Microbial DNA Extraction Research Articles

Grinding Beads Influence Microbial DNA Extraction from Organic-Rich Sub-Seafloor Sediment.

Sub-seafloor sediment is the largest microbial habitat on Earth. The study of microbes in sub-seafloor sediment is largely limited by the technical challenge of acquiring ambient microbial DNA because of sediment heterogeneity. Changes in the extraction method, even just by one step, can affect the extraction yields for complicated sediment samples. In this work, sub-seafloor sediment samples from the Baltic Sea with high organic carbon content were used to evaluate the influence of different grinding beads on DNA extraction. We found that the grinding beads can affect the DNA extraction from the organic-matter- and biosiliceous-clay-rich samples. A mixture of 0.5-mm and 0.1-mm grinding beads exhibited higher DNA yields and recovered more unique taxa than other bead combinations, such as Stenotrophomonas from Gammaproteobacteria and Leptotrichia from Fusobacteria; therefore, these beads are more suitable than the others for DNA extraction from the samples used in this study. This advantage might be magnified in samples with high biomass. On the contrary, the use of only small beads might lead to underestimation for certain Gram-positive strains. Overall, the discovery of abundant widespread deep biosphere clades in our samples indicated that our optimized DNA extraction method successfully recovered the in situ microbial community.

A protocol specialized for microbial DNA extraction from living poplar wood

Microbial DNA extraction is a critical step in metagenomic research. High contents of chemical substances in wood tissues always cause low microbial DNA yield and quality. Up to date, almost no specialized methods involved in microbial DNA extraction from living wood were reported. In this study, an improved protocol (M1) concerning microbial DNA extraction from living poplar wood was developed. We compared microbial DNA yield and quality by M1 with those by other seven methods, including PowerSoil DNA isolation kit (M2), two soil microbial DNA extraction methods (M3 and M4), poplar genomic DNA extraction method from wood (M5), and microbial DNA extraction method from herb stems (M6), isolating bacteria (M7) and isolating fungus (M8). Results showed that M1 yielded much better quality and concentration of microbial DNA than the other methods (M2-M8) from both poplar wetwood and sapwood tissues. Following M1 protocol, 1 g of wetwood sample could yield 272.27 ng/ul (vol=50 ul) pure microbial DNA with the absorption ratios of 1.87 (A260/A230) and 1.66 (A260/A280). For 1 g of sapwood sample, these values were 361.83 ng/ul, 1.85 and 2.24, respectively. These DNA could be stably visualized by agarose gel electrophoresis and amplified by primer sets of bacteria (16S V3-V4, 16S-V4, 16S V4-V5) and fungus (ITS1, ITS2). While, the other seven methods only obtained less or contaminated microbial DNA, which could not be amplified stably by aforementioned primer sets. Our protocol provided an approach for microbial community study in living poplar wood in a more accurate way by molecular biology techniques.

High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.

Characterization of rumen, fecal, and milk microbiota in lactating dairy cows.

Targeting the gastrointestinal microbiome for improvement of feed efficiency and reduction of production costs is a potential promising strategy. However little progress has been made in manipulation of the gut microbiomes in dairy cattle to improve milk yield and milk quality. Even less understood is the milk microbiome. Understanding the milk microbiome may provide insight into how the microbiota correlate with milk yield and milk quality. The objective of this study was to characterize similarities between rumen, fecal, and milk microbiota simultaneously, and to investigate associations between microbiota, milk somatic cell count (SCC), and milk yield. A total of 51 mid-lactation, multiparous Holstein dairy cattle were chosen for sampling of ruminal, fecal, and milk contents that were processed for microbial DNA extraction and sequencing. Cows were categorized based on low, medium, and high SCC; as well as low, medium, and high milk yield. Beta diversity indicated that ruminal, fecal, and milk populations were distinct (p < 0.001). Additionally, the Shannon index demonstrated that ruminal microbial populations were more diverse (p < 0.05) than were fecal and milk populations, and milk microbiota was the least diverse of all sample types (p < 0.001). While diversity indices were not linked (p > 0.1) with milk yield, milk microbial populations from cows with low SCC demonstrated a more evenly distributed microbiome in comparison to cows with high SCC values (p = 0.053). These data demonstrate the complexity of host microbiomes both in the gut and mammary gland. Further, we conclude that there is a significant relationship between mammary health (i.e., SCC) and the milk microbiome. Whether this microbiome could be utilized in efforts to protect the mammary gland remains unclear, but should be explored in future studies.

