scholarly journals Quantitative and qualitative evaluation of the impact of the G2 enhancer, bead sizes and lysing tubes on the bacterial community composition during DNA extraction from recalcitrant soil core samples based on community sequencing and qPCR

2018 ◽  
Author(s):  
Alex Gobbi ◽  
Rui G. Santini ◽  
Elisa Filippi ◽  
Lea Ellegaard- Jensen ◽  
Carsten S. Jacobsen ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Dna Extraction ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Soil Core ◽  
Soil Matrix ◽  
Soil Dna ◽  
Dna Recovery ◽  
The Impact

AbstractSoil DNA extraction encounters numerous challenges that can affect both yield and purity of the recovered DNA. Clay particles lead to reduced DNA extraction efficiency, and PCR inhibitors from the soil matrix can negatively affect downstream analyses when applying DNA sequencing. Further, these effects impede molecular analysis of bacterial community compositions in lower biomass samples, as often observed in deeper soil layers. Many studies avoid these complications by using indirect DNA extraction with prior separation of the cells from the matrix, but such methods introduce other biases that influence the resulting microbial community composition.To address these issues, a direct DNA extraction method was applied in combination with the use of a commercial product, the G2 DNA/RNA Enhancer®, marketed as being capable of improving the amount of DNA recovered after the lysis step. The results showed that application of G2 increased DNA yields from the studied clayey soils from layers between 1.00 and 2.20 m below ground level.Importantly, the use of G2 did not introduce bias, as it did not result in any significant differences in the biodiversity of the bacterial community measured in terms of alpha and beta diversity and taxonomical composition.Finally, this study considered a set of customised lysing tubes for evaluating possible influences on the DNA yield. Tubes customization included different bead sizes and amounts, along with lysing tubes coming from two suppliers. Results showed that the lysing tubes with mixed beads allowed greater DNA recovery compared to the use of either 0.1 or 1.4 mm beads, irrespective of the tube supplier.These outcomes may help to improve commercial products in DNA/RNA extraction kits, besides raising awareness about the optimal choice of additives, offering opportunities for acquiring a better understanding of topics such as vertical microbial characterisation and environmental DNA recovery in low biomass samples.

scholarly journals Impact of enzymatic digestion on bacterial community composition in CF airway samples

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3362 ◽  
Author(s):  
Kayla M. Williamson ◽  
Brandie D. Wagner ◽  
Charles E. Robertson ◽  
Emily J. Johnson ◽  
Edith T. Zemanick ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Dna Extraction ◽  
Community Composition ◽  
Bacterial Communities ◽  
Beta Diversity ◽  
Diversity Index ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Enzymatic Digestion ◽  
Rrna Gene

BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection ofStaphylococcus,an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the generaGemella(p < 0.01),Streptococcus(p < 0.01), andRothia(p < 0.01).Staphylococcus(p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD:Veillonella(p < 0.01),Granulicatella(p < 0.01),Prevotella(p < 0.01), andGemella(p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection ofStaphylococcusin CF OP samples. The enhanced identification ofStaphylococcus aureusis a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities.

scholarly journals Ecology and Host Identity Outweigh Evolutionary History in Shaping the Bat Microbiome

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Holly L. Lutz ◽  
Elliot W. Jackson ◽  
Paul W. Webala ◽  
Waswa S. Babyesiza ◽  
Julian C. Kerbis Peterhans ◽  
...  
Keyword(s):  
Microbial Community ◽  
Bacterial Community ◽  
Community Composition ◽  
Evolutionary History ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Anatomical Site ◽  
Skin Microbiota ◽  
Host Identity ◽  
Host Ecology

ABSTRACT Recent studies of mammalian microbiomes have identified strong phylogenetic effects on bacterial community composition. Bats (Mammalia: Chiroptera) are among the most speciose mammals on the planet and the only mammal capable of true flight. We examined 1,236 16S rRNA amplicon libraries of the gut, oral, and skin microbiota from 497 Afrotropical bats (representing 9 families, 20 genera, and 31 species) to assess the extent to which host ecology and phylogeny predict microbial community similarity in bats. In contrast to recent studies of host-microbe associations in other mammals, we found no correlation between chiropteran phylogeny and bacterial community dissimilarity across the three anatomical sites sampled. For all anatomical sites, we found host species identity and geographic locality to be strong predictors of microbial community composition and observed a positive correlation between elevation and bacterial richness. Last, we identified significantly different bacterial associations within the gut microbiota of insectivorous and frugivorous bats. We conclude that the gut, oral, and skin microbiota of bats are shaped predominantly by ecological factors and do not exhibit the same degree of phylosymbiosis observed in other mammals. IMPORTANCE This study is the first to provide a comprehensive survey of bacterial symbionts from multiple anatomical sites across a broad taxonomic range of Afrotropical bats, demonstrating significant associations between the bat microbiome and anatomical site, geographic locality, and host identity—but not evolutionary history. This study provides a framework for future systems biology approaches to examine host-symbiont relationships across broad taxonomic scales, emphasizing the need to elucidate the interplay between host ecology and evolutionary history in shaping the microbiome of different anatomical sites.

