14863952
Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome
BACKGROUND
DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.


METHODOLOGY/PRINCIPAL FINDINGS
In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.


CONCLUSIONS/SIGNIFICANCE
Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
1106834
An ecological and evolutionary perspective on human–microbe mutualism and disease
The microbial communities of humans are characteristic and complex mixtures of microorganisms that have co-evolved with their human hosts. The species that make up these communities vary between hosts as a result of restricted migration of microorganisms between hosts and strong ecological interactions within hosts, as well as host variability in terms of diet, genotype and colonization history. The shared evolutionary fate of humans and their symbiotic bacteria has selected for mutualistic interactions that are essential for human health, and ecological or genetic changes that uncouple this shared fate can result in disease. In this way, looking to ecological and evolutionary principles might provide new strategies for restoring and maintaining human health.
10323025
Influences of microbiota on intestinal immune system development.
The normal colonization of the mammalian intestine with commensal microbes is hypothesized to drive the development of the humoral and cellular mucosal immune systems during neonatal life and to maintain the physiologically normal steady state of inflammation in the gut throughout life. Neonatal conventionally reared mice and germ-free, deliberately colonized adult mice (gnotobiotic mice) were used to examine the efficacy of certain intestinal microbes.
27057225
The gut microbiota shapes intestinal immune responses during health and disease
Immunological dysregulation is the cause of many non-infectious human diseases such as autoimmunity, allergy and cancer. The gastrointestinal tract is the primary site of interaction between the host immune system and microorganisms, both symbiotic and pathogenic. In this Review we discuss findings indicating that developmental aspects of the adaptive immune system are influenced by bacterial colonization of the gut. We also highlight the molecular pathways that mediate host–symbiont interactions that regulate proper immune function. Finally, we present recent evidence to support that disturbances in the bacterial microbiota result in dysregulation of adaptive immune cells, and this may underlie disorders such as inflammatory bowel disease. This raises the possibility that the mammalian immune system, which seems to be designed to control microorganisms, is in fact controlled by microorganisms.
20844601
Human Immunodeficiency Virus Type 1 Is Trapped by Acidic but Not by Neutralized Human Cervicovaginal Mucus
ABSTRACT To reliably infect a primate model for human immunodeficiency virus (HIV), ∼10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ∼4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.
33008354
Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV
Background:Cross-sectional studies suggest an association between bacterial vaginosis (BV) and HIV-1 infection. However, an assessment of a temporal effect was not possible. Objectives:To determine the association of BV and other disturbances of vaginal flora with HIV seroconversion among pregnant and postnatal women in Malawi, Africa. Design:Longitudinal follow-up of pregnant and postpartum women. Methods:Women attending their first antenatal care visit were screened for HIV after counselling and obtaining informed consent. HIV-seronegative women were enrolled and followed during pregnancy and after delivery. These women were again tested for HIV at delivery and at 6-monthly visits postnatally. Clinical examinations and collection of laboratory specimens (for BV and sexually transmitted diseases) were conducted at screening and at the postnatal 6-monthly visits. The diagnosis of BV was based on clinical criteria. Associations of BV and other risk factors with HIV seroconversion, were examined using contingency tables and multiple logistic regression analyses on antenatal data, and Kaplan–Meier proportional hazards analyses on postnatal data. Results:Among 1196 HIV-seronegative women who were followed antenatally for a median of 3.4 months, 27 women seroconverted by time of delivery. Postnatally, 97 seroconversions occurred among 1169 seronegative women who were followed for a median of 2.5 years. Bacterial vaginosis was significantly associated with antenatal HIV seroconversion (adjusted odds ratio = 3.7) and postnatal HIV seroconversion (adjusted rate ratio = 2.3). There was a significant trend of increased risk of HIV seroconversion with increasing severity of vaginal disturbance among both antenatal and postnatal women. The approximate attributable risk of BV alone was 23% for antenatal HIV seroconversions and 14% for postnatal seroconversions. Conclusions:This prospective study suggests that progressively greater disturbances of vaginal flora, increase HIV acquisition during pregnancy and postnatally. The screening and treating of women with BV could restore normal flora and reduce their susceptibility to HIV.
