Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1–1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

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Metagenomic 16s rRNA investigation of microbial communities in the Black Sea estuaries in South-West of Ukraine.

Acta Biochimica Polonica ◽

10.18388/abp.2015_1145 ◽

2016 ◽

Vol 63 (2) ◽

Cited By ~ 5

Author(s):

Oleksandra Bobrova ◽

Jon Bent Kristoffersen ◽

Anastasis Oulas ◽

Volodymyr Ivanytsia

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

16S Rdna ◽

Black Sea ◽

Complex Analysis ◽

Bacterial Species ◽

Variable Region ◽

Illumina Miseq ◽

The Black Sea ◽

Rrna Gene

The Black Sea estuaries represent interfaces of the sea and river environments. Microorganisms that inhabit estuarine water play an integral role in all biochemical processes that occur there and form unique ecosystems. There are many estuaries located in the Southern-Western part of Ukraine and some of them are already separated from the sea. The aim of this research was to determine the composition of microbial communities in the Khadzhibey, Dniester and Sukhyi estuaries by metagenomic 16S rDNA analysis. This study is the first complex analysis of estuarine microbiota based on isolation of total DNA from a biome that was further subjected to sequencing. DNA was extracted from water samples and sequenced on the Illumina Miseq platform using primers to the V4 variable region of the 16S rRNA gene. Computer analysis of the obtained raw sequences was done with QIIME (Quantitative Insights Into Microbial Ecology) software. As the outcome, 57970 nucleotide sequences were retrieved. Bioinformatic analysis of bacterial community in the studied samples demonstrated a high taxonomic diversity of Prokaryotes at above genus level. It was shown that majority of 16S rDNA bacterial sequences detected in the estuarine samples belonged to phyla Cyanobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, Planctomycetes. The Khadhzibey estuary was dominated by the Proteobacteria phylum, while Dniester and Sukhyi estuaries were characterized by dominance of Cyanobacteria. The differences in bacterial populations between the Khadzhibey, Dniester and Sukhyi estuaries were demonstrated through the Beta-diversity analysis. It showed that the Khadzhibey estuary's microbial community significantly varies from the Sukhyi and Dniester estuaries. The majority of identified bacterial species is known as typical inhabitants of marine environments, however, for 2.5% of microbial population members in the studied estuaries no relatives were determined.

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Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

Applied and Environmental Microbiology ◽

10.1128/aem.03001-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 177-183 ◽

Cited By ~ 10

Author(s):

Lavane Kim ◽

Eulyn Pagaling ◽

Yi Y. Zuo ◽

Tao Yan

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Bacterial Species ◽

Community Analysis ◽

Microbial Community Analysis ◽

Rrna Gene ◽

Content Type ◽

Property Change ◽

And Control ◽

Start Ups

ABSTRACTThe impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected,BurkholderialesandRhodocyclalesof theBetaproteobacteriaclass were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.

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The bacteriome of the oral cavity in healthy dogs and dogs with periodontal disease

American Journal of Veterinary Research ◽

10.2460/ajvr.21.02.0027 ◽

2022 ◽

pp. 1-9

Author(s):

Brook A. Niemiec ◽

Jerzy Gawor ◽

Shuiquan Tang ◽

Aishani Prem ◽

Janina A. Krumbeck

Keyword(s):

Oral Cavity ◽

Periodontal Disease ◽

Bacterial Species ◽

Abundant Species ◽

Rrna Gene ◽

Core Microbiome ◽

The Core ◽

Severe Periodontal Disease ◽

Healthy Dogs ◽

V3 Region

Abstract OBJECTIVE To compare the bacteriome of the oral cavity in healthy dogs and dogs with various stages of periodontal disease. ANIMALS Dogs without periodontal disease (n = 12) or with mild (10), moderate (19), or severe (10) periodontal disease. PROCEDURES The maxillary arcade of each dog was sampled with a sterile swab, and swabs were submitted for next-generation DNA sequencing targeting the V1–V3 region of the 16S rRNA gene. RESULTS 714 bacterial species from 177 families were identified. The 3 most frequently found bacterial species were Actinomyces sp (48/51 samples), Porphyromonas cangingivalis (47/51 samples), and a Campylobacter sp (48/51 samples). The most abundant species were P cangingivalis, Porphyromonas gulae, and an undefined Porphyromonas sp. Porphyromonas cangingivalis and Campylobacter sp were part of the core microbiome shared among the 4 groups, and P gulae, which was significantly enriched in dogs with severe periodontal disease, was part of the core microbiome shared between all groups except dogs without periodontal disease. Christensenellaceae sp, Bacteroidales sp, Family XIII sp, Methanobrevibacter oralis, Peptostreptococcus canis, and Tannerella sp formed a unique core microbiome in dogs with severe periodontal disease. CONCLUSIONS AND CLINICAL RELEVANCE Results highlighted that in dogs, potential pathogens can be common members of the oral cavity bacteriome in the absence of disease, and changes in the relative abundance of certain members of the bacteriome can be associated with severity of periodontal disease. Future studies may aim to determine whether these changes are the cause or result of periodontal disease or the host immune response.

