PseudoMLSA: a database for multigenic sequence analysis of Pseudomonas species

<p>Abstract</p> <p>Background</p> <p>The genus <it>Pseudomonas </it>comprises more than 100 species of environmental, clinical, agricultural, and biotechnological interest. Although, the recommended method for discriminating bacterial species is DNA-DNA hybridisation, alternative techniques based on multigenic sequence analysis are becoming a common practice in bacterial species discrimination studies. Since there is not a general criterion for determining which genes are more useful for species resolution; the number of strains and genes analysed is increasing continuously. As a result, sequences of different genes are dispersed throughout several databases. This sequence information needs to be collected in a common database, in order to be useful for future identification-based projects.</p> <p>Description</p> <p>The PseudoMLSA Database is a comprehensive database of multiple gene sequences from strains of <it>Pseudomonas </it>species. The core of the database is composed of selected gene sequences from all <it>Pseudomonas </it>type strains validly assigned to the genus through 2008. The database is aimed to be useful for MultiLocus Sequence Analysis (MLSA) procedures, for the identification and characterisation of any <it>Pseudomonas </it>bacterial isolate. The sequences are available for download via a direct connection to the National Center for Biotechnology Information (NCBI). Additionally, the database includes an online BLAST interface for flexible nucleotide queries and similarity searches with the user's datasets, and provides a user-friendly output for easily parsing, navigating, and analysing BLAST results.</p> <p>Conclusions</p> <p>The PseudoMLSA database amasses strains and sequence information of validly described <it>Pseudomonas </it>species, and allows free querying of the database via a user-friendly, web-based interface available at <url>http://www.uib.es/microbiologiaBD/Welcome.html</url>. The web-based platform enables easy retrieval at strain or gene sequence information level; including references to published peer-reviewed articles, and direct external links to more specialized strain information databases (StrainInfo) and GeneBank (NCBI). The PseudoMLSA is intended to provide helpful strain-sequence information for a better and more comprehensive discriminative multigenic sequence based analysis of this special group of bacteria, contributing to enhance our understanding of the evolution of <it>Pseudomonas </it>species.</p

Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by 'universal' primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology

Metagenomic evidence for a polymicrobial signature of sepsis

Our understanding of the host component of sepsis has made significant progress. However, detailed study of the microorganisms causing sepsis, either as single pathogens or microbial assemblages, has received far less attention. Metagenomic data offer opportunities to characterize the microbial communities found in septic and healthy individuals. In this study we apply gradient-boosted tree classifiers and a novel computational decontamination technique built upon SHapley Additive exPlanations (SHAP) to identify microbial hallmarks which discriminate blood metagenomic samples of septic patients from that of healthy individuals. Classifiers had high performance when using the read assignments to microbial genera [area under the receiver operating characteristic (AUROC=0.995)], including after removal of species ‘culture-confirmed’ as the cause of sepsis through clinical testing (AUROC=0.915). Models trained on single genera were inferior to those employing a polymicrobial model and we identified multiple co-occurring bacterial genera absent from healthy controls. While prevailing diagnostic paradigms seek to identify single pathogens, our results point to the involvement of a polymicrobial community in sepsis. We demonstrate the importance of the microbial component in characterising sepsis, which may offer new biological insights into the aetiology of sepsis, and ultimately support the development of clinical diagnostic or even prognostic tools

Soil Microbial Diversity Across Different Agroecological Zones in New South Wales

A synergistic relationship between soil diversity (pedodiversity) and soil microbial diversity (biodiversity) seems axiomatic. Soil microbes contribute with biogeochemical cycles on which rely soil services (e.g. food production) and; vice-versa the soil matrix provides the living conditions that structure its microbial communities. A better insight would enable us to quantify/qualify and so sustain, protect, and improve those processes underpinned by soil microorganisms. We hypothesize that the structural patterns of soil microbes rely on multivariate soil units and gradients (e.g. soil horizons) instead of single discrete ‘factors’ (soil pH) and that the microbial patterns can become a well-defined property of determined soils. We began exploring this biotic-abiotic dynamic by modeling soil microbial α-diversity using two orthogonal transects (~900 km each) across NSW. Soils were sampled from paired conserved and disturbed ecosystems. Soil biophysicochemistry was characterized using 16SrDNA/ITS metabarcoding and pedometric approaches. Soil microbial patterns and physicochemical attributes were assessed using linear and non-linear relationships (bootstraped regression trees models) whose output enabled the microbial mapping at 1km across NSW. These maps showed a higher diversity of soil microbes in western than eastern NSW. Despite this gradient, fungi and archaea were respectively lower and higher in Vertosols, whereas bacteria distribution was less clear. Our results suggested that microbial structural patterns relate to most pedological attributes and, the extent of this relationship varies according to the structural parameters analyzed (taxa composition, abundance, diversity metric). Therefore, microbial patterns are more consistent with grouped features defining soil gradients (soil types) rather than on individual soil properties. These conclusions will be supported by analyzing microbial and pedological dissimilarities (β-diversity) in a further research