1765978
Defining the Normal Bacterial Flora of the Oral Cavity
ABSTRACT More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. Our purposes were (i) to utilize culture-independent molecular techniques to extend our knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and (ii) to determine the site and subject specificity of bacterial colonization. Nine sites from five clinically healthy subjects were analyzed. Sites included tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils. 16S rRNA genes from sample DNA were amplified, cloned, and transformed into Escherichia coli. Sequences of 16S rRNA genes were used to determine species identity or closest relatives. In 2,589 clones, 141 predominant species were detected, of which over 60% have not been cultivated. Thirteen new phylotypes were identified. Species common to all sites belonged to the genera Gemella, Granulicatella, Streptococcus, and Veillonella. While some species were subject specific and detected in most sites, other species were site specific. Most sites possessed 20 to 30 different predominant species, and the number of predominant species from all nine sites per individual ranged from 34 to 72. Species typically associated with periodontitis and caries were not detected. There is a distinctive predominant bacterial flora of the healthy oral cavity that is highly diverse and site and subject specific. It is important to fully define the human microflora of the healthy oral cavity before we can understand the role of bacteria in oral disease.
12230766
Molecular analysis of the bacterial microbiota in the human stomach.
The microbiota of the human stomach and the influence of Helicobacter pylori colonization on its composition remain largely unknown. We characterized bacterial diversity within the human gastric mucosa by using a small subunit 16S rDNA clone library approach and analyzed 1,833 sequences generated by broad-range bacterial PCR from 23 gastric endoscopic biopsy samples. A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described. The majority of sequences were assigned to the Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria phyla. Ten percent of the phylotypes were previously uncharacterized, including a Deinococcus-related organism, relatives of which have been found in extreme environments but not reported before in humans. The gastric clone libraries from 19 subjects contained H. pylori rDNA; however, only 12 of these subjects tested positive for H. pylori by conventional laboratory methods. Statistical analysis revealed a large degree of intersubject variability of the gastric ecosystem. The presence of H. pylori did not affect the composition of the gastric community. This gastric bacterial rDNA data set was significantly different from sequence collections of the human mouth and esophagus described in other studies, indicating that the human stomach may be home to a distinct microbial eco-system. The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease.
46686839
Bacterial biota in the human distal esophagus
The esophagus, like other luminal organs of the digestive system, provides a potential environment for bacterial colonization, but little is known about the presence of a bacterial biota or its nature. By using broad-range 16S rDNA PCR, biopsies were examined from the normal esophagus of four human adults. The 900 PCR products cloned represented 833 unique sequences belonging to 41 genera, or 95 species-level operational taxonomic units (SLOTU); 59 SLOTU were homologous with culture-defined bacterial species, 34 with 16S rDNA clones, and two were not homologous with any known bacterial 16S rDNA. Members of six phyla, Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Fusobacteria, and TM7, were represented. A large majority of clones belong to 13 of the 41 genera (783/900, 87%), or 14 SLOTU (574/900, 64%) that were shared by all four persons. Streptococcus (39%), Prevotella (17%), and Veilonella (14%) were most prevalent. The present study identified ≈56–79% of SLOTU in this bacterial ecosystem. Most SLOTU of esophageal biota are similar or identical to residents of the upstream oral biota, but the major distinction is that a large majority (82%) of the esophageal bacteria are known and cultivable. These findings provide evidence for a complex but conserved bacterial population in the normal distal esophagus.
17285720
Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.
The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases.
30897047
From Structure to Function: the Ecology of Host-Associated Microbial Communities
SUMMARY In the past several years, we have witnessed an increased interest in understanding the structure and function of the indigenous microbiota that inhabits the human body. It is hoped that this will yield novel insight into the role of these complex microbial communities in human health and disease. What is less appreciated is that this recent activity owes a great deal to the pioneering efforts of microbial ecologists who have been studying communities in non-host-associated environments. Interactions between environmental microbiologists and human microbiota researchers have already contributed to advances in our understanding of the human microbiome. We review the work that has led to these recent advances and illustrate some of the possible future directions for continued collaboration between these groups of researchers. We discuss how the application of ecological theory to the human-associated microbiota can lead us past descriptions of community structure and toward an understanding of the functions of the human microbiota. Such an approach may lead to a shift in the prevention and treatment of human diseases that involves conservation or restoration of the normal community structure and function of the host-associated microbiota.
