scholarly journals Systematic Bias Introduced by Genomic DNA Template Dilution in 16S rRNA Gene-Targeted Microbiota Profiling in Human Stool Homogenates

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Francesco Multinu ◽  
Sean C. Harrington ◽  
Jun Chen ◽  
Patricio R. Jeraldo ◽  
Stephen Johnson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Dna ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Unifrac Distance ◽  
Β Diversity ◽  
Unweighted Unifrac ◽  
Stool Samples ◽  
Community Representation

ABSTRACT Variability in representation of microbial communities can be caused by differences in microbial composition or artifacts introduced at sample collection or processing. Alterations in community representation introduced by variations in starting DNA concentrations have not been systematically investigated in stool samples. The goal of this study was to evaluate the effect of the genomic DNA (gDNA) concentration in the resulting 16S rRNA gene library composition and compare its effect to other sample processing variables in homogenized human fecal material. Compared to a gDNA input of 1 ng/μl, inputs of ≤1.6 × 10 −3  ng/μl resulted in a marked decrease in the concentration of the 16S rRNA gene amplicon ( P < 0.001). Low gDNA concentrations (≤1.6 × 10 −3  ng/μl) were also associated with a decrease ( P < 0.001) in the number of operational taxonomic units and significant divergence in β-diversity profiles (unweighted UniFrac distance, P < 0.001), as characterized by an overestimation of Proteobacteria and underestimation of Firmicutes . Even a gDNA concentration of 4 × 10 −2  ng/μl showed a significant impact on the β-diversity profile (unweighted UniFrac distance, P = 0.03). Overall, the gDNA concentration explained 22.4% to 38.1% of the microbiota variation based on various β-diversity measures ( P < 0.001). By comparison, the DNA extraction methods and PCR volumes tested did not significantly affect the microbial composition profile, and the PCR cycling method explained less than 3.7% of the microbiota variation (weighted UniFrac distance, P = 0.03). The 16S rRNA gene yield and the microbial community representation of human homogenized stool samples are significantly altered by gDNA template concentrations of ≤1.6 × 10 −3  ng/μl. In addition, data from studies with a gDNA input of ≤4 × 10 −2  ng/μl should be interpreted with caution. IMPORTANCE The genomic DNA input for stool samples utilized for microbiome composition has not been determined. In this study, we determined the reliable threshold level under which conclusions drawn from the data may be compromised. We also determined the type of microbial bias introduced by less-than-ideal genomic input.

scholarly journals Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

mSystems ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
William Walters ◽  
Embriette R. Hyde ◽  
Donna Berg-Lyons ◽  
Gail Ackermann ◽  
Greg Humphrey ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Human Body ◽  
Internal Transcribed Spacer ◽  
Genomic Dna ◽  
State Of The Art ◽  
Rrna Gene ◽  
Ice Caps ◽  
Microbiome Research ◽  
Its Primers

ABSTRACT We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art. Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

scholarly journals Allometry and Ecology of the Bilaterian Gut Microbiome

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00319-18 ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Kevin McCormick ◽  
Abigail Lauder ◽  
Aubrey Bailey ◽  
Laurie Zimmerman ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Island Biogeography ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Data Set ◽  
Community Construction ◽  
Wide Range ◽  
Niche Diversification ◽  
Bacterial Lineages

ABSTRACT Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals (Bilateria) ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction. IMPORTANCE The intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification.

scholarly journals Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983

2006 ◽  
Vol 56 (8) ◽  
pp. 1721-1724 ◽  
Author(s):  
Franco Dellaglio ◽  
Marc Vancanneyt ◽  
Akihito Endo ◽  
Peter Vandamme ◽  
Giovanna E. Felis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Dna ◽  
Taxonomic Status ◽  
Single Species ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Biochemical Characteristics ◽  
Binding Level

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8 % similarity over 1523 bp), partial recA gene sequences (99.5 % similarity over 600 bp) and partial hsp60 gene sequences (99.1 % similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA–DNA binding level (87 %) and similar genomic DNA G+C contents (41–44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synonym.

scholarly journals Sequencing-Based Analysis for the Identification of Bacteria Associated with Severe Early Childhood Caries (SECC)

