Profile der intestinalen Mikrobiota des Menschen während Antibiotikagabe und Resilienz

Each human being harbors an individual and quite stable community of microorganisms within the gastrointestinal tract, whereas the highest density can be found in the large intestine. The ability of the microbiota to recover after an external perturbation is referred to as the resilience phenomenon. The aim of this study was the investigation of resilience of the colonic microbiota after a three day course of antibiotic perturbation with paromomycin and a subsequent therapy with probiotic VSL#3 or placebo over a period of 43 days. For this observation 16S rRNA gene libraries of the luminal as well as 16S rRNA and 16S rRNA gene amplicon libraries of the mucosa-associated microbiota were generated and sequenced via sequencing techniques according to the method of Sanger and pyrosequencing, respectively. This enabled the establishment of microbial profiles at different timepoints throughout the whole study. A quantitative real-time PCR was performed additionally. Antibiotic treatment resulted in decrease of richness and diversity within the luminal as well as the present and active mucosa-associated microbiota. This was underlined by a decrease in abundance of Operational Taxonomic Units (OTUs) homologous to Lachnospiraceae, Faecalibacterium and Blautia, which were identified as indicator species. Subsequent resilience was characterised by a complete recovery of richness and diversity of the mucosa-associated microbiota. Diversity of the luminal microbiota was not restored to pretreatment levels until the end of the study. Abundances of OTUs altered due to antibiotc treatment recovered in the course of the study in the luminal as well as the mucosa-associated microbiota. Probiotc VSL#3 had no influence on resilience of diversity and richness. Apart from a significant increase of luminal VSL#3 constituent Streptococcus thermophilus no further changes of microbial profiles were observed in either the luminal or the mucosa-associated microbiota due to probiotic therapy. Probiotic VSL#3 did not result in accelerated resilience of microbial composition after antibiotic perturbation. It must be emphasized that VSL#3 was shown to improve bowel function and symptoms in diseased individuals as well as result in an increase of short chain fatty acids and beneficial effects on molecular level within other studies. VSL#3 is therefore assumed to influence function rather than composition of the colonic microbiota. Future studies should address functional-related questions, thereby elucidating possible mechanisms by which probiotics confer a health benefit to the human host.Jeder Mensch besitzt eine individuelle und relativ stabile Gemeinschaft von Mikroorganismen innerhalb des Gastrointestinaltraktes, wobei die höchste Dichte innerhalb des Dickdarmes zu finden ist. Die Fähigkeit der Mikrobiota nach einer Schädigung zu regenerieren wird als Resilienz-Phänomen bezeichnet. Ziel dieser Studie war die Untersuchung der Resilienz der humanen Dickdarmmikrobiota nach einer drei-tägigen Perturbation mit dem Antibiotikum Paromomycin und anschließender 43-tägiger Probiotika- oder Plazebobehandlung. Dafür wurden 16S rRNA-Genbibliotheken der luminalen sowie 16S rRNA und 16S rRNA-Gen Amplikonbibliotheken der mukosa-assoziierten Mikrobiota generiert und mittels Sanger- bzw. Pyrosequenzierung analysiert. Dies ermöglichte die Erstellung von mikrobiellen Profilen zu unterschiedlichen Zeitpunkten der Studie. Zusätzlich wurde eine quantitative real-time PCR durchgeführt. Die Antibiotikabehandlung resultierte in einer Abnahme des Artenreichtums und der -diversität der luminalen sowie der mukosa-assoziierten Mikrobiota. Dies wurde durch eine Abnahme in der Abundanz von Operational Taxonomic Units (OTUs) mit Homologie zu Lachnospiraceae, Faecalibacterium und Blautia, die auch als Indikator Spezies identifiziert wurden, unterstrichen. Die nachfolgende Resilienz war durch eine vollständige Regeneration von Artenreichtum und -diversität der mukosa-assoziierten Mikrobiota gekennzeichnet. Die Diversität der luminalen Mikrobiota konnte bis zum Ende der Studie das anfänglichen Niveau nicht erreichen. Die durch die Antibiotikagabe veränderten Abundanzen der OTUs der luminalen wie auch der mukosa-assoziierten Mikrobiota erholten sich im Laufe der Studie. Das Probiotikum VSL#3 hatte keinen Einfluss auf die Resilienz von Artenreichtum und -diversität. Außer einem signifikanten Anstieg des im VSL#3 enthaltenen Streptococcus thermophilus im Lumen, konnten keine weiteren Veränderungen in den mikrobiellen Profilen durch die Probiotikagabe beobachtet werden. Das Probiotikum VSL#3 hatte keinen Einfluss auf die Resilienz der mikrobiellen Zusammensetzung nach Antibiotikagabe. Allerdings wurde gezeigt, dass VSL#3 Gabe eine Verbesserung von Darmfunktion und Symptomen bei erkrankten Individuen sowie einen Anstieg von kurzkettigen Fettsäuren und günstige Einflüsse auf molekularer Ebene zur Folge hatte. Daher wird angenommen, dass VSL#3 eher die Funktionen als die Zusammensetzung der Mikrobiota beeinflusst. Zukünftige Studien sollten funktionell bedingte Fragestellungen beantworten, die es ermöglichen die Mechanismen, durch die Probiotika ihre vorteilhafte Wirkung hervorrufen, aufzuklären