5922034
Molecular analysis of human forearm superficial skin bacterial biota
The microbial ecology of human skin is complex, but little is known about its species composition. We examined the diversity of the skin biota from the superficial volar left and right forearms in six healthy subjects using broad-range small subunit rRNA genes (16S rDNA) PCR-based sequencing of randomly selected clones. For the initial 1,221 clones analyzed, 182 species-level operational taxonomic units (SLOTUs) belonging to eight phyla were identified, estimated as 74.0% [95% confidence interval (C.I.), ≈64.8–77.9%] of the SLOTUs in this ecosystem; 48.0 ± 12.2 SLOTUs were found in each subject. Three phyla (Actinobacteria, Firmicutes, and Proteobacteria) accounted for 94.6% of the clones. Most (85.3%) of the bacterial sequences corresponded to known and cultivated species, but 98 (8.0%) clones, comprising 30 phylotypes, had <97% similarity to prior database sequences. Only 6 (6.6%) of the 91 genera and 4 (2.2%) of the 182 SLOTUs, respectively, were found in all six subjects. Analysis of 817 clones obtained 8–10 months later from four subjects showed additional phyla (numbering 2), genera (numbering 28), and SLOTUs (numbering 65). Only four (3.4%) of the 119 genera (Propionibacteria, Corynebacteria, Staphylococcus, and Streptococcus) were observed in each subject tested twice, but these genera represented 54.4% of all clones. These results show that the bacterial biota in normal superficial skin is highly diverse, with few well conserved and well represented genera, but otherwise low-level interpersonal consensus.
6429422
DNA extraction method affects microbial community profiles from soils and sediment
To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types.
38529975
Quantification of Bias Related to the Extraction of DNA Directly from Soils
ABSTRACT In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.
44736518
Comparison of different methods for the isolation and purification of total community DNA from soil.
The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.
16610626
Metagenomic Sequencing of an In Vitro-Simulated Microbial Community
Background Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. Methodology/Principal Findings We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. Conclusions/Significance We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with different protocols are not suitable for comparative metagenomics.
24583687
Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.
Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.
7626402
Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens
BackgroundThe influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean® Fecal DNA Isolation Kit, M; QIAamp® DNA Stool Mini Kit, Q; FastDNA® SPIN Kit, FSp; FastDNA® SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.MethodTotal DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.ResultsRegardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.ConclusionWe conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.
24193626
Comparison of DNA extraction methods for analysis of turkey cecal microbiota
Aim:  As a prelude to long‐term studies to characterize the microbiota of the turkey ceca, 14 DNA isolation protocols were evaluated for their ability to reproducibly characterize microbial diversity.
42846516
Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil
ABSTRACT Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods. Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.
36467198
A strategy for optimizing quality and quantity of DNA extracted from soil.
The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.
657446
Molecular microbial ecology: land of the one-eyed king.
Studies of microbial biodiversity have made astounding discoveries of late due to the use of methodologies based on phylogenetic analyses of small subunit ribosomal RNA sequences. Although there are limitations to these methods, they can nonetheless be very useful if these limitations are kept in mind. These limitations range from technical problems such as obtaining representative genomic DNA and suitable primers, to conceptual problems such as defining and using meaningful taxonomic units of diversity (species). Here we discuss several of the limitations inherent in studies of microbial diversity that must be considered when interpreting the results obtained using these approaches.
22422923
Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria
The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.
41616916
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis.
After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
1334898
The Human Microbiome Project
A strategy to understand the microbial components of the human genetic and metabolic landscape and how they contribute to normal physiology and predisposition to disease.
5272418
The NIH Human Microbiome Project.
The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 "normal" volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.
30384130
High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction.