Evaluation of the oesophagogastric cancer associated microbiome: a systematic review and quality assessment

Objective. Oesophagogastric cancer is the fifth most common cancer worldwide, with poor survival outcomes. The role of bacteria in the pathogenesis of oesophagogastric cancer remains poorly understood. Design. A systematic search identified studies assessing the oesophagogastric cancer microbiome. The primary outcome was to identify bacterial enrichment specific to oesophagogastric cancer. Secondary outcomes included appraisal of the methodology, diagnostic performance of cancer bacteria and the relationship between oral and tissue microbiome. Results. A total of 9295 articles were identified, and 87 studies were selected for analysis. Five genera were enriched in gastric cancer: Lactobacillus, Streptococcus, Prevotella, Fusobacterium and Veillonella. No clear trends were observed in oesophageal adenocarcinoma. Streptococcus, Prevotella and Fusobacterium were abundant in oesophageal squamous cell carcinoma. Functional analysis supports the role of immune cells, localised inflammation and cancer-specific pathways mediating carcinogenesis. STORMS reporting assessment identified experimental deficiencies, considering batch effects and sources of contamination prevalent in low-biomass samples. Conclusions. Functional analysis of cancer pathways can infer tumorigenesis within the cancer–microbe–immune axis. There is evidence that study design, experimental protocols and analytical techniques could be improved to achieve more accurate and representative results. Whole-genome sequencing is recommended to identify key metabolic and functional capabilities of candidate bacteria biomarkers

Characterisation of diverse microbial communities and application of novel detection techniques

