Free DNA and Metagenomics Analyses: Evaluation of Free DNA Inactivation Protocols for Shotgun Metagenomics Analysis of Human Biological Matrices

Culture-independent approaches now represent the gold standard for the investigation of both environmental and host-associated complex microbial communities. Nevertheless, despite the great advantages offered by these novel methodologies based on the use of next-generation DNA sequencing approaches, a number of bias sources have been identified. Among the latter, free DNA contained in biological matrices is one of the main sources of inaccuracy in reconstructing the resident microbial population of viable cells. For this reason, the photoreactive DNA-binding dye propidium monoazide (PMAxx™) has been developed by improving standard PMA. This compound binds and inactivates free DNA, thus preventing its amplification and sequencing. While the performances of PMA have been previously investigated, the efficiency with PMAxx™ has been tested mainly for amplicon-based profiling approaches on a limited number of biological matrices. In this study, we validated the performance of PMAxx™ for shotgun metagenomics approaches employing various human-associated matrices. Notably, results revealed that the effectiveness of PMAxx™ in inactivating free DNA of prokaryotes and eukaryotes tends to vary significantly based on the biological matrices analyzed.

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Integration of culture-dependent and independent methods provides a more coherent picture of the pig gut microbiome

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa022 ◽

2020 ◽

Vol 96 (3) ◽

Cited By ~ 2

Author(s):

Gavin J Fenske ◽

Sudeep Ghimire ◽

Linto Antony ◽

Jane Christopher-Hennings ◽

Joy Scaria

Keyword(s):

Bacterial Communities ◽

Bacterial Species ◽

Shotgun Metagenomics ◽

Culture Techniques ◽

Microbiome Composition ◽

Culture Independent ◽

Health And Disease ◽

The Media ◽

And Function ◽

Culture Dependent

ABSTRACT Bacterial communities resident in the hindgut of pigs, have profound impacts on health and disease. Investigations into the pig microbiome have utilized either culture-dependent, or far more commonly, culture-independent techniques using next generation sequencing. We contend that a combination of both approaches generates a more coherent view of microbiome composition. In this study, we surveyed the microbiome of Tamworth breed and feral pigs through the integration high throughput culturing and shotgun metagenomics. A single culture medium was used for culturing. Selective screens were added to the media to increase culture diversity. In total, 46 distinct bacterial species were isolated from the Tamworth and feral samples. Selective screens successfully shifted the diversity of bacteria on agar plates. Tamworth pigs are highly dominated by Bacteroidetes primarily composed of the genus Prevotella whereas feral samples were more diverse with almost equal proportions of Firmicutes and Bacteroidetes. The combination of metagenomics and culture techniques facilitated a greater retrieval of annotated genes than either method alone. The single medium based pig microbiota library we report is a resource to better understand pig gut microbial ecology and function. It allows for assemblage of defined bacterial communities for studies in bioreactors or germfree animal models.

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Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

mSystems ◽

10.1128/msystems.00367-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Ganesh Babu Malli Mohan ◽

Ceth W. Parker ◽

Camilla Urbaniak ◽

Nitin K. Singh ◽

Anthony Hood ◽

...

Keyword(s):

16S Rrna ◽

Metal Surfaces ◽

Microbial Population ◽

Microbial Populations ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Particle Board ◽

Shotgun Metagenomics ◽

Glass Surfaces ◽

Glass Metal

ABSTRACT Microbial contamination during long-term confinements of space exploration presents potential risks for both crew members and spacecraft life support systems. A novel swab kit was used to sample various surfaces from a submerged, closed, analog habitat to characterize the microbial populations. Samples were collected from various locations across the habitat which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal), and microbial populations were examined by culture, quantitative PCR (qPCR), microbiome 16S rRNA gene sequencing, and shotgun metagenomics. Propidium monoazide (PMA)-treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit to 106 CFU/sample, and their identity was characterized using Sanger sequencing. Both 16S rRNA amplicon and shotgun sequencing were used to characterize the microbial dynamics, community profiles, and functional attributes (metabolism, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable (after PMA treatment) Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia, and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus, and Oceanobacillus), and Proteobacteria (especially Acinetobacter) on linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Nonmetric multidimensional scaling determined from both 16S rRNA and metagenomic analyses revealed differential microbial species on LDP surfaces and glass/metal surfaces. The shotgun metagenomic sequencing of samples after PMA treatment showed bacterial predominance of viable Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%), while fungal analyses revealed Aspergillus and Penicillium dominance. IMPORTANCE This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The results of the analyses presented herein suggest that the surface material plays a role in microbial community structure, as the microbial populations differed between LDP and metal/glass surfaces. The metal/glass surfaces had less-complex community, lower bioburden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged analog habitat differs greatly from that of previously studied analog habitats.

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Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques

Food Research International ◽

10.1016/j.foodres.2009.11.014 ◽

2010 ◽

Vol 43 (3) ◽

pp. 773-779 ◽

Cited By ~ 47

Author(s):

Imma Andorrà ◽

Sara Landi ◽

Albert Mas ◽

Braulio Esteve-Zarzoso ◽

José M. Guillamón

Keyword(s):

Microbial Population ◽

Population Evolution ◽

Fermentation Temperature ◽

Culture Independent

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Comparative analysis of bacterial community composition in bulk tank raw milk by culture-dependent and culture-independent methods using the viability dye propidium monoazide

Journal of Dairy Science ◽

10.3168/jds.2014-8340 ◽

2014 ◽

Vol 97 (11) ◽

pp. 6761-6776 ◽

Cited By ~ 18

Author(s):

Mareike Weber ◽

Janina Geißert ◽

Myriam Kruse ◽

André Lipski

Keyword(s):

Comparative Analysis ◽

Bacterial Community ◽

Community Composition ◽

Bacterial Community Composition ◽

Raw Milk ◽

Propidium Monoazide ◽

Culture Independent ◽

Bulk Tank ◽

Culture Dependent

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Metagenomic investigation of the equine faecal microbiome reveals extensive taxonomic and functional diversity

10.1101/2021.04.30.442084 ◽

2021 ◽

Author(s):

Rachel Gilroy ◽

Joy Leng ◽

Anuradha Ravi ◽

Evelien M Adriaenssens ◽

Aharon Oren ◽

...