Soil is a complex environment considered as one of the main reservoirs of microbial diversity. However, the inability to cultivate most soil bacteria hampered fundamental attempts to determine the diversity of the prokaryotic world and limited its industrial exploitation. In the last 20 years, new methods have been developed to overcome these limitations based on the direct extraction of DNA from bacteria in their natural environment. In addition to fundamental research, the cloning of the extracted DNA for the development of metagenomic DNA clone libraries offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria in surrogate bacterial hosts. However, such objectives require adapting DNA extraction methods and cloning strategies in order that entire gene clusters encoding biosynthetic pathway for secondary metabolites can be cloned. In this paper, we report that the size of DNA fragments extracted from soil varied in a range between less than 100 kb and more than 400 kb depending on the soil. The relatively limited size of DNA fragments extracted from some soil was not only due to mechanical, chemical or enzymatic shearing of the DNA during the extraction process but partly to the microbial growth status. Stimulating bacteria in situ by providing nutrients to the soil improved the size of extracted DNA, but it modified the bacterial community structure.
27898354
A comparison of five methods for extraction of bacterial DNA from human faecal samples.
The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.
24823604
Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting
In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNA yield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.
8555525
Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota.
Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.
10168112
Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples
ABSTRACT We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.
24980023
Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.
The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs.
11396992
Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods
The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene. Microbial community fingerprints were assessed by automated ribosomal intergenic spacer analysis. The resulting community profiles were analyzed with canonical correspondence analysis. Our results clearly demonstrate that direct DNA extraction methods can significantly influence the DNA quantity, purity, and observed community patterns of microbiota in activated sludge. Fast and Mobio generated high amounts of good quality DNA compared to Bead and Qiagen. Mobio also resulted in the detection of the highest number of species while Fast scored the best in discriminating between the community patterns of different activated sludge types. With respect to the characterization of community profiles, our analyses demonstrated a strong sludge type dependent variability among methods. Taking into account our results, we recommend Fast as the most suitable DNA extraction method for activated sludge samples used for bacterial community studies.
52991802
An Improved DNA Isolation Method for Metagenomic Analysis of the Microbial Flora of the Human Intestine
The efficiency with which lysis of five strictly anaerobic and six facultatively anaerobic bacterial species, all well-known human colonic commensals, were lysed was tested using a reference method for general metagenomic analysis and an improved method that involves higher levels of lysozyme and proteinase K, as well as the addition of achromopeptidase. Ten species were lysed with an efficiency of >80% by the reference method, while the lytic efficiency for Clostridium ramosum JCM 1298T was C. ramosum JCM 1298T was 82.5%. Similarly, five samples of human feces were tested with these methods, as well as with the QIAamp DNA stool mini kit. Although the efficiency of lysis of the microbes recovered from the fecal samples fluctuated depending on the sample in the cases of the reference method (13.3-84.6%) and QIAamp DNA stool mini kit (38.8-69.2%), the improved method gave stable and high-level lysis (>90%) for all the fecal samples. Accordingly, since the DNA samples isolated by the improved method can reflect nearly true genomic information in the microbial flora, our improved method should be applicable to metagenomic analyses, not only for bacteria in the human intestine but also for bacteria in other environments.
42793400
Chromosome replication and the division cycle of Escherichia coli B/r.
It is suggested that the replication of the bacterial genome during the division cycle of Escherichia coli Br growing with doubling times between approximately 20 and 60 minutes can be described by two constants: C, the time for a replication point to traverse the genome, and D, the time between the end of a round of replication and cell division. Direct measurements of C, D and C + D agree with the model. The predictions of the model are consistent with autoradiographic and biochemical data, and with the finding of “gaps” in DNA synthesis during slow growth and multiple replication forks during rapid growth. Measurements of the DNA content of cells with predicted chromosome configurations led to an estimate of the molecular weight of the bacterial genome (non-replicating) of 2.6 × 109 daltons, in good agreement with other, independent measurements.
33851102
Co‐ordinate regulation of the Escherichia coli cell cycle or The cloud of unknowing
A discussion of some aspects of the regulation of chromosome replication, segregation and cell division in Escherichia coli.
22338648
Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species
In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.
5068928
Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes
ABSTRACT rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.
23478746
Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig
Aims: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA.