Effects of probiotics and antibiotics on the intestinal homeostasis in a computer controlled model of the large intestine

Background: Antibiotic associated diarrhea and Clostridium difficile infection are frequent complications of broad spectrum antibiotic therapy. Probiotic bacteria are used as therapeutic and preventive agents in these disorders, but the exact functional mechanisms and the mode of action are poorly understood. The effects of clindamycin and the probiotic mixture VSL#3 (containing the 8 bacterial strains Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii subsp. Bulgaricus) consecutively or in combination were investigated and compared to controls without therapy using a standardized human fecal microbiota in a computer-controlled in vitro model of large intestine. Microbial metabolites (short chain fatty acids, lactate, branched chain fatty acids, and ammonia) and the intestinal microbiota were analyzed. Results: Compared to controls and combination therapy, short chain fatty acids and lactate, but also ammonia and branched chain fatty acids, were increased under probiotic therapy. The metabolic pattern under combined therapy with antibiotics and probiotics had the most beneficial and consistent effect on intestinal metabolic profiles. The intestinal microbiota showed a decrease in several indigenous bacterial groups under antibiotic therapy, there was no significant recovery of these groups when the antibiotic therapy was followed by administration of probiotics. Simultaneous application of anti- and probiotics had a stabilizing effect on the intestinal microbiota with increased bifidobacteria and lactobacilli. Conclusions: Administration of VSL#3 parallel with the clindamycin therapy had a beneficial and stabilizing effect on the intestinal metabolic homeostasis by decreasing toxic metabolites and protecting the endogenic microbiota from destruction. Probiotics could be a reasonable strategy in prevention of antibiotic associated disturbances of the intestinal homeostasis and disorders. © 2012 Rehman et al; licensee BioMed Central Lt

Paternal chronic colitis causes epigenetic inheritance of susceptibility to colitis.

