Gut colonization by a novel Clostridium species is associated with the onset of epizootic rabbit enteropathy

Epizootic rabbit enteropathy (ERE) represents one of the most devastating diseases affecting rabbit farms. Previous studies showing transmissibility of disease symptoms through oral inoculation of intestinal contents from sick animals suggested a bacterial infectious origin for ERE. However, no etiological agent has been identified yet. On the other hand, ERE is associated with major changes in intestinal microbial communities, pinpointing dysbiosis as an alterna‑ tive cause for the disease. To better understand the role of intestinal bacteria in ERE development, we have performed a prospective longitudinal study in which intestinal samples collected from the same animals before, during and after disease onset were analyzed using high‑throughput sequencing. Changes in hundreds of bacterial groups were detected after the initiation of ERE. In contrast, before ERE onset, the microbiota from rabbits that developed ERE did not differ from those that remained healthy. Notably, an expansion of a single novel Clostridium species (Clostridium cuniculi) was detected the day of ERE onset. C. cuniculi encodes several putative toxins and it is phylogenetically related to the two well‑characterized pathogens C. botulinum and C. perfringens. Our results are consistent with a bac‑ terial infectious origin of ERE and discard dysbiosis as the initial trigger of the disease. Although experimental valida‑ tion is required, results derived from sequencing analysis, propose a key role of C. cuniculi in ERE initiation

Methodological preparation and characteritzation of the microbial ecology on the skin

The study of skin microbiota has always been focused from a clinical point of view. However, an ecological approach to the skin microbiota is impeded by different methodological limitations, including the high host/microbial DNA ratio or the low microbial content. In contrast with the burgeoning field of gut metagenomics, skin metagenomics has been hindered by the absence of an efficient methodology to work with skin microbial DNA. This thesis aims to settle the basis for further human skin microbiome studies from a systems approach. I have set four different important approaches for a ecological and systematic view of skin: 1) I have tested and compared a method to construct NGS libraries from trace amounts of DNA, allowing to work with very rare samples, and performing multiple functional experiments on the same sample; 2) I have defined a method to isolate the microbial DNA from a skin sample, to perform actual metagenomic studies. We have proved the utility of the method by constructing 2 metagenomic libraries from mouse skin biopsies; 3) I have tested the relationship between microbial diversity and unrelated phenotypes in mouse skin samples; 4) I have analyzed the host phenotypical spectra, characterized what it is metabolic health, and assessed the relationship between health and microbiota.L’estudi de la microbiota de la pell ha estat sempre enfocat cap a un punt de vista clínic. No obstant, una aproximació ecològica a la microbiota cutània és impossibilitada per multiples limitacions metodològiques, que inclouen la baixa ràtio de DNA hospedador/DNA comensal o la baixa quantitat absoluta de DNA microbià. En contrast al pròsper camp de la metagenòmica intestinal, la metagenòmica cutània ha estat obstaculitzada metodològicament, i per això, aquesta tesi intenta sentar les bases per al futur de l’estudi del microbioma cutàni i la aplicació d’una aproximació de sistemes. He aplicat quatre aproximacions importants per a la observació sistemàtica i ecològica de la pell: 1) He testat i comparat un mètode per construir llibreries de NGS a partir de traces de DNA, permetent treballar amb mostres de difícil obtenció i aplicar-hi multiples experiments funcionals a la mateixa mostra, sense haver d’usar tot el material en la seqüenciació; 2) He definit un mètode per a aïllar el DNA microbià d’una mostra de pell completa per tal de dur a terme una anàlisi metagenòmica de la mostra. Hem demostrat la seva utilitat construint dues llibreries independents a partir de pell de ratolí; 3) He analitzat la relació entre la diversitat microbiana i un fenotip aïllat de l’hospedador; 4) He analitzat l’espectre fenotípic de l’hoste des d’un punt de vista sistèmic, he caracteritzat l’estat de salut metabòlica i la seua relació amb la microbiota

Staphylococcus prevails in the skin microbiota of long-term immunodeficient mice

Special Issue: Ecology, Evolution and Population Genetics of Pathogenic Microbes.Host-commensal relationships in the skin are a complex system governed by variables related to the host, the bacteria and the environment. A disruption of this system may lead to new steady states, which, in turn, may lead to disease. We have studied one such disruption by characterizing the skin microbiota in healthy and immunodepressed (ID) mice. A detailed anatomopathological study failed to reveal any difference between the skin of healthy and ID mice. We sequenced the 16S rDNA V1-V2 gene region to saturation in 10 healthy and 10 ID 8 week-old mice, and found than all of the healthy and two of the ID mice had bacterial communities that were similar in composition to that of human skin, although, presumably because of the uniform raising conditions, less interindividual variation was found in mice. However, eight ID mice showed microbiota dominated by Staphylococcus epidermidis. Quantitative PCR amplification of 16S rDNA gene and of the Staphylococcus-specific TstaG region confirmed the previous results and indicated that the quantitative levels of Staphylococcus were similar in both groups while the total number of 16S copies was greater in the healthy mice. Thus, it is possible that, under long-term immunodeficiency, which removes the acquired but not the native immune system, S. epidermidis may inhibit the growth of other bacteria but does not cause a pathogenic state. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.This work was financed by the MICINN (Spanish Ministry of Science and Innovation) Grant SAF2010-16240. M. G. G. was supported by a predoctoral fellowship from MICINN.Peer Reviewe

