Amélia Camarinha-Silva

Comparing the anterior nare bacterial community of two discrete human populations using Illumina amplicon sequencing

The anterior nares are an important reservoir for opportunistic pathogens and commensal microorganisms. A barcoded Illumina paired-end sequencing method targeting the 16S ribosomal RNA V1-2 hypervariable region was developed to compare the bacterial diversity of the anterior nares across distinct human populations (volunteers from Germany vs a Babongo Pygmy tribe, Africa). Of the 251 phylotypes detected, 231 could be classified to the genus level and 109 to the species level, including the unambiguous identification of the ubiquitous Staphylococcus aureus and Moraxella catarrhalis. The global bacterial community of both adult populations revealed that they shared 85% of the phylotypes, suggesting that our global bacterial communities have likely been with us for thousands of years. Of the 34 phylotypes unique to the non-westernized population, most were related to members within the suborder Micrococcineae. There was an even more overwelming distinction between children and adults of the same population, suggesting a progression of a childhood community of high-diversity comprising species of Moraxellaceae and Streptococcaceae to an adult community of lower diversity comprising species of Propionibacteriaceae, Clostridiales Incertae Sedis XI, Corynebacteriaceae and Staphylococcaceae. Thus, age was a stronger factor for accounting for differing bacterial assemblages than the origin of the human population sampled.

News in livestock research — use of Omics-technologies to study the microbiota in the gastrointestinal tract of farm animals

Technical progress in the field of next-generation sequencing, mass spectrometry and bioinformatics facilitates the study of highly complex biological samples such as taxonomic and functional characterization of microbial communities that virtually colonize all present ecological niches. Compared to the structural information obtained by metagenomic analyses, metaproteomic approaches provide, in addition, functional data about the investigated microbiota. In general, integration of the main Omics-technologies (genomics, transcriptomics, proteomics and metabolomics) in live science promises highly detailed information about the specific research object and helps to understand molecular changes in response to internal and external environmental factors. The microbial communities settled in the mammalian gastrointestinal tract are essential for the host metabolism and have a major impact on its physiology and health. The microbiotas of livestock like chicken, pig and ruminants are becoming a focus of interest for veterinaries, animal nutritionists and microbiologists. While pig is more often used as an animal model for human-related studies, the rumen microbiota harbors a diversity of enzymes converting complex carbohydrates into monomers which bears high potential for biotechnological applications. This review will provide a general overview about the recent Omics-based research of the microbiota in livestock including its major findings. Differences concerning the results of pre-Omics-approaches in livestock as well as the perspectives of this relatively new Omics-platform will be highlighted.

Intestinal Microbiota and Microbial Metabolites Are Changed in a Pig Model Fed a High-Fat/Low-Fiber or a Low-Fat/High-Fiber Diet

The intestinal microbiota and its metabolites appear to be an important factor for gastrointestinal function and health. However, research is still needed to further elaborate potential relationships between nutrition, gut microbiota and host’s health by means of a suitable animal model. The present study examined the effect of two different diets on microbial composition and activity by using the pig as a model for humans. Eight pigs were equally allotted to two treatments, either fed a low-fat/high-fiber (LF), or a high-fat/low-fiber (HF) diet for 7 weeks. Feces were sampled at day 7 of every experimental week. Diet effects on fecal microbiota were assessed using quantitative real-time PCR, DNA fingerprinting and metaproteomics. Furthermore, fecal short-chain fatty acid (SCFA) profiles and ammonia concentrations were determined. Gene copy numbers of lactobacilli, bifidobacteria (P<0.001) and Faecalibacterium prausnitzii (P<0.05) were higher in the LF pigs, while Enterobacteriaceae were more abundant in the HF pigs (P<0.001). Higher numbers of proteins affiliated to Enterobacteriaceae were also present in the HF samples. Proteins for polysaccharide breakdown did almost exclusively originate from Prevotellaceae. Total and individual fecal SCFA concentrations were higher for pigs of the LF treatment (P<0.05), whereas fecal ammonia concentrations did not differ between treatments (P>0.05). Results provide evidence that beginning from the start of the experiment, the LF diet stimulated beneficial bacteria and SCFA production, especially butyrate (P<0.05), while the HF diet fostered those bacterial groups which have been associated with a negative impact on health conditions. These findings correspond to results in humans and might strengthen the hypothesis that the response of the porcine gut microbiota to a specific dietary modulation is in support of using the pig as suitable animal model for humans to assess diet-gut-microbiota interactions. Data are available via ProteomeXchange with identifier PXD003447.