Inflammatory bowel disease (IBD) arises by unknown environmental triggers in genetically susceptible individuals. Epigenetic regulation of gene expression may integrate internal and external influences and may thereby modulate disease susceptibility. Epigenetic modification may also affect the germ-line and in certain contexts can be inherited to offspring. This study investigates epigenetic alterations consequent to experimental murine colitis induced by dextran sodium sulphate (DSS), and their paternal transmission to offspring. Genome-wide methylome- and transcriptome-profiling of intestinal epithelial cells (IECs) and sperm cells of males of the F0 generation, which received either DSS and consequently developed colitis (F0(DSS)), or non-supplemented tap water (F0(Ctrl)) and hence remained healthy, and of their F1 offspring was performed using reduced representation bisulfite sequencing (RRBS) and RNA-sequencing (RNA-Seq), respectively. Offspring of F0(DSS) males exhibited aberrant methylation and expression patterns of multiple genes, including Igf1r and Nr4a2, which are involved in energy metabolism. Importantly, DSS colitis in F0(DSS) mice was associated with decreased body weight at baseline of their F1 offspring, and these F1 mice exhibited increased susceptibility to DSS-induced colitis compared to offspring from F0(Ctrl) males. This study hence demonstrates epigenetic transmissibility of metabolic and inflammatory traits resulting from experimental colitis.This study was carried out as part of the Research Training Group “Genes, Environment and Inflammation”, supported by the Deutsche Forschungsgemeinschaft (RTG 1743/1) of which A.F. is the spokesperson, the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007–2013)/ERC Grant agreement no. 260961 (A.K.), the Austrian Science Fund and Ministry of Science P21530-B18 and START Y446-B18 (A.K.), the Wellcome Trust (investigator award 106260/Z/14/Z) to A.K., the Cambridge Biomedical Research Centre (A.K.), a fellowship from the European Crohn’s and Colitis Organisation (M.T. and T.E.A.) and a DOC fellowship from the Austrian Academy of Sciences (J.K.).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep3164

Effects of b-lactam antibiotics and fluoroquinolones on human gut microbiota in relation to clostridium difficile associated diarrhea

Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome.This work was supported by the ERANET Project PathoGenoMics program grant number 0315441A.Peer Reviewe

Rhinovirus infections change DNA methylation and mRNA expression in children with asthma.

Human rhinovirus infection (HRVI) plays an important role in asthma exacerbations and is thought to be involved in asthma development during early childhood. We hypothesized that HRVI causes differential DNA methylation and subsequently differential mRNA expression in epithelial cells of children with asthma. Primary nasal epithelial cells from children with (n = 10) and without (n = 10) asthma were cultivated up to passage two and infected with Rhinovirus-16 (RV-16). HRVI-induced genome-wide differences of DNA methylation in asthmatics (vs. controls) and resulting mRNA expression were analyzed by the HumanMethylation450 BeadChip Kit (Illumina) and RNA sequencing. These results were further verified by pyrosequencing and quantitative PCR, respectively. 471 CpGs belonging to 268 genes were identified to have HRVI-induced asthma-specifically modified DNA methylation and mRNA expression. A minimum-change criteria was applied to restrict assessment of genes with changes in DNA methylation and mRNA expression of at least 3% and least 0.1 reads/kb per million mapped reads, respectively. Using this approach we identified 16 CpGs, including HLA-B-associated transcript 3 (BAT3) and Neuraminidase 1 (NEU1), involved in host immune response against HRVI. HRVI in nasal epithelial cells leads to specific modifications of DNA methylation with altered mRNA expression in children with asthma. The HRVI-induced alterations in DNA methylation occurred in genes involved in the host immune response against viral infections and asthma pathogenesis. The findings of our pilot study may partially explain how HRVI contribute to the persistence and progression of asthma, and aid to identify possible new therapeutic targets. The promising findings of this pilot study would benefit from replication in a larger cohort

Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

Background The interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution, and development of both players. These interdependencies inspired a new view of multicellular organisms as “metaorganisms.” The goal of the Collaborative Research Center “Origin and Function of Metaorganisms” is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants. Methods In order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample. Conclusion While 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Overall, we recommend single- over multi-step amplification procedures, and although exceptions and trade-offs exist, the V3 V4 over the V1 V2 region of the 16S rRNA gene. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition

Frequencies of <i>C4B*Q0</i> carriers in healthy German individuals of different age-groups stratified for (a) smokers (current smokers and quitters <3 years) and (b) non-smokers (never smokers and quitters for ≥3 years).

<p>For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086188#pone-0086188-g001" target="_blank">Figure 1</a>. Due to the small number of centenarians in the replication sample we did not subdivide the case sample into a nonagenarian and centenarian subgroup.</p