Molecular analysis of intestinal microbiota composition and function in individuals with differen diets, age and disease status

Die Fragestellung der vorliegenden Doktoratsarbeit war, ob epidemiologisch definierte Bevoelkerungsgruppen wie VegetarierInnen, MischkoestlerInnen, alte und junge Menschen sich hinsichtlich ihrer Mikrobiota-Zusammensetzung unterscheiden. Weiters war es ein Ziel die Einfluesse von Chemotherapie und Antibiotika auf die GI-Mikrobiota im Vergleich zu gesunden Kontrollen zu charakterisieren. Stuhlproben von jungen Omnivoren, Vegetarierinnen, AltersheimbewohnerInnen und Individuen, die immunsuppressive Chemotherapie erhalten, wurden untersucht. Ernaehrungsgewohnheiten und Lebensstilfaktoren wurden in Interviews anhand eines Fragebogens erfasst. Moegliche Unterschiede sollten anhand der Abundanz und Fuelle an Spezies (richness) der dominierenden Bakterien und Bifidobakterien, Clostridium leptum cluster, clostridium coccoides cluster und Bacteroides beschrieben werden. Ausschliesslich molekulare Methoden, naemlich PCR-DGGE fingerprinting, quantitative PCR und stable isotope probing wurden angewandt. Um sinnvolle Interpretation der hoch diversen DGGE fingerprinting Daten zu erlauben, wurde Clustering basierend auf Pearson Korrelation und Hauptkomponentenanalyse basierend auf Kovarianz angewandt. Als ein Schritt in Richtung funktioneller Charakterisierung wurde das Butyryl-CoA CoA Transferase Gen zusaetzlich zur Quantifizierung des 16S rRNA Gens, anhand dessen phylogenetische Identifizierung vorgenommen wurde, durchgefuehrt. RNA-stable isotope probing wurde als vielversprechende Methode identifiziert, um phylogenetische Information mit funktionellen Charakteristika von mikrobiellen Gemeinschaften des Darms zu verbinden.The aim of this Ph.D. thesis was to test if epidemiologically defined population groups such as vegetarians, omnivores, the young and the elderly differ in their microbiota composition. Furthermore, the influences of chemotherapy and antibiotic treatment on the GI microbiota should be monitored during the course of treatment. Faecal samples of young healthy omnivores and vegetarians, institutionalized elderly and individuals undergoing immune-suppressive chemotherapy were analysed. Diet and lifestyle were assessed using an interviewer-conducted questionnaire. The objective was to compare abundance and species richness of Bacteria as well as subgroups such as bifidobacteria, Bacteroides, Clostridium leptum cluster, Clostridium coccoides cluster in faecal samples of omnivores, vegetarians, elderly and individuals undergoing chemotherapy. Molecular methods such as PCR-DGGE fingerprinting and qPCR as well as stable isotope probing were applied. To allow meaningful interpretation of the highly diverse DGGE fingerprinting datasets, clustering based on Pearson correlation and principal components analysis based on covariance were applied. As a step towards a functional characterization, the butyryl-CoA CoA transferase gene was targeted in addition to the 16S rRNA gene used for phylogenetic identification. RNA-stable isotope probing was explored as a means of linking phylogenetic information with functional characteristics of gut microbial communities

Isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage mould Penicillium commune and their potential as biopreservatives in cheese

Moulds are the most common cheese spoilage organisms which can lead to economic loss as well as raising public health concerns due to the production of mycotoxins. In this study, 897 lactic acid bacteria (LAB) isolated from different herbs, fruits and vegetables were screened for their antifungal activity in an agar plate overlay assay. Thirty-six isolates had weak activity, 11 had moderate activity and 12 were confirmed as having strong activity. The strong antifungal isolates were obtained from a range of different sources but were all identified by 16S rDNA sequencing as being Lactobacillus plantarum. The antiftmgal spectra for these 12 isolates were determined against eight other moulds commonly associated with cheese spoilage and all isolates were found to possess inhibition against Penicillium solitum, Aspergillus versicolor and Cladosporium herbarum, but not against Penicillium roqueforti, Penicillium glabrum, Mucor circinelloides, Geotrichum candidum or Byssochlamys nivea. The absence of sodium acetate from MRS agar resulted in no inhibition of Penicilium commune, suggesting the synergistic effect of acetic acid with the antifungal LAB, similarly to that previously reported. To determine their potential as biopreservatives in cheese, LAB isolates were inoculated into cottage cheese prior to the addition of P. commune. All Lb. plantarum isolates were found to prevent the visible growth of P. commune on cottage cheese by between 14 and >25 days longer than cottage cheese that contained either no added LAB or LAB that did not have antifungal activity (Lactococcus lactis, Weissella soli, Leuconostoc inhae and Leuconostoc mesenteroides isolates). The results of this study shows that LAB isolated from various herbs, fruits and vegetables possess antifungal activity and have potential for use as biopreservatives in cheese. (C) 2014 Elsevier Ltd. All rights reserved