2018 ◽  
Vol 17 (2) ◽  
Author(s):  
Zarina Zainuddin ◽  
Abdul Qahhar Bin Paiman ◽  
Yunita Dewi Ardini
Keyword(s):  
Early Childhood ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Dna ◽  
Early Childhood Caries ◽  
Bacterial Species ◽  
World Health ◽  
Rrna Gene ◽  
Severe Early Childhood Caries ◽  
Childhood Caries

Severe early childhood caries (SECC) is a microbialinfection that severely compromises the dentition ofyoung children.The aim of this study is toidentify bacteria associated with SECC and compare bacterial species between SECC and caries-free children. Bacterial samples were isolated from dental plaque samples of 3 to 6-yearold preschool children and the status of dental caries at the surface level was evaluated using World Health Organization (WHO) diagnostic criteria for decayed, missing and filled tooth surfaces (DFMS). 30 samples from the interproximal part were collected and genomic DNA was extracted using MasterPure™ Gram Positive DNA Purification Kit. Polymerase Reaction (PCR) was conducted on the genomic DNA extracted to amplify 16S rRNA gene and successfully amplified 16S rRNA gene from the samples was sent for sequencing.This research was approved by the IIUM Research Ethic Committee (IREC). Using the selected kit, genomic DNA was successfully extracted and PCR amplification of the bacterial 16S rRNAgeneusing universal bacterial primers was achieved giving a product of 1500 bp. Results from sequencing when analysedusing BLAST from the NCBI website, identified bacteria from the genus Vibrio, Haemophilus and Aggregatibacter from SECC samples while for caries-free samples Granulicatella was identified.

scholarly journals 2576. The Microbiome of Recurrent Bacterial Vaginosis Compared with Asymptomatic Controls

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S895-S895
Author(s):  
Elizabeth O Shay ◽  
Oluwatosin Goje ◽  
Roshan Padmanabhan ◽  
Charis Eng
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Vaginosis ◽  
Alpha Diversity ◽  
Dna Probe ◽  
Gardnerella Vaginalis ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Β Diversity ◽  
Probe Test

Abstract Background Bacterial vaginosis (BV) affects nearly 1 in 3 women in the United States and is poorly understood. The study of the vaginal microbiome, using 16S rRNA-gene amplicon sequencing, has increased our knowledge of BV. We aimed to characterize the vaginal microbiome of women with recurrent BV firstly in comparison to controls, and secondly in comparison to a sub-population of our asymptomatic controls, positive for Gardnerella vaginalis via a vaginal pathogens DNA direct probe test (DNA probe). Methods Women aged 18–40 years, with recurrent BV, and asymptomatic controls were prospectively enrolled. Vaginal samples were collected from each participant. DNA was extracted, amplified using primers targeting the V3-V4 variable region of the 16S rRNA-gene, and then sequenced and processed through a hybrid Qiime MICCA bioinformatics pipeline. We also tested for G. vaginalis using the DNA probe. Results Seventeen recurrent BV patients and 46 controls were enrolled. Β diversity (P = 0.045), but not alpha diversity (P = 0.076) differed between groups. The genera Gardnerella and Prevotella were relatively more abundant, while Lactobacillus was relatively less abundant in recurrent BV vs. control groups. Of the patients for whom results of the DNA probe for Gardnerella vaginalis were available, 11 (69%) recurrent BV patients and 14 (35%) controls were positive. Control patients, negative by the DNA probe test, showed decreased alpha diversity (P = 0.0001) and significantly different β diversity (P = 0.001) compared with recurrent BV patients. Neither alpha (P = 0.31) nor β (P = 0.096) diversity differed between recurrent BV patients and controls that were G. vaginalis positive. Conclusion The microbiome of recurrent BV patients is distinct from that of asymptomatic controls; recurrent BV patients exhibit different β diversity, less Lactobacillus and more Gardnerella and Prevotella. Asymptomatic Gardnerella vaginalis-colonized controls demonstrate similar microbiome profiles to those of recurrent BV patients. These findings suggest that individual factors may influence whether or not a patient with a BV microbiomic profile experiences symptoms. Further investigation into these mechanisms could yield insights into the treatment of recurrent BV. Disclosures All authors: No reported disclosures.