Association between rumen microbiota and marbling grade in Hu sheep.

The marbling fat regulates the flavor of mutton and measures the fat density in the loin eye and is the most important parameter of carcass grading. The objective of this study was to explore the relationship of rumen microbiota and mutton marbling grade. One hundred and eighty-seven feedlot-finished Hu male lambs (Age: 180 day; Final BW: 46.32 ± 6.03 kg) were slaughtered, and ruminal contents and marbling grade were collected. Ruminal microbial DNA extraction and 16S rRNA gene sequencing was performed to investigate microbial composition and to predict microbial metabolic pathways. The animal cohort was then grouped based on marbling grades [low marbling (LM), marbling grade ≤ 1; Medium marbling (MM), 1 < marbling grade ≤ 3; High Marbling (HM), 3 < marbling grade ≤ 5] and intramuscular fat-associated microorganisms were pinpointed using LEfSe and random forest classification model. Intramuscular fat content had significantly differences among the three groups (P < 0.05), and was significantly correlated with VFAs profiling. HM sheep showed a higher abundance of one bacterial taxon (Kandleria), and two taxa were overrepresented in the MM sheep (Pseudobutyrivibrio and Monoglobus), respectively. In addition, the main intramuscular fat deposition pathway was found to involve peroxisome proliferator-activated receptor (PPAR) fatty acid synthesis. By studying the effect of the ruminal microbiome on the marbling of sheep, the present study provides insights into the production of high-quality mutton.

Guideline for the analysis of the microbial communities of the human upper airways

ABSTRACT The recent COVID-19 pandemic prompted a rapid-growing interest in the investigation of the human microbiota of the upper airways. In fact, the resident microbial community of this body district may have an influence on the onset of SARS-CoV-2 infection and its clinical course in terms of presence, symptom severity, and outcomes. However, several microbiological methodologies are available to study the human microbiota, reflecting the extensive fragmentation of methodological approaches. We investigate the impact of two critical steps that can induce biases in the downstream analyses, i.e. sampling method and microbial DNA extraction kit employed. We observed major discrepancies regarding the total amount of prokaryotic DNA that could be retrieved from a biological sample and the proportion between bacterial DNA and human host DNA. Moreover, shotgun DNA sequencing and taxonomic profile reconstruction also revealed correlations between sampling methods and the procedures applied for microbial DNA extraction. Based on all the data collected in this study, we formulate indications regarding the most efficient and reliable methodological procedures for the metagenomic analyses of the upper airways’ microbiota to maximize accuracy and reproducibility.

Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library

The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products.

Taxonomic and Functional Fecal Microbiota Signatures Associated With Insulin Resistance in Non-Diabetic Subjects With Overweight/Obesity Within the Frame of the PREDIMED-Plus Study.

ObjectiveAn altered gut microbiota has been associated with insulin resistance, a metabolic dysfunction consisting of cellular insulin signaling impairment. The aim of the present study is to determine the taxonomic and functional fecal microbiota signatures associated with HOMA-IR index in a population with high cardiovascular risk.MethodsA total of 279 non-diabetic individuals (55–75 years aged) with overweight/obesity and metabolic syndrome were stratified according to tertiles of HOMA-IR index. Blood biochemical parameters, anthropometric measurements and fecal samples were collected at baseline. Fecal microbial DNA extraction, 16S amplicon sequencing and bioinformatics analysis were performed.Results Desulfovibrio, Odoribacter and Oscillospiraceae UCG-002 were negatively associated with HOMA-IR index, whereas predicted total functional abundances revealed gut metabolic modules mainly linked to amino acid degradation. Butyricicoccus, Erysipelotrichaceae UCG-003, Faecalibacterium were positively associated with HOMA-IR index, whereas predicted total functional abundances revealed gut metabolic modules mainly linked to saccharide degradation. These bacteria contribute differentially to the gut metabolic modules, being the degree of contribution dependent on insulin resistance. Both taxa and gut metabolic modules negatively associated to HOMA-IR index were linked to mechanisms involving sulfate reducing bacteria, improvement of intestinal gluconeogenesis and production of acetate. Furthermore, both taxa and gut metabolic modules positively associated to HOMA-IR index were linked to production and mechanisms of action of butyrate.ConclusionsSpecific taxonomic and functional fecal microbiota signatures associated with insulin resistance were identified in a non-diabetic population with overweight/obesity at high cardiovascular risk. These findings suggest that tailoring therapies based on specific fecal microbiota profiles could be a potential strategy to improve insulin sensitivity.

