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      Associations between the human intestinal microbiota, Lactobacillus rhamnosus GG and serum lipids indicated by integrated analysis of high-throughput profiling data

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          Abstract

          Accumulating evidence indicates that the intestinal microbiota regulates our physiology and metabolism. Bacteria marketed as probiotics confer health benefits that may arise from their ability to affect the microbiota. Here high-throughput screening of the intestinal microbiota was carried out and integrated with serum lipidomic profiling data to study the impact of probiotic intervention on the intestinal ecosystem, and to explore the associations between the intestinal bacteria and serum lipids. We performed a comprehensive intestinal microbiota analysis using a phylogenetic microarray before and after Lactobacillus rhamnosus GG intervention. While a specific increase in the L. rhamnosus-related bacteria was observed during the intervention, no other changes in the composition or stability of the microbiota were detected. After the intervention, lactobacilli returned to their initial levels. As previously reported, also the serum lipid profiles remained unaltered during the intervention. Based on a high-resolution microbiota analysis, intake of L. rhamnosus GG did not modify the composition of the intestinal ecosystem in healthy adults, indicating that probiotics confer their health effects by other mechanisms. The most prevailing association between the gut microbiota and lipid profiles was a strong positive correlation between uncultured phylotypes of Ruminococcus gnavus-group and polyunsaturated serum triglycerides of dietary origin. Moreover, a positive correlation was detected between serum cholesterol and Collinsella (Coriobacteriaceae). These associations identified with the spectrometric lipidome profiling were corroborated by enzymatically determined cholesterol and triglyceride levels. Actinomycetaceae correlated negatively with triglycerides of highly unsaturated fatty acids while a set of Proteobacteria showed negative correlation with ether phosphatidylcholines. Our results suggest that several members of the Firmicutes, Actinobacteria and Proteobacteria may be involved in the metabolism of dietary and endogenous lipids, and provide a scientific rationale for further human studies to explore the role of intestinal microbes in host lipid metabolism.

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          R: A language and environmental for statistical computing

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            Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine

            Background Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. Methods and Findings Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. Conclusions The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.
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              Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein.

              To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.
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                Author and article information

                Contributors
                Journal
                Peerj
                Peerj
                PeerJ
                PeerJ
                PeerJ
                PeerJ Inc. (San Francisco, USA )
                2167-8359
                26 February 2013
                2013
                : 1
                Affiliations
                [1 ]Department of Veterinary Biosciences, University of Helsinki , Finland
                [2 ]Laboratory of Microbiology, Wageningen University , Wageningen, Netherlands
                [3 ]Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki , Finland
                [4 ]Valio R&D , Helsinki, Finland
                [5 ]Quantitative Biology and Bioinformatics, VTT Technical Research Centre of Finland , Espoo, Finland
                Article
                32
                10.7717/peerj.32
                3628737
                23638368
                © 2013 Lahti et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Product
                Funding
                Funded by: Finnish Funding Agency for Technology and Innovation
                Award ID: (TEKES; grant number 40274/06)
                Funded by: European Research Council
                Award ID: (ERC; grant 250172)
                Funded by: Academy of Finland
                Award ID: (decisions 256950 to LL; 137389 to WMdV)
                This work was partly funded by the Finnish Funding Agency for Technology and Innovation (TEKES; grant number 40274/06), the ERC grant Microbes Inside (grant 250172), and the Academy of Finland (grant 137389) to WMdV. We gratefully acknowledge the HITChip team (Wageningen University, NL supported by the Spinoza Award of the Netherlands Foundation for Scientific Research to WMdV) for the HITChip analysis. The work at the University of Helsinki was performed at the Centre of Excellence on Microbial Food Safety Research, Academy of Finland. LL received funding from the Academy of Finland (decision 256950) and Alfred Kordelin Foundation. Support was received from Valio; a manufacturer of probiotics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Microbiology
                Clinical Trials
                Nutrition

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