Microbial DNA Extraction Research Articles

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere.

The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Modification of extraction method for community DNA isolation from salt affected compact wasteland soil samples

To overcome the issue of interferences by salt and compactness in release of bacterial cell required for lysis, method described by Yeates et al. (1998), was optimized for isolation of genomic material (Deoxyribo Nucleic Acid, DNA) from soil microbial community by addition of Al(NH4)SO4. Very low total viable count was observed in the samples tested and hence use of higher amount of soil is required primarily for DNA isolation from wasteland soils. The method proves itself efficient where commercially available bead beating and enzymatic lysis methods could not give isolation of any amount of community genomic DNA due to compact nature and salt concentrations present in soil.•The protocol was found efficient for soil samples with high clay content for microbial community DNA extraction.•Variation in lysis incubation and amount of soil may help with soil samples containing low microbial population.•Addition of Al(NH4)SO4 is crucial step in humic acid removal from extracted DNA samples for soil samples containing high salinity and clay particles.

Physiological effects of stress related to helicopter travel in Federal Emergency Management Agency search-and-rescue canines.

Working canines are deployed by the Federal Emergency Management Agency (FEMA), as part of a National Disaster Response Plan. Stress associated with helicopter flight and the resulting physical effects on the dog are unknown. Our objective was to test the hypotheses that (1) helicopter travel affects the physiology and faecal microbiota of working canines, but that (2) physiological consequences of helicopter travel will not negatively affect their work performance. A total of nine FEMA canines were loaded onto helicopters and flown for 30min in July 2015. Rectal temperature, behavioural stress indicators and saliva swabs (for cortisol) were collected at baseline, loading, mid-flight and post-flight. After flight, canines completed a standardised search exercise to monitor work performance. Faecal samples were collected for microbial DNA extraction and Illumina sequencing. All canines were on a standardised diet (CANIDAE® Grain Free PURE Land®) for 3 weeks prior to the study. Visible indicators of stress were observed at loading and at mid-flight and corresponded with an increase (P<0·05) in salivary cortisol from 5·4µg/l (baseline) to 6·4µg/l (loading). Additionally, rectal temperature increased (P <0·05) from 38·61°C (baseline) to 39·33°C (mid-flight) and 39·72°C (post-flight). Helicopter travel did not affect search performance (P>0·05). We found that α- and β-diversity measures of faecal microbiota were not affected (P>0·05). Our data suggest that although helicopter travel may cause physiological changes that have been associated with stress in working dogs, it does not make an impact on their search performance or the stability of faecal microbiota.

Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples.

The Role of Bifidobacteria in Ulcerative Colitis: Preliminary Results

Introduction: Ulcerative colitis (UC) has a multifactorial pathogenesis, including genetic susceptibility, environmental triggers, gut microbiota and microbial imbalance due to its immunomodulatory function. Previous studies showed that Bifidobacterium bifidum PRL2010 cells can effectively activate signals in macrophages, modulate metabolic activities and influence immune responses in vitro and when tested in a murine colitis model. The aim of this study was to characterize the microbiome of UC patients compared to healthy controls (HS), in order to evaluate the functional role of bifidobacteria as microbial biomarkers.Figure 1Methods: A stool sample was obtained from UC patients, HS and processed for microbial DNA extraction. The microbiota taxonomic profile and the bacterial population diversity index were determined by using a metagenomic approach. Patient demographics, level of disease activity and therapy were collected and analyzed. Data extracted from stool samples of UC patients were compared with those of HS. Results: Between April 2015 and April 2016, 28 patients were enrolled: 14 UC patients (mean age 39y), and 14 HS (mean age 33y). Among UC patients, 6 had active disease (3 were under therapy with infliximab, 1 with adalimumab, 1 with azathioprine), and 8 showed disease remission (3 were under therapy with azathioprine, 1 with adalimumab; 1 with mesalamine, 3 no therapy). The fecal microbiome of UC patients, independently from the type of therapy, showed a marked reduction in bacterial population diversity index. Bifidobacteria were reduced in abundance of >30 % in UC group compared to HS group. Among UC patients, those with active disease showed a significant reduction (4 fold; P< 0.05) of the bifidobacteria compared to those in remission. Conclusion:Bifidobacterium bifidum is a reliable microbial biomarker for UC. The reduction in its overall abundance and the decreased microbial diversity index appear to be related to the presence of disease and may explain, at least in part, the role of intestinal microbiome in the pathogenesis of ulcerative colitis. Moreover, the significantly different abundance of Bifidobacterium bifidum between patients with active disease and in remission, highlights that Bifidobacterium bifidum may be considered an activity index of the disease.