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Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA

Microorganisms ◽

10.3390/microorganisms9081721 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1721

Author(s):

Christian O’Dea ◽

Roger Huerlimann ◽

Nicole Masters ◽

Anna Kuballa ◽

Cameron Veal ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

Microbial Diversity ◽

Surface Waters ◽

Bacterial Species ◽

Rrna Gene ◽

Faecal Contamination ◽

Hypervariable Regions ◽

Geographical Regions ◽

V3 Region

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals’ faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1–V3, V3–V4, and V5–V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3–V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3–V4 (n = 11) and V5–V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

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Cave Drip Water-Related Samples as a Natural Environment for Aromatic Hydrocarbon-Degrading Bacteria

Microorganisms ◽

10.3390/microorganisms7020033 ◽

2019 ◽

Vol 7 (2) ◽

pp. 33 ◽

Cited By ~ 4

Author(s):

Eric Marques ◽

Gislaine Silva ◽

João Dias ◽

Eduardo Gross ◽

Moara Costa ◽

...

Keyword(s):

Aromatic Hydrocarbon ◽

Microbial Communities ◽

16S Rdna ◽

Aromatic Compounds ◽

Denaturing Gradient Gel Electrophoresis ◽

Amplicon Sequencing ◽

Drip Water ◽

Rdna Sequencing ◽

Degrading Bacteria ◽

Cave Drip Water

Restricted contact with the external environment has allowed the development of microbial communities adapted to the oligotrophy of caves. However, nutrients can be transported to caves by drip water and affect the microbial communities inside the cave. To evaluate the influence of aromatic compounds carried by drip water on the microbial community, two limestone caves were selected in Brazil. Drip-water-saturated and unsaturated sediment, and dripping water itself, were collected from each cave and bacterial 16S rDNA amplicon sequencing and denaturing gradient gel electrophoresis (DGGE) of naphthalene dioxygenase (ndo) genes were performed. Energy-dispersive X-ray spectroscopy (EDX) and atomic absorption spectroscopy (AAS) were performed to evaluate inorganic nutrients, and GC was performed to estimate aromatic compounds in the samples. The high frequency of Sphingomonadaceae in drip water samples indicates the presence of aromatic hydrocarbon-degrading bacteria. This finding was consistent with the detection of naphthalene and acenaphthene and the presence of ndo genes in drip-water-related samples. The aromatic compounds, aromatic hydrocarbon-degrading bacteria and 16S rDNA sequencing indicate that aromatic compounds may be one of the sources of energy and carbon to the system and the drip-water-associated bacterial community contains several potentially aromatic hydrocarbon-degrading bacteria. To the best of our knowledge, this is the first work to present compelling evidence for the presence of aromatic hydrocarbon-degrading bacteria in cave drip water.

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Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing

Polish Journal of Microbiology ◽

10.33073/pjm-2015-004 ◽

2015 ◽

Vol 64 (1) ◽

pp. 29-36 ◽

Cited By ~ 10

Author(s):

YA-BING CHEN ◽

DAO-LIANG LAN ◽

CHENG TANG ◽

XIAO-NONG YANG ◽

JIAN LI

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

Microbial Communities ◽

16S Rdna ◽

Dna Extraction ◽

Extraction Methods ◽

16S Rdna Sequencing ◽

Bead Beating ◽

Rdna Sequencing ◽

Dna Extraction Methods

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

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Distinct Microbial Communities Colonize Tonsillar Squamous Cell Carcinoma

10.21203/rs.3.rs-223228/v1 ◽

2021 ◽

Author(s):

Burkhard Ludewig ◽

Angelina De Martin ◽

Mechthild Lütge ◽

Yves Stanossek ◽

Céline Engetschwiler ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Sleep Apnea ◽

Microbial Communities ◽

Cell Carcinoma ◽

Squamous Cell ◽

Bacterial Species ◽

Microbial Colonization ◽

Rrna Gene ◽

Control Cohort ◽

Tonsil Cancer

Abstract Background: Squamous cell carcinoma of the tonsil is one of the most frequent cancers of the oropharynx. The escalating rate of tonsil cancer during the last decades is associated with the increase of high risk-human papilloma virus (HR-HPV) infections. While the microbiome in oropharyngeal malignant diseases has been characterized to some extent, the microbial colonization of HR-HPV-associated tonsil cancer remains largely unknown.Results: Using 16S rRNA gene amplicon amplicon sequencing, we have characterized the microbiome of human palatine tonsil crypts in patients suffering from HR-HPV-associated tonsil cancer in comparison to an age-matched control cohort of sleep apnea patients. We found an increased abundance of the phyla Firmicutes and Actinobacteria in tumor patients, whereas the abundance of Spirochaetes and Synergistetes was significantly higher in the control cohort. Furthermore, the accumulation of several genera such as Veillonella, Streptococcus and Prevotella_7 in tonsillar crypts was associated with tonsil cancer. In contrast, Fusobacterium, Prevotella and Treponema_2 were enriched in sleep apnea patients. Machine learning-based bacterial species analysis indicated that a particular bacterial composition in tonsillar crypts is tumor-predictive. Species-specific PCR-based validation in extended patient cohorts confirmed that differential abundance of Filifactor alocis and Prevotella melaninogenica is a distinct trait of tonsil cancer.Conclusion: This study shows that tonsil cancer patients harbor a characteristic microbiome in the crypt environment that differs from the microbiome of sleep apnea patients on all phylogenetic levels. Moreover, our analysis indicates that profiling of microbial communities in distinct tonsillar niches provides microbiome-based avenues for the diagnosis of tonsil cancer.

