scholarly journals Culture-Dependent and -Independent Characterization of Microbial Communities Associated with a Shallow Submarine Hydrothermal System Occurring within a Coral Reef off Taketomi Island, Japan

2007 ◽  
Vol 73 (23) ◽  
pp. 7642-7656 ◽  
Author(s):  
Hisako Hirayama ◽  
Michinari Sunamura ◽  
Ken Takai ◽  
Takuro Nunoura ◽  
Takuro Noguchi ◽  
...  
Keyword(s):  
Coral Reef ◽  
Microbial Communities ◽  
Hydrothermal System ◽  
Hydrothermal Activity ◽  
Molecular Techniques ◽  
Maximum Temperature ◽  
Rrna Gene ◽  
Type I ◽  
Optimum Growth ◽  
Culture Independent

ABSTRACT Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, ∼23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52°C) contained 175 μM H2S and gas bubbles mainly composed of CH4 (69%) and N2 (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37°C, and another novel type I methanotroph exhibited optimum growth at 45°C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.

scholarly journals The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

2020 ◽  
Vol 3 (2) ◽  
pp. 39 ◽  
Author(s):  
Anna Ojo-Okunola ◽  
Shantelle Claassen-Weitz ◽  
Kilaza S. Mwaikono ◽  
Sugnet Gardner-Lubbe ◽  
Heather J. Zar ◽  
...  
Keyword(s):  
Human Milk ◽  
Dna Extraction ◽  
Bacterial Population ◽  
Illumina Miseq ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Dna Recovery ◽  
Culture Independent ◽  
Zymo Research

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

scholarly journals Basalt-Hosted Microbial Communities in the Subsurface of the Young Volcanic Island of Surtsey, Iceland

2021 ◽  
Vol 12 ◽  
Author(s):  
Pauline Bergsten ◽  
Pauline Vannier ◽  
Alexandra María Klonowski ◽  
Stephen Knobloch ◽  
Magnús Tumi Gudmundsson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Drilling Fluid ◽  
Microbial Colonization ◽  
Maximum Temperature ◽  
Rrna Gene ◽  
Metabolic Potential ◽  
16S Rrna Gene Analysis ◽  
Drill Cores

The island of Surtsey was formed in 1963–1967 on the offshore Icelandic volcanic rift zone. It offers a unique opportunity to study the subsurface biosphere in newly formed oceanic crust and an associated hydrothermal-seawater system, whose maximum temperature is currently above 120°C at about 100m below surface. Here, we present new insights into the diversity, distribution, and abundance of microorganisms in the subsurface of the island, 50years after its creation. Samples, including basaltic tuff drill cores and associated fluids acquired at successive depths as well as surface fumes from fumaroles, were collected during expedition 5059 of the International Continental Scientific Drilling Program specifically designed to collect microbiological samples. Results of this microbial survey are investigated with 16S rRNA gene amplicon sequencing and scanning electron microscopy. To distinguish endemic microbial taxa of subsurface rocks from potential contaminants present in the drilling fluid, we use both methodological and computational strategies. Our 16S rRNA gene analysis results expose diverse and distinct microbial communities in the drill cores and the borehole fluid samples, which harbor thermophiles in high abundance. Whereas some taxonomic lineages detected across these habitats remain uncharacterized (e.g., Acetothermiia, Ammonifexales), our results highlight potential residents of the subsurface that could be identified at lower taxonomic rank such as Thermaerobacter, BRH-c8a (Desulfallas-Sporotomaculum), Thioalkalimicrobium, and Sulfurospirillum. Microscopy images reveal possible biotic structures attached to the basaltic substrate. Finally, microbial colonization of the newly formed basaltic crust and the metabolic potential are discussed on the basis of the data.