Evaluation of DNA extraction kits and phylogenetic diversity of the porcine gastrointestinal tract based on Illumina sequencing of two hypervariable regions.

A robust DNA extraction method is important to identify the majority of microorganisms present in environmental microbial communities and to enable a consistent comparison between different studies. Here, 15 manual and four automated commercial DNA extraction kits were evaluated for their efficiency to extract DNA from porcine feces and ileal digesta samples. DNA yield, integrity, and purity varied among the different methods. Terminal restriction fragment length polymorphism (T‐RFLP) and Illumina amplicon sequencing were used to characterize the diversity and composition of the microbial communities. We also compared phylogenetic profiles of two regions of the 16S rRNA gene, one of the most used region (V1–2) and the V5–6 region. A high correlation between community structures obtained by analyzing both regions was observed at genus and family level for ileum digesta and feces. Based on our findings, we want to recommend the FastDNA™ SPIN Kit for Soil (MP Biomedical) as a suitable kit for the analyses of porcine gastrointestinal tract samples.

Low diversity of planktonic bacteria in the tropical ocean.

The diversity of macro-organisms increases towards the equator, with almost no exceptions. It is the most conserved biogeographical pattern on earth and is thought to be related to the increase of temperature and productivity in the tropics. The extent and orientation of a latitudinal gradient of marine bacterioplankton diversity is controversial. Here we studied the euphotic zone of the Atlantic Ocean based on a transect covering ~12.000 km from 51°S to 47 °N. Water samples were collected at 26 stations at five depths between 20 and 200 m and sequentially filtered through 8 μm, 3 μm and 0,22 μm filters, resulting in a total of 359 samples. Illumina sequencing of the V5–V6 region of the 16S rRNA gene revealed a clear biogeographic pattern with a double inverted latitudinal gradient. Diversity was higher in mid-latitudinal regions of the Atlantic Ocean and decreased towards the equator. This pattern was conserved for bacteria from all three planktonic size fractions. Diversity showed a non-linear relationship with temperature and was negatively correlated with bacterial cell numbers in the upper depth layers (<100 m). The latitudinal gradients of marine bacterial diversity and the mechanisms that govern them are distinct from those found in macro-organisms.

Insights into Broilers' Gut Microbiota Fed with Phosphorus, Calcium, and Phytase Supplemented Diets

Phytase supplementation in broiler diets is a common practice to improve phosphorus (P) availability and to reduce P loss by excretion. An enhanced P availability, and its concomitant supplementation with calcium (Ca), can affect the structure of the microbial community in the digestive tract of broiler chickens. Here, we aim to distinguish the effects of mineral P, Ca, and phytase on the composition of microbial communities present in the content and the mucosa layer of the gastrointestinal tract (GIT) of broiler chickens. Significant differences were observed between digesta and mucosa samples for the GIT sections studied (p = 0.001). The analyses of 56 individual birds showed a high microbial composition variability within the replicates of the same diet. The average similarity within replicates of digesta and mucosa samples across all diets ranged from 29 to 82% in crop, 19–49% in ileum, and 17–39% in caeca. Broilers fed with a diet only supplemented with Ca had the lowest body weight gain and feed conversion values while diets supplemented with P showed the best performance results. An effect of each diet on crop mucosa samples was observed, however, similar results were not obtained from digesta samples. Microbial communities colonizing the ileum mucosa samples were affected by P supplementation. Caeca-derived samples showed the highest microbial diversity when compared to the other GIT sections and the most prominent phylotypes were related to genus Faecalibacterium and Pseudoflavonifractor, known for their influence on gut health and as butyrate producers. Lower microbial diversity in crop digesta was linked to lower growth performance of birds fed with a diet only supplemented with Ca. Each diet affected microbial communities within individual sections, however, no diet showed a comprehensive effect across all GIT sections, which can primarily be attributed to the great variability among replicates. The substantial community differences between digesta and mucosa derived samples indicate that both habitats have to be considered when the influence of diet on the gut microbiota, broiler growth performance, and animal health is investigated.