17937676
Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners
BackgroundMost studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats for Lactobacillus crispatus, L. gasseri and Gardnerella vaginalis and developed new formats for Atopobium vaginae, L. iners and L. jensenii to obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years.ResultsThe 71 vaginal microflora samples of these women were categorized, using the Ison and Hay criteria, as refined by Verhelst et al. (2005), as follows: grade Ia: 8 samples, grade Iab: 10, grade Ib: 13, grade I-like: 10, grade II: 11, grade III: 12 and grade IV: 7.L. crispatus was found in all but 5 samples and was the most frequent Lactobacillus species detected. A significantly lower concentration of L. crispatus was found in grades II (p < 0.0001) and III (p = 0.002) compared to grade I. L. jensenii was found in all grades but showed higher concentration in grade Iab than in grade Ia (p = 0.024). A. vaginae and G. vaginalis were present in high concentrations in grade III, with log10 median concentrations (log10 MC), respectively of 9.0 and 9.2 cells/ml. Twenty (38.5%) of the 52 G. vaginalis positive samples were also positive for A. vaginae. In grade II we found almost no L. iners (log10 MC: 0/ml) but a high concentration of L. gasseri ( log10 MC: 8.7/ml). By contrast, in grade III we found a high concentration of L. iners( log10 MC: 8.3/ml) and a low concentration of L. gasseri ( log10 MC: 0/ml). These results show a negative association between L. gasseri and L. iners (r = -0.397, p = 0.001) and between L. gasseri and A. vaginae (r = -0.408, p < 0.0001).ConclusionIn our study we found a clear negative association between L. iners and L. gasseri and between A. vaginae and L. gasseri. Our results do not provide support for the generally held proposition that grade II is an intermediate stage between grades I and III, because L. gasseri, abundant in grade II is not predominant in grade III, whereas L. iners, abundant in grade III is present only in low numbers in grade II samples.
41404361
O-acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis.
Bacterial cell walls and their structural units, particularly peptidoglycan, induce a vast variety of biological effects in host organisms. The pathobiological effects of peptidoglycan are greatly enhanced by various modifications and substitutions to its basic composition and structure. One such modification is the presence of acetyl moieties at the C-6 hydroxyl group of N-acetylmuramyl residues, and to date, 11 species of eubacteria, including some important human pathogens, such as Neisseria gonorrhoeae, Proteus mirabilis, and Staphylococcus aureus, are known to possess O-acetylated peptidoglycan. This review addresses the influence of O-acetylation of peptidoglycan on its resistance to degradation both in vitro and in vivo, the clinical importance of the modification, and the currently held views on the pathway for its biosynthesis.
22972934
Glucosamine substitution and muramidase susceptibility in Bacillus anthracis.
Cell walls of Bacillus anthracis were found to be resistant to lysozyme, and partially resistant to mutanolysin, a muramidase from Streptomyces globisporus. Following treatment with acetic anhydride, it was observed that the walls were highly susceptible to hydrolysis by lysozyme or mutanolysin. Analyses of cell walls, prior to and following derivatization with fluorodinitrobenzene, revealed that approximately 88% of the glucosamine residues and 34% of the muramic acid residues of the peptidoglycan contained unsubstituted amino groups, thereby providing an explanation for the resistance of the walls to lysozyme. The walls of B. anthracis were approximately 19% cross-linked, based on the findings that 81% of the diaminopimelic acid residues could be modified by fluorodinitrobenzene. Walls of B. thuringiensis 4040 and B. cereus ATCC 19637 also contained high percentages of unsubstituted amino sugars, and unless acetylated, were also relatively resistant to lysozyme and mutanolysin. When B. anthracis, B. cereus, or B. thuringiensis were grown in the presence of 100 micrograms/mL lysozyme, there was a decrease in the average number of cells per chain, but there was no decrease in growth rates, suggesting that the enzyme was acting at septa. It is unlikely that lysozyme and autolysins act synergistically in Bacillus, because azide anion, which activates autolysins, did not enhance the lytic action of lysozyme in B. anthracis, B. cereus, or B. thuringiensis.
21284518
Crystallization and preliminary X-ray analysis of a bacterial lysozyme produced by Streptomyces globisporus.