Consistency of metagenomic assignment programs in simulated and real data

Background: Metagenomics is the genomic study of uncultured environmental samples, which has been greatly facilitated by the advent of shotgun-sequencing technologies. One of the main focuses of metagenomics is the discovery of previously uncultured microorganisms, which makes the assignment of sequences to a particular taxon a challenge and a crucial step. Recently, several methods have been developed to perform this task, based on different methodologies such as sequence composition or sequence similarity. The sequence composition methods have the ability to completely assign the whole dataset. However, their use in metagenomics and the study of their performance with real data is limited. In this work, we assess the consistency of three different methods (BLAST + Lowest Common Ancestor, Phymm, and Naïve Bayesian Classifier) in assigning real and simulated sequence reads. Results: Both in real and in simulated data, BLAST + Lowest Common Ancestor (BLAST+LCA), Phymm, and Naïve Bayesian Classifier consistently assign a larger number of reads in higher taxonomic levels than in lower levels. However, discrepancies increase at lower taxonomic levels. In simulated data, consistent assignments between all three methods showed greater precision than assignments based on Phymm or Bayesian Classifier alone, since the BLAST + LCA algorithm performed best. In addition, assignment consistency in real data increased with sequence read length, in agreement with previously published simulation results. Conclusions: The use and combination of different approaches is advisable to assign metagenomic reads. Although the sensitivity could be reduced, the reliability can be increased by using the reads consistently assigned to the same taxa by, at least, two methods, and by training the programs using all available information.This work was financed by the MICINN (Spanish Ministry of Science and Innovation) grant SAF2010-16240. MGG was supported by a predoctoral fellowship from MICIN

Consistency of metagenomic assignment programs in simulated and real data

Background: Metagenomics is the genomic study of uncultured environmental samples, which has been greatly facilitated by the advent of shotgun-sequencing technologies. One of the main focuses of metagenomics is the discovery of previously uncultured microorganisms, which makes the assignment of sequences to a particular taxon a challenge and a crucial step. Recently, several methods have been developed to perform this task, based on different methodologies such as sequence composition or sequence similarity. The sequence composition methods have the ability to completely assign the whole dataset. However, their use in metagenomics and the study of their performance with real data is limited. In this work, we assess the consistency of three different methods (BLAST + Lowest Common Ancestor, Phymm, and Naïve Bayesian Classifier) in assigning real and simulated sequence reads. Results: Both in real and in simulated data, BLAST + Lowest Common Ancestor (BLAST+LCA), Phymm, and Naïve Bayesian Classifier consistently assign a larger number of reads in higher taxonomic levels than in lower levels. However, discrepancies increase at lower taxonomic levels. In simulated data, consistent assignments between all three methods showed greater precision than assignments based on Phymm or Bayesian Classifier alone, since the BLAST + LCA algorithm performed best. In addition, assignment consistency in real data increased with sequence read length, in agreement with previously published simulation results. Conclusions: The use and combination of different approaches is advisable to assign metagenomic reads. Although the sensitivity could be reduced, the reliability can be increased by using the reads consistently assigned to the same taxa by, at least, two methods, and by training the programs using all available information.This work was financed by the MICINN (Spanish Ministry of Science and Innovation) grant SAF2010-16240. MGG was supported by a predoctoral fellowship from MICIN

Abundance and co-occurrence of extracellular capsules increase environmental breadth: Implications for the emergence of pathogens.

Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them-including Archaea-encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system-often from different groups-and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of environmental adaptation. Understanding the role of capsules in natural environments might enlighten their function in pathogenesis

A new method for extracting skin microbes allows metagenomic analysis of whole-deep skin

In the last decade, an extensive effort has been made to characterize the human microbiota, due to its clinical and economic interests. However, a metagenomic approach to the skin microbiota is hampered by the high proportion of host DNA that is recovered. In contrast with the burgeoning field of gut metagenomics, skin metagenomics has been hindered by the absence of an efficient method to avoid sequencing the host DNA. We present here a method for recovering microbial DNA from skin samples, based on a combination of molecular techniques. We have applied this method to mouse skin, and have validated it by standard, quantitative PCR and amplicon sequencing of 16S rRNA. The taxonomic diversity recovered was not altered by this new method, as proved by comparing the phylogenetic structure revealed by 16S rRNA sequencing in untreated vs. treated samples. As proof of concept, we also present the first two mouse skin metagenomes, which allowed discovering new taxa (not only prokaryotes but also viruses and eukaryots) not reachable by 16S rRNA sequencing, as well as to characterize the skin microbiome functional landscape. Our method paves the way for the development of skin metagenomics, which will allow a much deeper knowledge of the skin microbiome and its relationship with the host, both in a healthy state and in relation to disease.This work was funded by the Spanish Ministry of Science and Innovation (MICINN)[grant numbers SAF2010-16240 and BFU2009-12895-CO2-01]. MGG was supported by a predoctoral fellowship from the Spanish Ministry of Scienceand Innovation [Grant number BES-2008-006029]

Canonical Correspondence Analysis (CCA) of the bacterial diversity in skin in Standard and Proposed methods.

<p>Methods are labelled B for Bacterial Enrichment extraction and T for Total DNA extraction.</p

Metagenomic profiles of Skin metagenomes, split by taxa.

<p>Classification was made first by taxonomic assignation using PhymmBL, and then functional classification was based on EggNOG using HMM classification. Counts were normalized by sample and taxa using log transformation. The color gradient indicates the level of representation for that taxa. Hierarchical clustering was performed by function and taxon.</p