothrix. Phylogenetic analysis of the 16S rRNA gene identified large sequence diversity among the Calothrix morphotype strains. Our results demonstrate that Calothrix, Gloeotrichia and Tolypothrix do not form a monophyletic group but instead display a high level of genetic diversity. The evolutionary distances between cyanobacteria, morphologically identified as Calothrix, suggest that they belong to at least five different genera. Our results also suggest that the genus Gloeotrichia is distantly related to the genus Calothrix. We found correlations between genetic grouping and morphology in redundancy analysis. However, morphology alone was not sufficiently reliable to distinguish strains from different 16S rRNA gene clusters. The high level of diversity that we observed confirms the hypothesis that the Rivulariaceae are species rich.

Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea.

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(-1) of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

Detection of microcystin-producing cyanobacteria in Finnish lakes with genus-specific microcystin synthetase gene E (mcyE) PCR and associations with environmental factors.

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.