scholarly journals Combined Phospholipid Biomarker-16S rRNA Gene Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Diversity and Physiological Status in an Intertidal Microbial Mat

2004 ◽  
Vol 70 (11) ◽  
pp. 6920-6926 ◽  
Author(s):  
Laura Villanueva ◽  
Antoni Navarrete ◽  
Jordi Urmeneta ◽  
David C. White ◽  
Ricardo Guerrero
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Mat ◽  
Physiological Status ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Lipid Biomarker ◽  
Gradient Gel Electrophoresis

ABSTRACT A combined lipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis was used to monitor changes in the physiological status, biomass, and microbial composition of a microbial mat. In the morning hours, an increase in the biomass of layers containing a high density of phototrophs and a decrease in the growth rate in the deep layers were observed. The combined approach also revealed differences in major groups of microorganisms, including green nonsulfur, gram-positive, and heterotrophic bacteria.

scholarly journals Characterization of alkaliphilic Bacillus strains used in industry: proposal of five novel species

2005 ◽  
Vol 55 (6) ◽  
pp. 2309-2315 ◽  
Author(s):  
Yuichi Nogi ◽  
Hideto Takami ◽  
Koki Horikoshi
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Type Strain ◽  
Novel Species ◽  
16S Rrna Gene Sequencing ◽  
Industrial Applications ◽  
Rrna Gene ◽  
Bacterial Strains

Twenty alkaliphilic bacterial strains from industrial applications or enzyme studies were subjected to a polyphasic taxonomic investigation, including 16S rRNA gene sequencing, determination of genomic DNA G+C content, DNA–DNA hybridization, fatty acid analysis and standard bacteriological characterization. By comparing the groupings obtained based on the genomic DNA G+C content and the construction of a phylogenetic tree based on the 16S rRNA gene sequence, 12 clusters of similar strains were recognized. DNA–DNA hybridization revealed that these clusters represented five novel genospecies. Further analysis supported the proposal of five novel species in the genus Bacillus: Bacillus wakoensis sp. nov. (type strain N-1T=JCM 9140T=DSM 2521T), Bacillus hemicellulosilyticus sp. nov. (type strain C-11T=JCM 9152T=DSM 16731T), Bacillus cellulosilyticus sp. nov. (type strain N-4T=JCM 9156T=DSM 2522T), Bacillus akibai sp. nov. (type strain 1139T=JCM 9157T=ATCC 43226T) and Bacillus mannanilyticus sp. nov. (type strain AM-001T=JCM 10596T=DSM 16130T).

scholarly journals Animal Rennets as Sources of Dairy Lactic Acid Bacteria

2014 ◽  
Vol 80 (7) ◽  
pp. 2050-2061 ◽  
Author(s):  
Margherita Cruciata ◽  
Ciro Sannino ◽  
Danilo Ercolini ◽  
Maria L. Scatassa ◽  
Francesca De Filippis ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Streptococcus Thermophilus ◽  
Species Level ◽  
Lactobacillus Delbrueckii ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Content Type

ABSTRACTThe microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB),Streptococcus thermophilusand some lactobacilli, mainlyLactobacillus crispatusandLactobacillus reuteri, were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, asEnterococcus casseliflavus,Enterococcus faecium,Enterococcus faecalis,Enterococcus lactis,Lactobacillus delbrueckii, andStreptococcus thermophilus, while the other strains, all belonging to the genusEnterococcus, could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.

scholarly journals Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids

2006 ◽  
Vol 73 (4) ◽  
pp. 1054-1064 ◽  
Author(s):  
Diana Z. Sousa ◽  
M. Alcina Pereira ◽  
Alfons J. M. Stams ◽  
M. Madalena Alves ◽  
Hauke Smidt
Keyword(s):  
Fatty Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Enrichment Culture ◽  
Methane Yield ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Dgge Profile ◽  
Enrichment Cultures ◽  
Long Chain

ABSTRACTAnaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria withinSyntrophomonadaceaeandSyntrophobacteraceaefamilies. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with theSyntrophomonasgenus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.

Sign in / Sign up

Export Citation Format

Share Document