Keyword(s):

Functional Diversity ◽

Gut Microbiome ◽

Metagenomic Sequencing ◽

Shotgun Metagenomics ◽

High Fiber ◽

Functional Studies ◽

Fiber Diet ◽

Faecal Samples ◽

Culture Independent ◽

Medium Quality

Background: The horse plays crucial roles across the globe, including in horseracing, as a working and companion animal and as a food animal. The horse hindgut microbiome makes a key contribution in turning a high fiber diet into body mass and horsepower. However, despite its importance, the horse hindgut microbiome remains largely undefined. Here, we applied culture-independent shotgun metagenomics to thoroughbred equine faecal samples to deliver novel insights into this complex microbial community. Results: We performed metagenomic sequencing on five equine faecal samples to construct 123 high- or medium-quality metagenome-assembled genomes from Bacteria and Archaea. In addition, we recovered nearly 200 bacteriophage genomes. We document surprising taxonomic and functional diversity, encompassing dozens of novel or unnamed bacterial genera and species, to which we have assigned new Candidatus names. Many of these genera are conserved across a range of mammalian gut microbiomes. Conclusions: Our metagenomic analyses provide new insights into the bacterial, archaeal and bacteriophage components of the horse gut microbiome. The resulting datasets provide a key resource for future high-resolution taxonomic and functional studies on the equine gut microbiome.

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UMGAP: the Unipept MetaGenomics Analysis Pipeline

10.1101/2021.05.18.444604 ◽

2021 ◽

Author(s):

Felix Van der Jeugt ◽

Rien Maertens ◽

Aranka Steyaert ◽

Pieter Verschaffelt ◽

Caroline De Tender ◽

...

Keyword(s):

State Of The Art ◽

Analysis Pipeline ◽

Shotgun Metagenomics ◽

Taxonomic Profiling ◽

Taxonomic Analysis ◽

Command Line Tool ◽

Interactive Visualizations ◽

Protein Code ◽

Fast Memory ◽

Metagenomics Analysis

Shotgun metagenomics is now commonplace to gain insights into communities from diverse environments, but fast, memory-friendly, and accurate tools are needed for deep taxonomic analysis of the metagenome data. To meet this need we developed UMGAP, a highly versatile open source command line tool implemented in Rust for taxonomic profiling of shotgun metagenomes. It differs from state-of-the-art tools in its use of protein code regions identified in short reads for robust taxonomic identifications, a broad-spectrum index that can identify both archaea, bacteria, eukaryotes and viruses, a non-monolithic design, and support for interactive visualizations of complex biodiversities.

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Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

Plant Disease ◽

10.1094/pdis-11-19-2397-re ◽

2020 ◽

Vol 104 (8) ◽

pp. 2225-2232

Author(s):

A-li Chai ◽

Hai-yan Ben ◽

Wei-tao Guo ◽

Yan-xia Shi ◽

Xue-wen Xie ◽

...

Keyword(s):

Real Time ◽

Pseudomonas Syringae ◽

Real Time Pcr ◽

Light Exposure ◽

Qpcr Assay ◽

Bacterial Suspension ◽

Propidium Monoazide ◽

Chromosomal Dna ◽

Tomato Seed ◽

Viable Cells

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter−1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml−1 and 11.86 CFU g−1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g−1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

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Effect of Amplicon Length in Propidium Monoazide Quantitative PCR for the Enumeration of Viable Cells of Salmonella in Cooked Ham

Food Analytical Methods ◽

10.1007/s12161-012-9460-0 ◽

2012 ◽

Vol 6 (2) ◽

pp. 683-690 ◽

Cited By ~ 34

Author(s):

Belen Martin ◽

Sergi Raurich ◽

Margarita Garriga ◽

Teresa Aymerich

Keyword(s):

Quantitative Pcr ◽

Propidium Monoazide ◽

Viable Cells ◽

Amplicon Length ◽

Cooked Ham

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Mesophilic Sporeformers Identified in Whey Powder by Using Shotgun Metagenomic Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.01305-18 ◽

2018 ◽

Vol 84 (20) ◽

Cited By ~ 5

Author(s):

Aoife J. McHugh ◽

Conor Feehily ◽

John T. Tobin ◽

Mark A. Fenelon ◽

Colin Hill ◽

...

Keyword(s):

Dairy Product ◽

Metagenomic Sequencing ◽

Shotgun Metagenomics ◽

Food Ingredients ◽

Content Type ◽

Whey Powder ◽

Shotgun Metagenomic Sequencing ◽

Spoilage Bacteria ◽

Culture Independent ◽

Spore Forming Bacteria

ABSTRACT Spoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted. IMPORTANCE The ability of sporeformers to remain dormant in a desiccated state is of concern from a safety and spoilage perspective in dairy powder. Traditional culturing techniques are slow and provide little information without further investigation. We describe the identification of mesophilic sporeformers present in powders produced over 1 year, using novel shotgun metagenomic sequencing. This method allows detection and identification of possible pathogens and spoilage bacteria in parallel. Strain-level analysis and functional gene analysis, such as identification of toxin genes, were also performed. This approach has the potential to be of great value with respect to the detection of spore-forming bacteria and could allow a processor to make an informed decision surrounding process changes to reduce the risk of spore contamination.

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