Microbes are essential for all life on Earth. They are found in all viable habitats from deep sea sediments and bedrock to high up in the atmosphere with a variety that exceeds by far the eukaryotic diversity. Ecosystem services provided by microorganisms, such as degradation of organic material and mediation of biogeochemical cycles are fundamentally important for the whole biosphere and its inhabitants. Microbes also form symbiotic relationships with multicellular organisms, and play important roles in nutrition and disease. Recent developments in molecular techniques, especially the next generation sequencing technologies and microarray applications, have opened new possibilities in studying diverse microbial communities. In this thesis, the aim was to determine the diversity and community structure of environmental samples collected from the northern Baltic Sea water column and anaerobic digestion reactor, and to assess how the prevailing abiotic factors affect the microbial community structure. We applied 16S rRNA and ITS gene amplicon sequencing method with 454 sequencing technology to form a detailed taxonomic description of studied communities. The produced sequence data was further utilised in designing probes for a new padlock probe based ligation detection reaction (LDR) microarray that could be employed for specific and sensitive taxonomic identification of microbial groups in diverse communities. The functionality, specificity and sensitivity of the microarray were assessed using artificial and real environmental samples. Additionally, selected amplicon sequencing data analysis methods were compared in order to discover which algorithms work most reliably. In this subproject, we aimed to clarify how significantly the selected analysis methods, specifically denoising and clustering algorithms, affect the results and how comparable the results derived from different analysis pipelines are. Amplicon sequencing revealed diverse microbial communities in the northern Baltic Sea water column and anaerobic digestion reactor. The pelagic bacterial communities in the northern Baltic Sea were strongly stratified, with aerobic Bacteria such as Pseudomonas and Flavobacterium dominating in the surface layer and Oleispira and sulfate-reducing bacteria in the anoxic deep waters. Based on the sequence data the diversity was assessed one order of magnitude less diverse compared to Atlantic and Pacific ocean bacterial communities. The anaerobic digestion reactor communities were dominated by Bacteria belonging to phyla Bacteroidetes, Firmicutes and Thermotogae and methanogenic Archaea, all essential and typical degraders in anaerobic digestion. The process also supported a diverse fungal community of phyla Ascomycota and Basidiomycota, including several taxa capable of degrading organic material in anaerobic conditions. The LDR microarray technology proved sensitive, specific and semiquantitative method for identifying microbes in diverse communities. The proof of principle tests and experiments with real environmental samples showed that if the probes are designed carefully, the detection is comparable to qPCR and amplicon sequencing. The detection limit was 0.01 fmol/µl/template. Data analysis method comparisons revealed prominent differences in observed operational taxonomic units and relative abundance of identified taxa. The majority of tested methods assessed the species richness too high. Using a functioning denoising method evened out the differences in the number of observed OTUs caused by various clustering algorithms. The ability to filter out the spurious taxa produced by amplification and sequencing, but still retain all the real diversity varied between methods. This study shows both the potential and the challenges in the use of amplicon sequencing and microarray technologies in studying diverse microbial communities. The results indicate that the padlock based LDR microarray can be designed for very accurate and sensitive identification of microbial groups of interest. The data suggest that amplicon sequencing is a powerful tool in identifying microbes and assessing the diversity but distinguishing between spurious and true community members remain a challenge. There is still work to be done in the development and application of data analysis tools.Mikrobit ovat mikroskooppisen pieniä eliöitä, jotka elävät lähes kaikenlaisissa ympäristöissä ja olosuhteissa aina merien syvänteiden sedimenteistä ylempiin ilmakehän kerroksiin asti. Ne ovat täysin välttämättömiä elämälle maapallolla: mikrobeilla on keskeinen rooli aineiden kierrossa, ne muodostavat symbioottisia suhteita monisoluisten eliöiden kanssa, ja vaikuttavat merkittävästi ravitsemukseen sekä sairauksiin. Uuden sukupolven sekvensointitekniikoiden sekä mikrosirusovellusten nopea kehitys on luonut uusia mahdollisuuksia mikrobien ja monimuotoisten mikrobiyhteisöjen tutkimiseen. Tässä väitöskirjatyössä tutkittiin pohjoisen Itämeren sekä mädättämöprosessin mikrobiyhteisöjen rakennetta ja monimuotoisuutta 454-sekvensointimenetelmällä ja kehitettiin uusi ligaatioreaktioon perustuva DNA-mikrosiru kohdesekvenssien tarkkaan tunnistamiseen monimuotoisissa mikrobiyhteisöissä. Mikrosirun herkkyyttä ja tarkkuutta arvioitiin sekä keinotekoisten että oikeiden ympäristönäytteiden avulla. Sekvenssianalyysityökalujen luotettavuutta tutkittiin vertailemalla yleisesti käytettyjä algoritmeja. Pyrimme selvittämään, kuinka merkittävästi eri analyysityökalut vääristävät tuloksia ja voidaanko eri menetelmiä käyttäen saatuja tuloksia vertailla keskenään. Tutkitut ympäristönäytteet osoittautuivat mikrobiyhteisöiltään monimuotoisiksi. Pohjoisen Itämeren bakteeriyhteisöt olivat voimakkaasti kerrostuneita: pintavedessä viihtyivät aerobiset bakteerit kuten Pseudomonas ja Flavobacterium, kun taas läheltä pohjaa hapettomista oloista tunnistimme mm. öljyä hajottavia Oleispira-bakteereja sekä sulfaattia pelkistäviä bakteerisukuja. Aineistomme perusteella Itämeren bakteerien monimuotoisuus vaikuttaa kuitenkin valtameriä vaatimattomammalta. Mädättämöjen yleisimpiin mikrobeihin lukeutuivat mm. Bacteroidetes-, Firmicutes- ja Thermotogae-pääjaksojen bakteerit sekä metaania tuottavat arkit, joiden tiedetään olevan tyypillisiä mädättämöjen asukkeja. Prosessissa oli läsnä myös hyvin monimuotoinen sieniyhteisö, jonka jäsenien tiedetään kykenevän orgaanisen aineksen hajotukseen hapettomissa olosuhteissa. Kokeidemme perustella uusi mikrosirumenetelmä toimi herkästi ja tarkasti. Mikrosirun avulla kyettiin havaitsemaan mitkä mikrobiryhmät ovat läsnä näytteissä ja signaalin voimakkuuden perusteella voitiin päätellä oliko kyseessä yleinen vai vähän harvinaisempi mikrobi. Menetelmän tunnistusrajaksi määrittyi 0.01 fmol/µl/näytettä ja hyvin suunnitelluilla koettimilla mikrobien tunnistuksessa voidaan päästä kvantitatiivisen PCR-menetelmän ja amplikonisekvensoinnin tasolle. Eri sekvenssianalyysityökaluilla saadut tulokset poikkesivat merkittävästi toisistaan. Erityisesti erilaisten mikrobityyppien määrää arvioitaessa on oltava varovainen, sillä osa menetelmistä arvioi monimuotoisuuden jopa kertaluokan todellisuutta suuremmaksi ja erot menetelmien välillä olivat huomattavat. Myös mikrobiryhmien taksonomisesta luokittelusta löytyi tilastollisesti merkitseviä eroja menetelmien välillä. Tämä väitöskirjatyö tuo esille amplikonisekvensoinnin ja mikrosiruteknologian mahdollisuudet sekä haasteet monimuotoisten mikrobiyhteisöjen tutkimisessa. Menetelmät sopivat hyvin mikrobien tunnistamiseen monimuotoisista ympäristönäytteistä, mutta analyysityökalujen kehittämisessä riittää vielä työtä