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Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.66.3.1167-1174.2000 ◽

2000 ◽

Vol 66 (3) ◽

pp. 1167-1174 ◽

Cited By ~ 76

Author(s):

A. C. Layton ◽

P. N. Karanth ◽

C. A. Lajoie ◽

A. J. Meyers ◽

I. R. Gregory ◽

...

Keyword(s):

Wastewater Treatment ◽

16S Rrna ◽

Activated Sludge ◽

16S Rdna ◽

Industrial Wastewater ◽

Bacterial Species ◽

Treatment Plant ◽

Rrna Gene ◽

16S Rrna Analysis ◽

Industrial Wastewater Treatment

ABSTRACT The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work onHyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained fromHyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869T inHyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those ofHyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specificHyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed thatHyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed forHyphomicrobium cluster I and Hyphomicrobiumcluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

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Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

Journal of Clinical Microbiology ◽

10.1128/jcm.38.3.1094-1104.2000 ◽

2000 ◽

Vol 38 (3) ◽

pp. 1094-1104 ◽

Cited By ~ 142

Author(s):

Andreas Roth ◽

Udo Reischl ◽

Anna Streubel ◽

Ludmila Naumann ◽

Reiner M. Kroppenstedt ◽

...

Keyword(s):

16S Rdna ◽

Restriction Analysis ◽

Diagnostic Algorithm ◽

Species Level ◽

23S Rrna ◽

Rrna Gene ◽

Correct Identification ◽

Mycobacterium Fortuitum ◽

Mycobacterium Chelonae ◽

Rdna Sequencing

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence,HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found inMycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium aviumcomplex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, orAvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus,M. chelonae, Mycobacterium farcinogenes,Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

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Microbial community analysis with a specific statistical approach after a record breaking snowfall in Southern Italy

Annals of Microbiology ◽

10.1186/s13213-020-01604-6 ◽

2020 ◽

Vol 70 (1) ◽

Author(s):

Pamela Monaco ◽

Fabio Divino ◽

Gino Naclerio ◽

Antonio Bucci

Keyword(s):

Microbial Communities ◽

Statistical Approach ◽

Biological Diversity ◽

Southern Italy ◽

Bacterial Species ◽

Species Level ◽

Microbial Community Analysis ◽

Rrna Gene ◽

Additional Tool ◽

Snow Samples

Abstract Purpose Snow and ice ecosystems present unexpectedly high microbial abundance and diversity. Although arctic and alpine snow environments have been intensively investigated from a microbiological point of view, few studies have been conducted in the Apennines. Accordingly, the main purpose of this research was to analyze the microbial communities of the snow collected in two different locations of Capracotta municipality (Southern Italy) after a snowfall record occurred on March 2015 (256 cm of snow in less than 24 h). Methods Bacterial communities were analyzed by the Next-Generation Sequencing techniques. Furthermore, a specific statistical approach for taxonomic hierarchy data was introduced, both for the assessment of diversity within microbial communities and the comparison between different microbiotas. In general, diversity and similarity indices are more informative when computed at the lowest level of the taxonomic hierarchy, the species level. This is not the case with microbial data, for which the species level is not necessarily the most informative. Indeed, the possibility to detect a large number of unclassified records at every level of the hierarchy (even at the top) is very realistic due to both the partial knowledge about the cultivable fraction of microbial communities and limitations to taxonomic assignment connected to the quality and completeness of the 16S rRNA gene reference databases. Thus, a global approach considering information from the whole taxonomic hierarchy was adopted in order to obtain a more consistent assessment of the biodiversity. Result The main phyla retrieved in the investigated snow samples were Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Interestingly, DNA from bacteria adapted to thrive at low temperatures, but also from microorganisms normally associated with other habitats, whose presence in the snow could be justified by wind-transport, was found. Biomolecular investigations and statistical data analysis showed relevant differences in terms of biodiversity, composition, and distribution of bacterial species between the studied snow samples. Conclusion The relevance of this research lies in the expansion of knowledge about microorganisms associated with cold environments in contexts poorly investigated such as the Italian Apennines, and in the development of a global statistical approach for the assessment of biological diversity and similarity of microbial communities as an additional tool to be usefully combined with the barcoding methods.

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