Alterations in the Stool Microbiome in Newborns Undergoing Mild Therapeutic Hypothermia after Hypoxic-Ischemic Encephalopathy

Background: Hypoxic-ischemic encephalopathy (HIE) is associated with brain injury in newborns and may lead to disability or death. Mild therapeutic hypothermia (TH) is an effective neuroprotective intervention and an established standard of care in western countries. The gut microbiome, the genomic and physicochemical contribution of the gut microbiota, serves important functions and is increasingly recognized as a major influencer on development. The impact of HIE and TH on the evolving gut microbiota of the newborn remains to be elucidated. Objective: The objective of this study was to carry out an exploratory study on the effects of HIE and TH on the gut microbiome in term neonates. Methods and Results: Stool samples were obtained from 28 newborns with HIE (median age 68 h) undergoing TH on the neonatal unit (HIE TH group), with a follow-on stool sample available for 20 of these babies (median age 151 h). For comparison, a single stool specimen was obtained from 19 healthy newborns on the postnatal ward (median age 34 h). The microbiota composition was determined using established microbial DNA extraction and 16S rRNA gene sequencing methodology. There was no difference in the mode of delivery or the method of feeding the newborns, once established, between the 2 groups. All the infants in the HIE TH group had received antibiotics compared to only one of the controls. A lower α-diversity, quantified by the Shannon diversity index, was noted in the microbiota of the HIE TH group in comparison to the control group. The HIE TH group had a higher mean relative abundance (MRA) of facultative anaerobes and aerobes such as Staphylococcus species and a lower MRA of strict anaerobes, such as members of the Bacteroides genus, compared to the control. Also, there was a significant reduction in the MRA of the genus Bifidobacterium in the HIE TH group. Although the mode of delivery exerts a profound influence on the gut microbiota of the newborn, distance-based redundancy analysis showed that TH may exert an independent influence. This study could not determine the independent contribution of the use of antibiotics or the neonatal intensive care unit environment. Conclusion: In this study, we demonstrate an alteration in the microbiota composition in newborns undergoing TH for HIE.

Identifying Predictive Bacterial Markers from Cervical Swab Microbiota on Pregnancy Outcome in Woman Undergoing Assisted Reproductive Technologies.

Background and aims: Failure of the embryo to implant causes about three-fourths of lost pregnancies. Female genital tract microbiota has been associated to Assisted Reproductive Technologies (ART) outcomes. The objective of this study was to analyze the microbiota of human cervical swab and to correlate these findings with the ART outcomes. Materials and Methods: In this study, 88 cervical swabs were collected from women undergoing ART cycles, with various causes of infertility, at the beginning of the ART protocols. After microbial DNA extraction, V3–V4 variable regions of the 16S rRNA gene were amplified and sequenced on the Illumina MiSeq platform. PEnalized LOgistic Regression Analysis (PELORA) was performed to identify clusters of bacterial populations with differential abundances between patients with unfavorable and favorable pregnancy outcome groups, respectively. Results: We identified a core of microorganisms at lower taxonomic levels that were predictive of women’s pregnancy outcomes. Statistically significant differences were identified at species levels with Lactobacillus salivarius, Lactobacillus rhamnosus among others. Moreover the abundance of Lactobacillus crispatus and iners, respectively increased and decreased in favorable group as compared to unfavorable group, resulted within the core of microorganisms associated to positive ART outcome. Although the predominance of lactobacilli is generally considered to be advantageous for ART outcome, we found that also the presence of Bifidobacterium (together with the other lactobacilli) was more abundant in the favorable group. Discussion: Cervix is colonized by microorganisms which can play a role in ART outcomes as seen by an overall decrease in embryo attachment rates and pregnancy rates in both fertile and infertile women. If confirmed in a larger cohort, the abundance of these bacteria can be useful not only as a marker of unfavorable pregnancy outcome but also they may open the way to new interventional strategies based on genital tract microbiota manipulation in order to increase the pregnancy rates in woman undergoing assisted reproductive technologies.