Metagenomic Characterization of Microbial Communities In Situ Within the Deeper Layers of the Ileum in Crohn's Disease.

Background & AimsMicrobial dysbiosis and aberrant host–microbe interactions in the gut are believed to contribute to the development and progression of Crohn’s disease (CD). Microbiome studies in CD typically have focused on microbiota in feces or superficial mucosal layers of the colon because accessing DNA from deeper layers of the bowel is challenging. In this study, we analyzed the deep tissue microbiome in patients who underwent surgical resection of the small intestine.MethodsParaffin blocks were obtained from 12 CD patients undergoing ileocecal resection, and healthy ileum samples (inflammatory bowel disease–free controls) were obtained from 12 patients undergoing surgery for right-sided colon cancer. Diseased and healthy-appearing ileum was identified using microscopy, and paraffin blocks were macrodissected using a core needle to specifically isolate DNA. Illumina Whole Genome Sequencing was used for microbial sequence identification and subsequent taxonomic classification using the PathSeq tool.ResultsWe observed significant differences between the microbiome of CD samples vs inflammatory bowel disease–free controls, including depletion of Bacteroidetes and Clostridia. Notably, microbial composition at the phyla level did not differ markedly between healthy and diseased areas of CD patients. However, we observed enrichment of potentially pathogenic organisms at the species level.ConclusionsOur study showed dysbiosis within deeper layers of the ileum of CD patients, specifically enrichment of enterotoxigenic Staphylococcus aureus and an environmental Mycobacterium species not described previously. Future studies with larger cohort sizes are warranted to confirm these findings. Studies would benefit from effective microbial DNA extraction methods from paraffin sections and host nucleic acid depletion approaches to increase microbial read coverage.

The effect of dietary Chlorella vulgaris supplementation on micro-organism community, enzyme activities and fatty acid profile in the rumen liquid of goats.

Microalgae might be considered as an alternative source of fat and/or protein for ruminant's diets. However, changes in populations of ruminal micro-organisms associated with biohydrogenation process, methane and ammonia production in response to microalgae dietary supplementation have not been well characterized. Thus, 16 cross-bred goats were divided into two groups. Each goat of both groups was fed individually with alfalfa hay and concentrates separately. The concentrates of the control group had no microalgae while those of the treated group were supplemented with 10g lyophilized Chlorella vulgaris/kg concentrate (chlor). On the 30th experimental day, samples of rumen fluid were collected for microbial DNA extraction, fatty acid profile and enzyme activity analyses. The results showed that the chlor diet compared with the control increased significantly the populations of Methanosphaera stadtmanae, Methanobrevibacter ruminantium and Methanogens bacteria and protozoa in the rumen of goats. A significant reduction in the cellulase activity and in the abundance of Ruminococcus albus, and a significant increase in the protease activity and in the abundance of Clostridium sticklandii in the rumen liquid of goats fed with the chlor diet, compared with the control, were found. Chlorella vulgaris supplementation promoted the formation of trans C18:1 , trans-11 C18:1 and monounsaturated fatty acids (MUFA), while the proportions of C18:0 and long-chain fatty acids (LCFA) reduced significantly in the rumen liquid of goats. This shift in ruminal biohydrogenation pathway was accompanied by a significant increase in Butyrivibrio fibrisolvens trans C18:1 -producing bacteria. In conclusion, the supplementation of diets with microalgae needs further investigation because it enhances the populations of methane-producing bacteria and protozoa.