The extracellular bacteriolytic enzyme produced by Streptomyces globisporus shows a beta-1,4-N,6-O-diacetylmuramidase activity as well as a beta-1,4-N-acetylmuramidase activity. Crystals of this enzyme have been obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. They belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 63.11 (4), c = 121.1 (1) A, diffract to at least 2.0 A resolution and are suitable for high-resolution structure analysis. The crystal structure was solved by molecular replacement using lysozyme produced by S. erythraeus as a search model. The structure refinement is now in progress.
36825802
LYSOSTAPHIN: A NEW BACTERIOLYTIC AGENT FOR THE STAPHYLOCOCCUS.
3Wilt, F. H., Biochem. Biophys. Res. Commun., 11, 447 (1963). 7Brachet, J., et al., Biochim. Biophys. Acta, 72, 662 (1963). 8 Gross, P. R., and G. H. Cousineau, Exptl. Cell Res., in press. I Hultin, T., Exptl. Cell Res., 25, 405 (1961). 10 Wilt, F. H., and T. Hultin, Biochem. Biophys. Res. Commun., 9, 313 (1962). 11 Melton, C. R., Genetics, 48, 901 (1963). 12Tyler, A., Am. Zoologist, 3, 109 (1963). 13 Gross, P. R., W. Spindel, and G. H. Cousineau, Biocheem. Biophys. Res. Commun., 13, 405 (1963). 14 Cavanaugh, G., ed., Formulae and Methods of the Marine Biological Laboratory, IV (Woods Hole, 1956). 15 Herberg, R., Science, 128, 199 (1958). 16Bray, G., Anal. Biochem., 1, 279 (1960). 17 Lowry, O., et al., J. Biol. Chem., 193, 265 (1951). 18 Scherrer, K., and J. Darnell, Biochem. Biophys. Res. Commun., 7, 486 (1962). 19 Fraenkel-Conrat, H., B. Singer, and A. Tsugita, Virology, 14, 54 (1961). 20 Hurwitz, J., et al., these PROCEEDINGS, 48, 1222 (1962). 21 Reich, E., I. H. Goldberg, and M. Rabinowitz, Nature, 196, 743 (1962). 22 Levinthal, C., A. Keynan, and A. Higa, these PROCEEDINGS, 48, 1631 (1962). 23 Monier, R., et al., J. Mol. Biol., 5, 311 (1962). 24 Nemer, M., and S. G. Bard, Science, 140, 664 (1963). 25 Maggio, R., and C. Catalano, in preparation. 26 Hultin, T., Experientia, 7, 410 (1961). 27 Gross, P. R., and G. H. Cousineau, J. Cell Biol., 19, 260 (1963).
16022673
Efficient recovery of environmental DNA for expression cloning by indirect extraction methods.
Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability for expression cloning in Escherichia coli. Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used (<8% of eukaryotic DNA by indirect methods versus 61-93% by direct lysis protocols). Considering the higher purity, i.e. higher cloning efficiency of DNA isolated by indirect methods, similar numbers of clones carrying prokaryotic inserts could be produced by either strategy. Gene banks prepared from directly extracted DNA, however, are expected to contain large portions of clones with eukaryotic inserts, whereas those constructed from indirectly isolated DNA should mainly contain inserts of bacterial origin. As eukaryotic genetic information is generally not expressed in bacterial host organisms but increases the library size, our findings suggest that the use of indirect DNA isolation methods allows the construction of environmental gene banks of superior quality.
43275986
Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.
Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.
11395613
Metagenomic Analysis of the Human Distal Gut Microbiome
The human intestinal microbiota is composed of 1013 to 1014 microorganisms whose collective genome (“microbiome”) contains at least 100 times as many genes as our own genome. We analyzed ∼78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction–amplified 16S ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-erythritol 4-phosphate pathway–mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.
17203181
Ecological and Evolutionary Forces Shaping Microbial Diversity in the Human Intestine
The human gut is populated with as many as 100 trillion cells, whose collective genome, the microbiome, is a reflection of evolutionary selection pressures acting at the level of the host and at the level of the microbial cell. The ecological rules that govern the shape of microbial diversity in the gut apply to mutualists and pathogens alike.