Optimization of microbial DNA extraction from human skin samples for CRISPR typing

The human skin microbiome, a complex microbial community harbored by the human skin surface, has been proposed as a new forensic genetic marker for human identification to complement short tandem repeat typing. A recent study utilized Streptococcus clustered regularly interspaced short palindromic repeat (CRISPR) polymorphism of the skin microbiome to characterize individuality of the skin microbiome; however, further validation is required for forensic purposes. Optimization of the DNA extraction protocol is critical for both forensic human DNA and microbiome analyses. Previous reports have demonstrated that DNA extraction protocols may alter the results of microbiome analysis, suggesting careful selection of DNA extraction protocols. In this study, we evaluated protocols for DNA extraction from skin swab samples by comparing two DNA extraction protocols, EZ1 DNA Investigator Kit and DNeasy PowerSoil Kit, in order to optimize the metagenomic CRISPR typing method. Quantification of 16S rRNA gene copy numbers and qualification of CRISPR diversities suggested that microbial DNA yield and composition of the CRISPRs were not altered by the two DNA extraction methods. Furthermore, the accuracy of the individual prediction models built using each sample set was comparable. Thus, our results support that the EZ1 platform, an automated DNA extraction protocol used in forensic laboratories, can also be used for metagenomic CRISPR typing intended for forensic human identification.

Freeze-Thaw Pretreatment Can Improve Efficiency of Bacterial DNA Extraction From Meconium.

Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at −80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1–3 times at −20°C, −80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at −20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at −20°C, −80°C, and in liquid nitrogen. (4) Among the conditions of −20°C, −80°C, and liquid nitrogen, −20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

An Abnormal Host/Microbiomes Signature of Plasma-Derived Extracellular Vesicles Is Associated to Polycythemia Vera.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

Optimizing an enclosed bead beating extraction method for microbial and fish environmental DNA

AbstractDNA extraction is a critical step in processing environmental DNA (eDNA), influencing biodiversity estimates and ecosystem monitoring. Sterivex filters have become popular for collecting aquatic eDNA, allowing for on‐site filtration and reduced contamination. Yet, extracting eDNA from an enclosed filter remains laborious. Recent methods have improved extraction of microbial DNA from Sterivex by adding lysis buffer and beads directly to the filter; however, it remains unclear how bead size affects eDNA recovery across multiple trophic levels. Further, the method has not been scaled up for 96‐well magnetic bead extraction kits and robotic manipulation. We collected surface water from Bear Cut (Biscayne Bay, Florida) and performed enclosed Sterivex DNA extractions with different bead size combinations (0.1, 0.5, 0.1 + 0.5 mm, and no beads) and two magnetic kits (Zymo and NucleoMag). We employed 16S and 12S rRNA gene metabarcoding to assess extraction effects on microbes (bacteria, archaea, and eukaryotes) and bony fishes. We polled local experts to verify fish eDNA, revealing several non‐native taxa originating from a nearby aquarium. DNA yield, microbial richness, and diversity were not influenced by beads. Bacterial relative abundance (family to ASV) was conserved across bead treatments, while several hard‐to‐lyse eukaryotes (diatoms and chlorophytes) were less abundant (or absent) when extracted without beads. Higher fish richness was detected with larger beads; however, relative abundance and composition were not associated with beads, likely reflecting heterogeneity or low concentrations of fish compared to microbial eDNA. We recommend using larger or differently sized beads for Sterivex extractions to detect a range of marine life, with increased water volume (&gt;2 L per filter) or replication as necessary to enhance fish eDNA capture.

An improved method for extraction of microbial DNA from alkaline-saline soil

A modified method for the direct extraction of DNA from alkaline-saline soils with minimum DNA fragmentation and a possible reduction in chimera formation during polymerase chain reaction (PCR) was developed. The commercial extraction kit Power Soil DNA (Mo Bio™ Laboratories, Inc.) was used as a reference technique. The method reported here was based on cell lysis employing ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and cell disruption with mechanical force with FastPrep-24™ equip followed by one cycle of freezing at -40 °C for 60 min and thawing at 65 °C for 20 min. The extraction method was tested for allophonic soils with large concentrations of organic matter, fulvic and humic acids, electrolytic conductivity (EC) ranging between 2.6 dS m-1 and 39.9 dS m-1, and pH between 8.8 and 10.9. The yield of DNA extracted depended on soil type, i.e., DNA extracted from soil varied between 2.35 (Texcoco-2) to 3.66 (Texcoco-1) μg DNA g-1 soil. The proposed method in this study produced enough DNA with yield and quality for PCR amplification of 16S rDNA when bovine serum albumin (BSA) was added to the reaction buffer. The DNA obtained had sufficient quality and yield for later use for 16S sequencing or possible use in other sequencing technologies, e.g. whole metagenome shotgun sequencing.