scholarly journals Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

1999 ◽  
Vol 65 (11) ◽  
pp. 4887-4897 ◽  
Author(s):  
Mark G. Wise ◽  
J Vaun McArthur ◽  
Lawrence J. Shimkets
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Dna ◽  
Rrna Gene ◽  
Type I ◽  
Type Ii ◽  
Partial Sequencing ◽  
16S Ribosomal Dna ◽  
Culture Independent ◽  
The 16S Rrna Gene

ABSTRACT The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobiumgroup, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly publishedMethylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

scholarly journals Chronic Rhinosinusitis and the Evolving Understanding of Microbial Ecology in Chronic Inflammatory Mucosal Disease

2016 ◽  
Vol 30 (1) ◽  
pp. 321-348 ◽  
Author(s):  
Michael Hoggard ◽  
Brett Wagner Mackenzie ◽  
Ravi Jain ◽  
Michael W. Taylor ◽  
Kristi Biswas ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Chronic Rhinosinusitis ◽  
Inflammatory Diseases ◽  
Modern Technology ◽  
Molecular Techniques ◽  
Modern Culture ◽  
Culture Independent ◽  
New Perspective ◽  
Microbiological Research

SUMMARY Chronic rhinosinusitis (CRS) encompasses a heterogeneous group of debilitating chronic inflammatory sinonasal diseases. Despite considerable research, the etiology of CRS remains poorly understood, and debate on potential roles of microbial communities is unresolved. Modern culture-independent (molecular) techniques have vastly improved our understanding of the microbiology of the human body. Recent studies that better capture the full complexity of the microbial communities associated with CRS reintroduce the possible importance of the microbiota either as a direct driver of disease or as being potentially involved in its exacerbation. This review presents a comprehensive discussion of the current understanding of bacterial, fungal, and viral associations with CRS, with a specific focus on the transition to the new perspective offered in recent years by modern technology in microbiological research. Clinical implications of this new perspective, including the role of antimicrobials, are discussed in depth. While principally framed within the context of CRS, this discussion also provides an analogue for reframing our understanding of many similarly complex and poorly understood chronic inflammatory diseases for which roles of microbes have been suggested but specific mechanisms of disease remain unclear. Finally, further technological advancements on the horizon, and current pressing questions for CRS microbiological research, are considered.

Design of PCR Primers for the mcrA Genes Detection of Methanogen Communities

2011 ◽  
Vol 135-136 ◽  
pp. 408-413 ◽  
Author(s):  
Nguyen Ngoc Tuan ◽  
Shir Ly Huang
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Biogas Production ◽  
Pcr Primers ◽  
Molecular Techniques ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Mcra Gene ◽  
Catabolic Genes ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

Methanogens play an important role to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent green house gases, during organic matter degradation in anaerobic environments. Therefore, it is necessary to better understand microorganisms that produce natural gas. Indeed, methanogens are difficult to perform through culture based methods. In addition, the culture independent methods using the 16S rRNA gene also revealed some disadvantages. For these reasons, the culture independent molecular techniques using the specific catabolic genes such as methyl coenzyme M reductase (MCR) were studied. In this study, a primer set which can amplify specific fragments from a wide variety of mcrA gene was designed based on the homologous regions of 100 mcrA genes listed in the GenBank. PCR with the mcrA primers amplified DNA fragments of the expected size from all the six samples which obtained from biogas production reactors. In addition, denaturing gradient gel electrophoresis PCR analysis using our designed primers also revealed the diversity of mcrA gene in each sample. These results revealed that our primers were successfully to detect the mcrA genes and it is also helpful to know the diversity of mcrA genes in methanogen communities.

scholarly journals Living Lithic and Sublithic Bacterial Communities in Namibian Drylands

2021 ◽  
Vol 9 (2) ◽  
pp. 235
Author(s):  
Steffi Genderjahn ◽  
Simon Lewin ◽  
Fabian Horn ◽  
Anja M. Schleicher ◽  
Kai Mangelsdorf ◽  
...  
Keyword(s):  
Community Structure ◽  
Microbial Communities ◽  
Soil Formation ◽  
Amplicon Sequencing ◽  
Mineral Weathering ◽  
Rrna Gene ◽  
Rock Weathering ◽  
Weathering Processes ◽  
Rock Types ◽  
Culture Independent