8345877
Evolution of Mammals and Their Gut Microbes
Mammals are metagenomic in that they are composed of not only their own gene complements but also those of all of their associated microbes. To understand the coevolution of the mammals and their indigenous microbial communities, we conducted a network-based analysis of bacterial 16S ribosomal RNA gene sequences from the fecal microbiota of humans and 59 other mammalian species living in two zoos and in the wild. The results indicate that host diet and phylogeny both influence bacterial diversity, which increases from carnivory to omnivory to herbivory; that bacterial communities codiversified with their hosts; and that the gut microbiota of humans living a modern life-style is typical of omnivorous primates.
10262374
Microbes on the human vaginal epithelium
Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated.
8143221
Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women
The maintenance of a low pH in the vagina through the microbial production of lactic acid is known to be an important defense against infectious disease in reproductive age women. Previous studies have shown that this is largely accomplished through the metabolism of lactic acid bacteria, primarily species of Lactobacillus. Despite the importance of this defense mechanism to women's health, differences in the species composition of vaginal bacterial communities among women have not been well defined, nor is it known if and how these differences might be linked to differences in the risk of infection. In this study, we defined and compared the species composition of vaginal bacterial communities in 144 Caucasian and black women in North America. This was carried out based on the profiles of terminal restriction fragments of 16S rRNA genes, and phylogenetic analysis of 16S rRNA gene sequences of the numerically dominant microbial populations. Among all the women sampled, there were eight major kinds of vaginal communities (‘supergroups’) that occurred in the general populace at a frequency of at least 0.05 (P=0.99). From the distribution of these supergroups among women, it was possible to draw several conclusions. First, there were striking, statistically significant differences (P=0.0) in the rank abundance of community types among women in these racial groups. Second, the incidence of vaginal communities in which lactobacilli were not dominant was higher in black women (33%) as compared to Caucasian women (7%). Communities not dominated by lactobacilli had Atopobium and a diverse array of phylotypes from the order Clostridiales. Third, communities dominated by roughly equal numbers of more than one species of Lactobacillus were rare in black women, but common in Caucasian women. We postulate that because of these differences in composition, not all vaginal communities are equally resilient, and that differences in the vaginal microbiota of Caucasian and black women may at least partly account for known disparities in the susceptibility of women in these racial groups to bacterial vaginosis and sexually transmitted diseases.
1197652
Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis
BackgroundBacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess.MethodsNucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score ≤ 3) and abnormal flora (Nugent score ≥ 4). These women ranged in age from 14 to 48 and 82% were African American.ResultsThe DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (~300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant.ConclusionThe results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant.
22687734
Molecular identification of bacteria associated with bacterial vaginosis.
BACKGROUND
Bacterial vaginosis affects millions of women and is associated with several serious health conditions. The cause of bacterial vaginosis remains poorly understood despite numerous studies based on cultures. Bacteria in microbial communities can be identified without cultivation by characterizing their ribosomal DNA (rDNA) sequences.


METHODS
We identified bacteria in samples of vaginal fluid with a combination of broad-range polymerase-chain-reaction (PCR) amplification of 16S rDNA with clone analysis, bacterium-specific PCR assay of 16S rDNA, and fluorescence in situ hybridization (FISH) performed directly on vaginal fluid from 27 subjects with bacterial vaginosis and 46 without the condition. Twenty-one subjects were studied with the use of broad-range PCR of 16S rDNA, and 73 subjects were studied with the use of bacterium-specific PCR.


RESULTS
Women without bacterial vaginosis had 1 to 6 vaginal bacterial species (phylotypes) in each sample (mean, 3.3), as detected by broad-range PCR of 16S rDNA, and lactobacillus species were the predominant bacteria noted (83 to 100 percent of clones). Women with bacterial vaginosis had greater bacterial diversity (P<0.001), with 9 to 17 phylotypes (mean, 12.6) detected per sample and newly recognized species present in 32 to 89 percent of clones per sample library (mean, 58 percent). Thirty-five unique bacterial species were detected in the women with bacterial vaginosis, including several species with no close cultivated relatives. Bacterium-specific PCR assays showed that several bacteria that had not been previously described were highly prevalent in subjects with bacterial vaginosis but rare in healthy controls. FISH confirmed that newly recognized bacteria detected by PCR corresponded to specific bacterial morphotypes visible in vaginal fluid.