Performance comparison of different microbial DNA extraction methods on bird feces

BackgroundAs an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet.MethodsIn this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild.ResultsBead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity.ConclusionsDNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

Multifunctional magnetic nanoparticle cloud assemblies for in situ capture of bacteria and isolation of microbial DNA.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 103 cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 106 cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample.

Characterization of Gut Microbiota in Prenatal Cold Stress Offspring Rats by 16S rRNA Sequencing.

Simple SummaryPrenatal stress, including prenatal cold stress has long-term effects on offspring’s physical and mental health. Our previous study showed a reduction of anxiety-like behavior in offspring rats suffered from prenatal cold stress. It is well-known that gut microbiota was involved in a variety of physiological activities, such as emotion, cognition, and behavior. However, information on the comparison between prenatal cold stress and gut microbiota in offspring is limited. The current study compared the gut microbiota composition of the prenatal cold stress and non-stress offspring rats. Cold stressed during gestation period showed to change the offspring gut microbiota composition, and Bacteroides and Lactobacillus were significantly increased in prenatal cold stress offspring rat guts. With the hope, cold stress-induced negative effects of animals can be prevented by microbiological interventions.Our previous study showed a reduction of anxiety-like behavior in offspring rats suffered from prenatal cold stress; whether this was related to changes in the offspring gut microbiota is unclear. To obtain the evidence for the role of the gut microbiota in prenatal cold stress offspring, 16S rRNA sequencing technology was used. Male and female offspring rat feces were collected from a room temperature group and a prenatal cold stress group (n ≥ 8) for microbial DNA extraction, followed by 16S rRNA sequencing. The results indicated that prenatal cold stress could change the offspring’s gut microbiota composition. Prenatal cold stress significantly upregulates Lactobacillus, Lactobacillus_gasseri, Bacteroides, and Bacteroides-acidifaciens in female offspring, whereas prenatal cold stress significantly reduced Lachnospiraceae and Prevotellaceae in male offspring. These data showed the characterization of gut microbiota in prenatal cold stress offspring rats, and these data suggest that microbiological intervention in the future can potentially prevent the negative effects caused by cold stress to animals.

DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing

Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non- or slow-growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant-related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies’ (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10-fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection-causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible.

Perilla frutescens Leaf Alters the Rumen Microbial Community of Lactating Dairy Cows.

Perilla frutescens (L.) Britt., an annual herbaceous plant, has antibacterial, anti-inflammation, and antioxidant properties. To understand the effects of P. frutescens leaf on the ruminal microbial ecology of cattle, Illumina MiSeq 16S rRNA sequencing technology was used. Fourteen cows were used in a randomized complete block design trial. Two diets were fed to these cattle: a control diet (CON); and CON supplemented with 300 g/d P. frutescens leaf (PFL) per cow. Ruminal fluid was sampled at the end of the experiment for microbial DNA extraction. Overall, our findings revealed that supplementation with PFL could increase ruminal fluid pH value. The ruminal bacterial community of cattle was dominated by Bacteroidetes, Firmicutes, and Proteobacteria. The addition of PFL had a positive effect on Firmicutes, Actinobacteria, and Spirochaetes, but had no effect on Bacteroidetes and Proteobacteria compared with the CON. The supplementation with PFL significantly increased the abundance of Marvinbryantia, Acetitomaculum, Ruminococcus gauvreauii, Eubacterium coprostanoligenes, Selenomonas_1, Pseudoscardovia, norank_f__Muribaculaceae, and Sharpea, and decreased the abundance of Treponema_2 compared to CON. Eubacterium coprostanoligenes, and norank_f__Muribaculaceae were positively correlated with ruminal pH value. It was found that norank_f__Muribaculaceae and Acetitomaculum were positively correlated with milk yield, indicating that these different genera are PFL associated bacteria. This study suggests that PFL supplementation could increase the ruminal pH value and induce shifts in the ruminal bacterial composition.