scholarly journals Anthropogenic Impact on Tropical Perennial River in South India: Snapshot of Carbon Dynamics and Bacterial Community Composition

Water ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1354
Author(s):  
Katrin Premke ◽  
Gunasekaran Dharanivasan ◽  
Kristin Steger ◽  
Kai Nils Nitzsche ◽  
Vijayan Jayavignesh ◽  
...  
Keyword(s):  
Water Quality ◽  
Bacterial Community ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Cauvery River ◽  
Main River ◽  
Warning Limits ◽  
Induced Changes ◽  
Riverine Systems ◽  
The Impact

Riverine systems play an important role in the global carbon cycle, and they are considered hotspots for bacterial activities such as organic matter decomposition. However, our knowledge about these processes in tropical or subtropical regions is limited. The aim of this study was to investigate anthropogenically induced changes of water quality, the distribution of selected pharmaceuticals, and the effects of pollution on greenhouse gas concentrations and bacterial community composition along the 800 km long Cauvery river, the main river serving as a potable and irrigation water supply in Southern India. We found that in situ measured pCO2 and pCH4 concentrations were supersaturated relative to the atmosphere and ranged from 7.9 to 168.7 μmol L−1, and from 0.01 to 2.76 μmol L−1, respectively. Pharmaceuticals like triclosan, carbamazepine, ibuprofen, naproxen, propylparaben, and diclofenac exceeded warning limits along the Cauvery. Proteobacteria was the major phylum in all samples, ranging between 26.1% and 82.2% relative abundance, and it coincided with the accumulation of nutrients in the flowing water. Results emphasized the impact of industrialization and increased population density on changes in water quality, riverine carbon fluxes, and bacterial community structure.

scholarly journals DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

2001 ◽  
Vol 67 (5) ◽  
pp. 2354-2359 ◽  
Author(s):  
F. Martin-Laurent ◽  
L. Philippot ◽  
S. Hallet ◽  
R. Chaussod ◽  
J. C. Germon ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ribosomal Dna ◽  
Dna Analysis ◽  
Restriction Analysis ◽  
Farmyard Manure ◽  
Soil Biodiversity ◽  
Soil Dna ◽  
Recovery Method ◽  
Dna Recovery ◽  
The Impact

ABSTRACT The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

scholarly journals Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

2014 ◽  
Vol 81 (4) ◽  
pp. 1463-1471 ◽  
Author(s):  
Stefan Thiele ◽  
Bernhard M. Fuchs ◽  
Rudolf Amann ◽  
Morten H. Iversen
Keyword(s):  
Microbial Community ◽  
Bacterial Community ◽  
Microbial Communities ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Marine Snow ◽  
Free Living ◽  
Fluorescence Maximum

ABSTRACTDue to sampling difficulties, little is known about microbial communities associated with sinking marine snow in the twilight zone. A drifting sediment trap was equipped with a viscous cryogel and deployed to collect intact marine snow from depths of 100 and 400 m off Cape Blanc (Mauritania). Marine snow aggregates were fixed and washedin situto prevent changes in microbial community composition and to enable subsequent analysis using catalyzed reporter deposition fluorescencein situhybridization (CARD-FISH). The attached microbial communities collected at 100 m were similar to the free-living community at the depth of the fluorescence maximum (20 m) but different from those at other depths (150, 400, 550, and 700 m). Therefore, the attached microbial community seemed to be “inherited” from that at the fluorescence maximum. The attached microbial community structure at 400 m differed from that of the attached community at 100 m and from that of any free-living community at the tested depths, except that collected near the sediment at 700 m. The differences between the particle-associated communities at 400 m and 100 m appeared to be due to internal changes in the attached microbial community rather thande novocolonization, detachment, or grazing during the sinking of marine snow. The new sampling method presented here will facilitate future investigations into the mechanisms that shape the bacterial community within sinking marine snow, leading to better understanding of the mechanisms which regulate biogeochemical cycling of settling organic matter.

scholarly journals Sample Dilution and Bacterial Community Composition Influence Empirical Leucine-to-Carbon Conversion Factors in Surface Waters of the World's Oceans

2015 ◽  
Vol 81 (23) ◽  
pp. 8224-8232 ◽  
Author(s):  
Eva Teira ◽  
Víctor Hernando-Morales ◽  
Francisco M. Cornejo-Castillo ◽  
Laura Alonso-Sáez ◽  
Hugo Sarmento ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Nucleic Acid Content ◽  
Carbon Conversion ◽  
Production Rates ◽  
Conversion Factors ◽  
Content Type ◽  
Carbon Production ◽  
The Impact

ABSTRACTThe transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu−1and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyllaconcentration, picocyanobacterial abundance (mostlyProchlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that bothProchlorococcusand HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean.

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