Dryland xeric conditions exert a deterministic effect on microbial communities, forcing life into refuge niches. Deposited rocks can form a lithic niche for microorganisms in desert regions. Mineral weathering is a key process in soil formation and the importance of microbial-driven mineral weathering for nutrient extraction is increasingly accepted. Advances in geobiology provide insight into the interactions between microorganisms and minerals that play an important role in weathering processes. In this study, we present the examination of the microbial diversity in dryland rocks from the Tsauchab River banks in Namibia. We paired culture-independent 16S rRNA gene amplicon sequencing with culture-dependent (isolation of bacteria) techniques to assess the community structure and diversity patterns. Bacteria isolated from dryland rocks are typical of xeric environments and are described as being involved in rock weathering processes. For the first time, we extracted extra- and intracellular DNA from rocks to enhance our understanding of potentially rock-weathering microorganisms. We compared the microbial community structure in different rock types (limestone, quartz-rich sandstone and quartz-rich shale) with adjacent soils below the rocks. Our results indicate differences in the living lithic and sublithic microbial communities.

scholarly journals The Microbiome of the Reef Macroalga Sargassum ilicifolium in Singapore

2021 ◽  
Vol 9 (5) ◽  
pp. 898
Author(s):  
Ren Min Oh ◽  
Elena Bollati ◽  
Prasha Maithani ◽  
Danwei Huang ◽  
Benjamin J. Wainwright
Keyword(s):  
Coral Reef ◽  
Microbial Communities ◽  
Bacterial Communities ◽  
Microbial Community Composition ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Benthic Organisms ◽  
Polymerase Chain ◽  
Microbial Change ◽  
The 16S Rrna Gene

The large canopy-forming macroalga, Sargassum ilicifolium, provides shelter and food for numerous coral reef species, but it can also be detrimental at high abundances where it outcompetes other benthic organisms for light and space. Here, we investigate the microbial communities associated with S. ilicifolium in Singapore, where it is an abundant and important member of coral reef communities. We collected eight complete S. ilicifolium thalli from eight island locations along an approximate 14 km east-to-west transect. Each thallus was dissected into three separate parts: holdfast, vesicles, and leaves. We then characterized the bacterial communities associated with each part via polymerase chain reaction (PCR) amplification of the 16S rRNA gene V4 region. We then inferred predicted metagenome functions using METAGENassist. Despite the comparatively short distances between sample sites, we show significant differences in microbial community composition, with communities further differentiated by part sampled. Holdfast, vesicles and leaves all harbor distinct microbial communities. Functional predictions reveal some separation between holdfast and leaf communities, with higher representation of sulphur cycling taxa in the holdfast and higher representation of nitrogen cycling taxa in the leaves. This study provides valuable baseline data that can be used to monitor microbial change, and helps lay the foundation upon which we can begin to understand the complexities of reef-associated microbial communities and the roles they play in the functioning and diversity of marine ecosystems.

scholarly journals A Longitudinal Study of the Human Oropharynx Microbiota Over Time Reveals a Common Core and Significant Variations With Self-Reported Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Lydia Luise Bach ◽  
Asha Ram ◽  
Umer Z. Ijaz ◽  
Thomas J. Evans ◽  
Jan Lindström
Keyword(s):  
Microbial Communities ◽  
Community Composition ◽  
Common Core ◽  
Temporal Stability ◽  
Alpha Diversity ◽  
Molecular Techniques ◽  
Human Pathogens ◽  
Healthy Human ◽  
Culture Independent ◽  
Over Time

