Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder that has been associated with aberrant microbiota. This review focuses on the recent molecular insights generated by analysing ...the intestinal microbiota in subjects suffering from IBS. Special emphasis is given to studies that compare and contrast the microbiota of healthy subjects with that of IBS patients classified into different subgroups based on their predominant bowel pattern as defined by the Rome criteria. The current data available from a limited number of patients do not reveal pronounced and reproducible IBS-related deviations of entire phylogenetic or functional microbial groups, but rather support the concept that IBS patients have alterations in the proportions of commensals with interrelated changes in the metabolic output and overall microbial ecology. The lack of apparent similarities in the taxonomy of microbiota in IBS patients may partially arise from the fact that the applied molecular methods, the nature and location of IBS subjects, and the statistical power of the studies have varied considerably. Most recent advances, especially the finding that several uncharacterized phylotypes show non-random segregation between healthy and IBS subjects, indicate the possibility of discovering bacteria specific for IBS. Moreover, tools are being developed for the functional analysis of the relationship between the intestinal microbiota and IBS. These approaches may be instrumental in the evaluation of the ecological dysbiosis hypothesis in the gut ecosystem. Finally, we discuss the future outlook for research avenues and candidate microbial biomarkers that may eventually be used in IBS diagnosis.
Aims: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA‐targeted species‐ and group‐specific primers ...for more accurate quantification of bacteria from faecal samples with real‐time PCR.
Methods and Results: A linear range of quantification between 0·1–10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30–4500 to 1·9 × 106–6·0 × 106 target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification.
Conclusions: Real‐time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples.
Significance and Impact of the Study: To design and optimize an extensive set of real‐time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.
Summary
Background A multispecies probiotic has shown beneficial effects in irritable bowel syndrome. In addition, certain other probiotics have demonstrated advantageous effects, but the mechanisms ...behind this are poorly understood.
Aim To investigate the mode of action of a multispecies probiotic consisting of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 by monitoring its effects on intestinal microbiota and markers of microbial activity.
Methods A total of 55 irritable bowel syndrome patients participated in this placebo‐controlled double‐blind trial. Subjects received either multispecies probiotic or placebo supplementation daily during a 6‐month period. The composition of intestinal microbiota was analysed with real‐time polymerase chain reaction, short‐chain fatty acids with gas chromatography and enzymes with spectrophotometer.
Results Each supplemented probiotic strain was detected in faecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group (P = 0.028). No changes in short‐chain fatty acids occurred. A decrease in ß‐glucuronidase activity was detected in 67% of the subjects in the probiotic group vs. 38% in the placebo group (P = 0.06).
Conclusions Factors other than the microbial groups and metabolites studied herein seem responsible for the alleviation of irritable bowel syndrome symptoms by the multispecies probiotic.
Aims: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional ...properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects.
Methods and Results: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR–ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed‐field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests.
Conclusions: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain‐dependent.
Significance and Impact of the Study: Applicability of ribotyping with the automated RiboPrinter® System for identification and genotyping of bifidobacteria was shown in the present study.
Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. ...This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.
Aims: The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and ...colorimetric hybridization (PCR–ELISA).
Methods and Results: Forty‐two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR–ELISA. Oligonucleotide probes used for hybridization in PCR–ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour‐developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum‐related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum.
Conclusions: Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR–ELISA.
Significance and Impact of the Study: PCR–ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.
Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In ...order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.
An adequate bowel cleansing is essential for a successful colonoscopy. Although purgative consumption is safe for the patient, there is little consensus on how the intestinal microbiota is affected ...by the procedure, especially regarding the potential long-term consequences.
23 healthy subjects were randomised into two study groups consuming a bowel preparation (Moviprep), either in two separate doses of 1 L or as a single 2-L dose. Participants donated faecal samples at the baseline, after bowel cleansing, 14 and 28 days after the treatment. The intestinal microbiota composition was determined with phylogenetic microarray as well as quantitative PCR analysis and correlated with the previously quantified faecal serine proteases.
The lavage introduced an instant and substantial change to the intestinal microbiota. The total microbial load was decreased by 31-fold and 22% of the participants lost the subject-specificity of their microbiota. While the bacterial levels and community composition were essentially restored within 14 days, the rate of recovery was dose dependent: consumption of the purgative in a single dose had a more severe effect on the microbiota composition than that of a double dose, and notably increased the levels of Proteobacteria, Fusobacteria and bacteria related to Dorea formicigenerans. The abundance of the latter also correlated with the amount of faecal serine proteases that were increased after purging.
Our results suggest that the bowel cleansing using two separate dosages introduces fewer alterations to the intestinal microbiota than a single dose and hence may be preferred in clinical practice.
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