Changes in Human Fecal Microbiota Due to Chemotherapy Analyzed by TaqMan-PCR, 454 Sequencing and PCR-DGGE Fingerprinting

BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037) following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics

A PCR-DGGE fingerprinting of 16S rRNA coding regions of dominant bacteria over time.

<p>Bands that become stronger or nearly disappear following a single chemotherapeutic treatment are indicated with arrows. B Principal components analysis (PCA) based on dominant bacteria PCR-DGGE fingerprinting. The two outliers in the lower right corner of the plot are two samples of P07 following blood stem cell transplantation. C PCA illustrating the development of <i>Clostridium</i> cluster <i>IV</i> diversity in the course of chemotherapy and antibiotic treatment. Cluster <i>IV</i> diversity drops right after chemotherapy, causing a grouping of samples. Samples under antibiotic treatment (indicated as grey dots) group even closer, indicating a strong influence of antibiotics on <i>Clostridium</i> cluster <i>IV</i> diversity. A, sample of P01 before chemotherapy B, C and D, samples of P01 after chemotherapy; E, healthy control; SL, unrelated standard lane; black symbols… patients under chemotherapy and antibiotic treatment.</p

Number of bands observed in PCR-DGGE fingerprinting in oncology patients before chemotherapy (T₀), immediately after chemotherapy (T₁) and 5–9 days after chemotherapy (T₂) and healthy controls averaged over all time points.

<p>Number of bands observed in PCR-DGGE fingerprinting in oncology patients before chemotherapy (T₀), immediately after chemotherapy (T₁) and 5–9 days after chemotherapy (T₂) and healthy controls averaged over all time points.</p

Phylogenetic tree showing the <i>Peptostreptococcaceae</i> found in samples from two oncology patients before and after chemotherapy.

<p>Identical sequences were grouped; the table on the right hand side shows their abundances in the 454 sequencing dataset. Sequences with >98.9% similarity to <i>Clostridium difficile</i> appeared only in samples taken immediately after chemotherapeutic cycles. Numbers indicate bootstrap values after 100 resamplings.</p

16S rRNA gene primers used for PCR-DGGE fingerprinting.

<p>16S rRNA gene primers used for PCR-DGGE fingerprinting.</p

Heatmap showing abundances within the 454 sequencing dataset on the genus level.

<p>High throughput sequencing of samples P09 and P11 before (T₀) and after therapy (T₁) further helped to characterize the influence of a single chemotherapeutic cycle on the GI-microbiota. P11 was treated with chemotherapy alone and P09 also received antibiotic treatment.</p

Abundances of bacterial 16S rRNA coding regions over time in oncology patients (P) and healthy controls (C).

<p>The declined abundances of bacteria, <i>Bacteroides</i>, <i>Clostridium</i> cluster <i>XIVa</i>, <i>Clostridium</i> cluster <i>IV</i> and bifidobacteria immediately after chemotherapy (T₁) were observed to recover several days after treatment (T₂). Patients P04, P08 and P13 had never received chemotherapy before; P04, P05, P07, P08, P09 and P10 took antibiotics. Values were z-scored for presentation in this heatmap showing changes over time rather than absolute abundances. T₀, before chemotherapy; T₁, 1–2 days after chemotherapy; T₂, 5–9 days after chemotherapy; F, fever; S, blood stem cell transplantation.</p