Approaches for diversity analysis of cultivable and non-cultivable bacteriain real soil

Until recently, investigators had no idea how accurately cultivated microorganisms represented the overall microbial diversity. The cultivation-dependent approach is limited by the fact that the overwhelming majority of microorganisms present in soil cannot be cultivated under laboratory conditions. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. There is a vast amount of information held within the genomes of cultivable and non-cultivable microorganisms, and new methods based on analysis of DNA allow to investigate this potential. In this work we show some aspects, advantages and disadvantages of classical and new approaches in taxonomical and functional description of bacteria present in natural microbial assemblages on the example of cultivable bacteria isolated from rhizosphere of plants, tobacco and black nightshade, planted in PCB contaminated soil. Biochemical analysis of isolates showed 8 different bacterial species. This identification was compared by discrimination using MALDI-TOF mass spectrometry and identity evaluation after sequencing of 16S rDNA. Six strains from original number of 8 were positively identified after 16S rDNA sequencing and their phylogenetic relations were compared. These analyses confirmed closed relations of all species (two of isolates exhibited the same characteristics and were discriminated as the same species <I>Pseudomonas stutzeri</I>) and also of <I>Burkholderia xenovorans</I> LB 400, a well-known PCB degrader. Nevertheless, only two isolates gave a positive reaction after amplification of the biphenyl dioxygenase gene and exhibited potential to degrade PCB. These results indicate that only a subset of the recovered molecular information, derived from active population based on molecular and functional analysis is relevant to microbial ecology.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.060616-0 ◽

2014 ◽

Vol 63 (3) ◽

pp. 433-440 ◽

Cited By ~ 7

Author(s):

Haiyin Wang ◽

Pengcheng Du ◽

Juan Li ◽

Yuanyuan Zhang ◽

Wen Zhang ◽

...

Keyword(s):

Microbial Communities ◽

16S Rdna ◽

Gold Standard ◽

Clinical Utility ◽

Bacterial Species ◽

Relative Proportion ◽

Rrna Gene ◽

Low Concentrations ◽

Rdna Sequencing ◽

V3 Region

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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Isolation and Characterization of Pseudomonas stutzeri as Lead Tolerant Bacteria from Water Bodies of Udaipur, India using 16S rDNA Sequencing Technique

Journal of Pure and Applied Microbiology ◽

10.22207/jpam.11.2.39 ◽

2017 ◽

Vol 11 (2) ◽

pp. 975-979 ◽

Cited By ~ 2

Author(s):

Garima Verma ◽

Nadim Chishty ◽

Chandra Veer

Keyword(s):

16S Rdna ◽

Pseudomonas Stutzeri ◽

Water Bodies ◽

16S Rdna Sequencing ◽

Isolation And Characterization ◽

Rdna Sequencing ◽

Sequencing Technique

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Bacterial Microbiota in Unfed Ticks (Dermacentor nuttalli) From Xinjiang Detected Through 16S rDNA Amplicon Sequencing and Culturomics

Zoonoses ◽

10.15212/zoonoses-2021-0007 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Kai Song ◽

Yuxin Ji ◽

Surong Sun ◽

Xihong Yue ◽

Cheng Wang ◽

...

Keyword(s):