Back to the Future of Soil Metagenomics.

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek (Table 1). The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?

Comparison of two commercial DNA extraction kits for the analysis of nasopharyngeal bacterial communities

Characterization of microbial communities via next-generation sequencing (NGS) requires an extraction ofmicrobial DNA. Methodological differences in DNA extraction protocols may bias results and complicate inter-study comparisons. Here we compare the effect of two commonly used commercial kits (Norgen and Qiagen)for the extraction of total DNA on estimatingnasopharyngeal microbiome diversity. The nasopharynxis a reservoir for pathogens associated with respiratory illnesses and a key player in understandingairway microbial dynamics. Total DNA from nasal washes corresponding to 30 asthmatic children was extracted using theQiagenQIAamp DNA and NorgenRNA/DNA Purification kits and analyzed via IlluminaMiSeq16S rRNA V4 ampliconsequencing. The Norgen samples included more sequence reads and OTUs per sample than the Qiagen samples, but OTU counts per sample varied proportionallybetween groups (r = 0.732).Microbial profiles varied slightly between sample pairs, but alpha- and beta-diversity indices (PCoAand clustering) showed highsimilarity between Norgen and Qiagenmicrobiomes. Moreover, no significant differences in community structure (PERMANOVA and adonis tests) and taxa proportions (Kruskal-Wallis test) were observed betweenkits. Finally, aProcrustes analysis also showed low dissimilarity (M 2 = 0.173; P < 0.001) between the PCoAs of the two DNA extraction kits. Contrary to what has been observed in previous studies comparing DNA extraction methods, our 16S NGS analysis of nasopharyngeal washes did not reveal significant differences in community composition or structure between kits. Our findingssuggest congruence between column-based chromatography kits and supportthe comparison of microbiomeprofilesacross nasopharyngeal metataxonomic studies.

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

Glycomacropeptide is a prebiotic that reduces Desulfovibrio bacteria, increases cecal short-chain fatty acids, and is anti-inflammatory in mice.

Glycomacropeptide (GMP) is a 64-amino acid (AA) glycophosphopeptide with application to the nutritional management of phenylketonuria (PKU), obesity, and inflammatory bowel disease (IBD). GMP is a putative prebiotic based on extensive glycosylation with sialic acid, galactose, and galactosamine. Our objective was to determine the prebiotic properties of GMP by characterizing cecal and fecal microbiota populations, short-chain fatty acids (SCFA), and immune responses. Weanling PKU (Pah(enu2)) and wild-type (WT) C57Bl/6 mice were fed isoenergetic AA, GMP, or casein diets for 8 wk. The cecal content and feces were collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing. SCFA were determined by gas chromatography, plasma cytokines via a Bio-Plex Pro assay, and splenocyte T cell populations by flow cytometry. Changes in cecal and fecal microbiota are primarily diet dependent. The GMP diet resulted in a reduction from 30-35 to 7% in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice with genotype-dependent changes in Bacteroidetes or Firmicutes. Cecal concentrations of the SCFA acetate, propionate, and butyrate were increased with GMP. The percentage of stimulated spleen cells producing interferon-γ (IFN-γ) was significantly reduced in mice fed GMP compared with casein. In summary, plasma concentrations of IFN-γ, TNF-α, IL-1β, and IL-2 were reduced in mice fed GMP. GMP is a prebiotic based on reduction in Desulfovibrio, increased SCFA, and lower indexes of inflammation compared with casein and AA diets in mice. Functional foods made with GMP may be beneficial in the management of PKU, obesity, and IBD.