A Structural and Functional Elucidation of the Rumen Microbiome Influenced by Various Diets and Microenvironments

The structure and function of the microbiome inhabiting the rumen are, amongst other factors, mainly shaped by the animals feed intake. Describing the influence of different diets on the inherent community arrangement and associated metabolic activities of the most active ruminal fractions (bacteria and archaea) is of great interest for animal nutrition, biotechnology, and climatology. Samples were obtained from three fistulated Jersey cows rotationally fed with corn silage, grass silage or grass hay, each supplemented with a concentrate mixture. Samples were fractionated into ruminal fluid, particle-associated rumen liquid, and solid matter. DNA, proteins and metabolites were analyzed subsequently. DNA extracts were used for Illumina sequencing of the 16S rRNA gene and the metabolomes of rumen fluids were determined by 500 MHz-NMR spectroscopy. Tryptic peptides derived from protein extracts were measured by LC-ESI-MS/MS and spectra were processed by a two-step database search for quantitative metaproteome characterization. Data are available via ProteomeXchange with the identifier PXD006070. Protein- and DNA-based datasets revealed significant differences between sample fractions and diets and affirmed similar trends concerning shifts in phylogenetic composition. Ribosomal genes and proteins belonging to the phylum of Proteobacteria, particularly Succinivibrionaceae, exhibited a higher abundance in corn silage-based samples while fiber-degraders of the Lachnospiraceae family emerged in great quantities throughout the solid phase fractions. The analysis of 8163 quantified bacterial proteins revealed the presence of 166 carbohydrate active enzymes in varying abundance. Cellulosome affiliated proteins were less expressed in the grass silage, glycoside hydrolases appeared in slightest numbers in the corn silage. Most expressed glycoside hydrolases belonged to families 57 and 2. Enzymes analogous to ABC transporters for amino acids and monosaccharides were more abundant in the corn silage whereas oligosaccharide transporters showed a higher abundance in the fiber-rich diets. Proteins involved in carbon metabolism were detected in high numbers and identification of metabolites like short-chain fatty acids, methylamines and phenylpropionate by NMR enabled linkage between producers and products. This study forms a solid basis to retrieve deeper insight into the complex network of microbial adaptation in the rumen.

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

Host Genome Influence on Gut Microbial Composition and Microbial Prediction of Complex Traits in Pigs

The aim of the present study was to analyze the interplay between gastrointestinal tract (GIT) microbiota, host genetics, and complex traits in pigs using extended quantitative-genetic methods. The study design consisted of 207 pigs that were housed and slaughtered under standardized conditions, and phenotyped for daily gain, feed intake, and feed conversion rate. The pigs were genotyped with a standard 60 K SNP chip. The GIT microbiota composition was analyzed by 16S rRNA gene amplicon sequencing technology. Eight from 49 investigated bacteria genera showed a significant narrow sense host heritability, ranging from 0.32 to 0.57. Microbial mixed linear models were applied to estimate the microbiota variance for each complex trait. The fraction of phenotypic variance explained by the microbial variance was 0.28, 0.21, and 0.16 for daily gain, feed conversion, and feed intake, respectively. The SNP data and the microbiota composition were used to predict the complex traits using genomic best linear unbiased prediction (G-BLUP) and microbial best linear unbiased prediction (M-BLUP) methods, respectively. The prediction accuracies of G-BLUP were 0.35, 0.23, and 0.20 for daily gain, feed conversion, and feed intake, respectively. The corresponding prediction accuracies of M-BLUP were 0.41, 0.33, and 0.33. Thus, in addition to SNP data, microbiota abundances are an informative source of complex trait predictions. Since the pig is a well-suited animal for modeling the human digestive tract, M-BLUP, in addition to G-BLUP, might be beneficial for predicting human predispositions to some diseases, and, consequently, for preventative and personalized medicine.

Linking microbial community and catabolic gene structures during the adaptation of three contaminated soils under continuous long term pollutant stress

ABSTRACT Three types of contaminated soil from three geographically different areas were subjected to a constant supply of benzene or benzene/toluene/ethylbenzene/xylenes (BTEX) for a period of 3 months. Different from the soil from Brazil (BRA) and Switzerland (SUI), the Czech Republic (CZE) soil which was previously subjected to intensive in situ bioremediation displayed only negligible changes in community structure. BRA and SUI soil samples showed a clear succession of phylotypes. A rapid response to benzene stress was observed, whereas the response to BTEX pollution was significantly slower. After extended incubation, actinobacterial phylotypes increased in relative abundance, indicating their superior fitness to pollution stress. Commonalities but also differences in the phylotypes were observed. Catabolic gene surveys confirmed the enrichment of actinobacteria by identifying the increase of actinobacterial genes involved in the degradation of pollutants. Proteobacterial phylotypes increased in relative abundance in SUI microcosms after short-term stress with benzene, and catabolic gene surveys indicated enriched metabolic routes. Interestingly, CZE soil, despite staying constant in community structure, showed a change in the catabolic gene structure. This indicates that a highly adapted community, which had to adjust its gene pool to meet novel challenges, has been enriched.