Population genetics and the microbiome: solving conservation problems

Conservation science has long focused on how a target species interacts with its biotic and abiotic environment, however to conserve a species it is important to consider its biotic interactions in their entirety, including the microbes living both on and within it. In particular, gut microbiome communities in the digestive tract of animals play a key role in digestive capabilities, as well as immune function and behavioural responses. This thesis examines how sequencing gut microbiomes of threatened species can inform conservation strategy. Firstly, I review the tools available to a researcher beginning a microbiome investigation in wildlife, with emphasis on how the different methods can be used to solve specific conservation problems. I then apply these methods to a free-ranging population of long-nosed potoroos (Potorous tridactylus), a marsupial in the family Potoroidae. We found that the potoroo gut microbiome is dominated by the bacterial phyla Firmicutes and Bacteroidota, with a mean Firmicutes to Bacteroidota ratio (F:B) of 2.4. We also observed that host inbreeding was associated with changes in the abundance of two bacterial genera within the potoroo gut microbiome. These observations suggest the possibility that inbreeding depression in a small population could feasibly manifest through changes in the microbiome, although the fitness ramifications cannot be determined from our data. Overall, this thesis advances the discussion around microbiome research in conservation and demonstrates the role of the microbiome in the biology of a unique host species. Going forward it is important that the potential of inbreeding depression to manifest via the gut microbiome is considered during population management strategies, through application of a wide variety of analytical tools, especially in species with unique or highly specialised diets

Diet tracing in ecology: Method comparison and selection

1. Determining diet is a key prerequisite for understanding species interactions, food web structure and ecological dynamics. In recent years, there has been considerable development in both the methodology and application of novel and more traditional dietary tracing methods, yet there is no comprehensive synthesis that systematically and quantitatively compares among the different approaches. 2. Here we conceptualize diet tracing in ecology, provide recommendations for method selection, and illustrate the advantages of method integration. We summarize empirical evidence on how different methods quantify diet mixtures, by contrasting estimates of dietary proportions from multiple methods applied to the same consumer-resource datasets, or from experimental studies with known diet compositions. 3. Our data synthesis revealed an urgent need for more experiential comparisons among the dietary methods. The comparison of diet quantifications from field observations showed that different techniques aligned well in cases with less than six diet items, but diverged considerably when applied to more complex diet mixtures. 4. Efforts are ongoing to further advance dietary estimation, including how reliably compound specific stable isotope analyses and fatty acid profiles can quantify more prey items than bulk stable isotope analyses. Similarly, DNA analyses, which can depict trophic interactions at a higher resolution than any other methods, are generating new ways to better quantify diets and differentiate among life-stages of prey. Such efforts, combined with more empirical testing of each dietary method and establishment of open data repositories for dietary data, promise to greatly advance community and ecosystem ecology