Assessment of microbial DNA extraction methods of cadaver soil samples for criminal investigations

Gravesoil beneath decomposing cadavers undergoes substantial biochemical changes that have the potential to aid in PMI estimation and identification of clandestine gravesites. The use of DNA extraction methods is necessary for culture-independent downstream molecular applications such as PCR and next-generation sequencing. In this study, a comparison of four methods was performed for cadaver-soil collected beneath the heads and feet of 11 cadavers decaying in a natural setting. The four methods isolated 3.6–263 ng/μl of genomic DNA as determined by optical density analysis. The purity of the extracted DNA according to A260/280 and A260/230 ratios was determined. The A260/280 and A260/230 ratios were 1.24–1.97 and 0.27–2.12, respectively. The optical density at 320 nm was measured for humic acid quantification of the lysates from the method that provided the most efficient removal of humic acid. The results demonstrated that this method provided a 98% reduction of humic acid. PCR of 16S rRNA genes followed by gel electrophoresis was performed. The statistical analysis revealed a significant correlation between the yields and days on/in the soil using a phenol-chloroform method for soil collected at the head and feet. No earlier published work has extensively elucidated the efficacy of DNA extraction methods for DNA obtained from cadaver-soil.

Application of a new purification method of West-Kazakhstan chestnut soil microbiota DNA for metagenomic analysis

A method for the extraction of soil microbial DNA has been tested on chestnut soils (Kastanozems) of the West Kazakhstan region. The taxonomic analysis of soil microbiome libraries has shown that the phyla Actinobacteria and Proteobacteria constitute the largest part of microbial communities in the analyzed soils. The Archaea form an appreciable part of the microbiome in the studied samples. In the underdeveloped dark chestnut soil, their portion is higher than 11%. This is of interest, as the proportion of Archaea in the soil communities of virgin lands usually does not exceed 5%. In addition to the phyla mentioned above, there are representatives of the phyla Acidobacteria, Bacteroidetes, Firmicutes, Gemmatimonadales, Planctomycetes, and Verrucomicrobia, which are all fairly common in soil communities.

Glycomacropeptide Shows Prebiotic and Immune Modulating Properties in Phenylketonuria and Wild Type Mice

BackgroundGlycomacropeptide (GMP) is a 64 amino acid (AA) glycophosphopeptide with application to the nutritional management of phenylketonuria (PKU), obesity and intestinal disorders. GMP is a putative prebiotic based on its carbohydrate content including sialic acid.ObjectiveTo determine the prebiotic properties of GMP by characterizing cecal and fecal microbiota populations and immune responses in C57Bl/6 mice.MethodsWeanling PKU (Pahenu2) and wild type (WT) mice were fed isoenergetic AA, GMP or casein diets for 8 weeks. The cecum content and feces were collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing. Plasma cytokines were determined using a Bio‐Plex Pro assay and splenocyte T cell populations were assessed using flow cytometry.ResultsPairwise comparison suggests that changes in cecal microbiota are primarily diet dependent. The GMP diet resulted in a significant reduction in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice with genotype‐dependent changes in Bacteroides or Firmicutes. The percentage of stimulated spleen cells producing interferon‐gamma (IF‐γ) was significantly reduced in mice fed GMP compared to casein. Plasma concentrations of IF‐γ and tumor necrosis factor‐α were lowest in mice fed GMP.ConclusionsGMP is a prebiotic based on modulation of the intestinal microbiota compared with casein and AA diets in mice. The shift in microbial populations with GMP appears to be beneficial based on lower indices of inflammation with GMP.

A single protocol for extraction of gDNA from bacteria and yeast.

Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification.

Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

Simultaneous extraction, separation and purification of microbial genomic DNA and total RNA from acidic habitat samples

DNA and RNA simultaneously extracted fromA. fusing the optimised method. (a) Total nucleic acid extracted fromA. f: lane M1, 1 kb ladder; lane M2,HindIII-cut lambda molecular size marker; lanes 1–4, biological replicates. (b) DNA precipitated by isopropanol. (c) RNA precipitated by LiCl.

Bacterial and fungal DNA extraction from blood samples: manual protocols.

A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

Bacterial and fungal DNA extraction from blood samples: automated protocols.

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol·L−1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol·L−1 Tris (pH 8.0), 1.5 mol·L−1 NaCl, 0.1 mol·L−1 NaH2PO4, and Na2HPO4 at 65°C with 180 r·min−1 for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil™ DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil™ DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.