CONCLUSIONS
Women with bacterial vaginosis have complex vaginal infections with many newly recognized species, including three bacteria in the Clostridiales order that were highly specific for bacterial vaginosis.
1291033
rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea
A dramatic exception to the general pattern of single-copy genes in bacterial and archaeal genomes is the presence of 1–15 copies of each ribosomal RNA encoding gene. The original version of the Ribosomal RNA Database (rrnDB) cataloged estimates of the number of 16S rRNA-encoding genes; the database now includes the number of genes encoding each of the rRNAs (5S, 16S and 23S), an internally transcribed spacer region, and the number of tRNA genes. The rrnDB has been used largely by microbiologists to predict the relative rate at which microbial populations respond to favorable growth conditions, and to interpret 16S rRNA-based surveys of microbial communities. To expand the functionality of the rrnDB (http://ribosome.mmg.msu.edu/rrndb/index.php), the search engine has been redesigned to allow database searches based on 16S rRNA gene copy number, specific organisms or taxonomic subsets of organisms. The revamped database also computes average gene copy numbers for any collection of entries selected. Curation tools now permit rapid updates, resulting in an expansion of the database to include data for 785 bacterial and 69 archaeal strains. The rrnDB continues to serve as the authoritative, curated source that documents the phylogenetic distribution of rRNA and tRNA genes in microbial genomes.
29044841
Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri
Aims:  To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons.
1373790
Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy
ABSTRACT The Ribosomal Database Project (RDP) Classifier, a naïve Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (≥95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/ .
83812524
A first course in design and analysis of experiments
86 Line –2 should end = √ 4 × 4.782 = 4.37 , 94 g − 2, g − 1, . . . , 2 should beg − 2, g − 3, . . . , 2. 117 The Seeded and Unseeded labels should be switched in Table 6. 1. 153 Lines 3 and 4. Initial sample size is 19.2, or 20 rounding up. 158 Example 7.5. The Type I error rate is .01, although it is not ex plicitly stated. 179 Lines 13 and 14. Some plus and minus signs should be switched t o r ad α̂β21 = 2.153 − (2.065 + .234 + −.134) = −.012 α̂β12 = 1.952 − (2.065 + −.234 + .134) = −.012 245 The sequential ANOVAs given in Exercise 10.1 are not adequat e to answer the question. The ANOVAs should be
136471
Bacterial succession in a glacier foreland of the High Arctic
Succession is defined as changes in biological communities over time. It has been extensively studied in plant communities, but little is known about bacterial succession, in particular in environments such as High Arctic glacier forelands. Bacteria carry out key processes in the development of soil, biogeochemical cycling and facilitating plant colonization. In this study we sampled two roughly parallel chronosequences in the foreland of Midre Lovén glacier on Svalbard, Norway and tested whether any of several factors were associated with changes in the structure of bacterial communities, including time after glacier retreat, horizontal variation caused by the distance between chronosequences and vertical variation at two soil depths. The structures of soil bacterial communities at different locations were compared using terminal restriction fragment length polymorphisms of 16S rRNA genes, and the data were analyzed by sequential analysis of log-linear statistical models. Although no significant differences in community structure were detected between the two chronosequences, statistically significant differences between sampling locations in the surface and mineral soils could be demonstrated even though glacier forelands are patchy and dynamic environments. These findings suggest that bacterial succession occurs in High Arctic glacier forelands but may differ in different soil depths.
147194189
Nonparametric Statistical Methods
This Second Edition of Myles Hollander and Douglas A. Wolfe's successful Nonparametric Statistical Methods meets the needs of a new generation of users, with completely up-to-date coverage of this important statistical area. Like its predecessor, the revised edition, along with its companion ftp site, aims to equip readers with the conceptual and technical skills necessary to select and apply the appropriate procedures for a given situation. An extensive array of examples drawn from actual experiments illustrates clearly how to use nonparametric approaches to handle one- or two-sample location and dispersion problems, dichotomous data, and one-way and two-way layout problems. An ideal text for an upper-level undergraduate or first-year graduate course, Nonparametric Statistical Methods, Second Edition is also an invaluable source for professionals who want to keep abreast of the latest developments within this dynamic branch of modern statistics.