Our understanding of human microbial communities, in particular in regard to diseases is advancing, yet the basic understanding of the microbiome in healthy subjects over time remains limited. The oropharynx is a key target for colonization by several important human pathogens. To understand how the oropharyngeal microbiome might limit infections, and how intercurrent infections might be associated with its composition, we characterized the oropharyngeal microbiome of 18 healthy adults, sampled weekly over a 40-weeks using culture-independent molecular techniques. We detected nine phyla, 202 genera and 1438 assignments on OTU level, dominated by Firmicutes, Bacteroidetes, and Proteobacteria on phylum level. Individual microbiomes of participants were characterized by levels of high alpha diversity (mean = 204.55 OTUs, sd = 35.64), evenness (19.83, sd = 9.74) and high temporal stability (mean Pearson’s correlation between samples of 0.52, sd = 0.060), with greater differences in microbiome community composition between than within individuals. Significant changes in community composition were associated with disease states, suggesting that it is possible to detect specific changes in OTU abundance and community composition during illness. We defined the common core microbiota by varying occurrence and abundance thresholds showing that individual core microbiomes share a substantial number of OTUs across participants, chiefly Streptococci and Veillonella. Our results provide insights into the microbial communities that characterize the healthy human oropharynx, community structure and variability, and provide new approaches to define individual and shared cores. The wider implications of this result include the potential for modeling the general dynamics of oropharynx microbiota both in health and in response to antimicrobial treatments or probiotics.

scholarly journals Characterization of Endophytic Microbial Communities in Store-Bought Kale Evaluated by Different Plant Tissue Homogenization Methods

2020 ◽  
Vol 4 (3) ◽  
pp. 211-216
Author(s):  
C. Ruth McNees ◽  
Audrey D. Law ◽  
Luke A. Moe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Plant Tissue ◽  
Fruits And Vegetables ◽  
Enzyme Digestion ◽  
Human Consumption ◽  
Rrna Gene ◽  
Culture Independent ◽  
Tissue Maceration

Endophytic microorganisms live in intercellular and vascular spaces of plants and span the continuum from beneficial to pathogenic in both plants and animals. Increasing human consumption of fruits and vegetables has placed an emphasis on identifying and studying those microbes that colonize the internal tissues of plants, with a goal of limiting populations of enteric pathogens in store-bought foods meant for raw consumption, such as leafy greens. Culture-independent (i.e., metagenomic) methods are increasingly used to obtain an accurate snapshot of plant microbial communities, yet technical hurdles limit the accuracy and throughput of these methods. This includes the low-throughput nature of plant tissue maceration, and the prevalence of plant plastid DNA in metagenomic DNA extracts, which is typically coamplified via PCR strategies that target the bacterial 16S rRNA gene. In this study, we use kale (Brassica oleracea) as a model to explore the leafy green endophytic microbiome and to compare how two tissue maceration techniques used in traditional endophyte research compare in culture-independent microbiome studies using the Illumina Miseq platform. Three different brands of store-bought kale were surface sterilized and subjected to two tissue maceration strategies: enzyme digestion and blender processing. Analysis of 16S rRNA gene amplicon libraries revealed two highly abundant operational taxonomic units present in all libraries, one classified to the genus Pseudomonas and one to the family Enterobacteriaceae. Community structure and membership were highly similar among brands and between tissue maceration strategies, suggesting that both enzyme digestion and blender processing are suitable methods. Nonetheless, enzyme digestion may increase sample throughput and minimize steps involved in sample processing.

scholarly journals Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting

2008 ◽  
Vol 74 (8) ◽  
pp. 2414-2423 ◽  
Author(s):  
Ilse Scheirlinck ◽  
Roel Van der Meulen ◽  
Ann Van Schoor ◽  
Marc Vancanneyt ◽  
Luc De Vuyst ◽  
...  
Keyword(s):  
16S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Type I ◽  
Technological Parameters ◽  
Structure And Stability ◽  
Two Samples ◽  
Culture Independent ◽  
Culture Dependent

ABSTRACT A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

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