16S Rdna ◽

Bacterial Species ◽

Amplicon Sequencing ◽

Zoonotic Diseases ◽

Comprehensive Understanding ◽

Bacterial Microbiota ◽

Western Tianshan ◽

Rdna Sequencing ◽

16S Rdna Amplicon Sequencing ◽

Acinetobacter Pittii

Background: Ticks are a major arthropod vector of zoonotic diseases affecting both humans and domestic animals worldwide. Thus, studying tick microbiota would aid in understanding of the potential threats posed by ticks. Methods: Approximately 8,000 unfed ticks, identified as Dermacentor nuttalli, were collected from the sylvosteppe in the western Tianshan mountains. To investigate their potential pathogens, we divided the ticks into 36 groups of 200–300 individuals each for examination with culturomics and 16S rDNA amplicon sequencing. Results: A total of 237 bacterial genera were identified with the two methods. Culturomics identified 46 bacterial species from 23 genera, predominantly Pseudomonas, Pantoea, and Bacillus, whereas 16S rDNA sequencing identified 461 OTUs from 233 genera, predominantly Pseudomonas (53.8%), Coxiella (17.2%), and Pantoea (6.4%). Coxiella, Rickettsia, and ten other genera were discovered only by sequencing, because optimal cultivating conditions were not used for their isolation, whereas Arthrobacter and three other genera were discovered only through culturomics. Conclusions: Several of the identified bacteria, such as line-related sepsis-causing Delftia acidovorans and the pneumonia agent Acinetobacter pittii, can cause human diseases. Thus, both sequencing and culturomics methods are crucial for comprehensive understanding of the microbiota of D. nuttalli.

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Faculty Opinions recommendation of Early impact of 10-valent pneumococcal conjugate vaccine in childhood pneumonia hospitalizations using primary data from an active population-based surveillance.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726041128.793522903 ◽

2016 ◽

Author(s):

Charles Feldman

Keyword(s):

Conjugate Vaccine ◽

Pneumococcal Conjugate Vaccine ◽

Population Based ◽

Primary Data ◽

Childhood Pneumonia ◽

Active Population

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Metabolite fingerprinting of novel Streptomyces UK-238 from the Himalayan forest

Current Pharmaceutical Analysis ◽

10.2174/1573412916666200206160836 ◽

2020 ◽

Vol 16 ◽

Author(s):

Nidhi Srivastava ◽

Indira P. Sarethy

Keyword(s):

Ethyl Acetate ◽

16S Rdna ◽

Retention Index ◽

Ethyl Acetate Extract ◽

Similarity Index ◽

Antimicrobial Drug ◽

16S Rdna Sequencing ◽

Metabolite Fingerprinting ◽

Streptomyces Sp ◽

Rdna Sequencing

Aims: Characterization of antimicrobial metabolites of novel Streptomyces sp. UK-238. Background: Novel antimicrobial drug discovery is urgently needed due to emerging multi antimicrobial drug resistance among pathogens. Since many years, natural products have provided the basic skeletons for many therapeutic compounds including antibiotics. Bioprospection of un/under explored habitats and focussing on selective isolation of actinobacteria as major reservoir of bio and chemodiversity has yielded good results. Objective: The main objectives of the study were the identification of UK-238 by 16S rDNA sequencing and antimicrobial metabolite fingerprinting of culture extracts. Method: In the present study, a promising isolate, UK-238, has been screened for antimicrobial activity and metabolite fingerprinting from the Himalayan Thano Reserve forest. It was identified by 16S rDNA sequencing. Ethyl acetate extract was partially purified by column chromatography. The pooled active fractions were fingerprinted by GC-MS and compounds were tentatively identified by collated data analysis based on Similarity Index, observed Retention Index from Databases and calculated Retention Index. Results: UK-238 was identified as Streptomyces sp. with 98.4% similarity to S. niveiscabiei. It exhibited broad-spectrum antibacterial and antifungal activity. GC-MS analysis of active fractions of ethyl acetate extract showed the presence of eighteen novel antimicrobial compounds belonging to four major categories- alcohols, alkaloid, esters and peptide. Conclusion: The study confirms that bioprospection of underexplored habitats can elaborate novel bio and chemodiversity.

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2-Way k-Means as a Model for Microbiome Samples

Journal of Healthcare Engineering ◽

10.1155/2017/5284145 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Weston J. Jackson ◽

Ipsita Agarwal ◽

Itsik Pe’er

Keyword(s):

Unsupervised Learning ◽

16S Rdna ◽

Mixture Model ◽

Vaginal Microbiome ◽

16S Rdna Sequencing ◽

Sequencing Data ◽

Cluster Assignment ◽

Rdna Sequencing

Motivation. Microbiome sequencing allows defining clusters of samples with shared composition. However, this paradigm poorly accounts for samples whose composition is a mixture of cluster-characterizing ones and which therefore lie in between them in the cluster space. This paper addresses unsupervised learning of 2-way clusters. It defines a mixture model that allows 2-way cluster assignment and describes a variant of generalized k-means for learning such a model. We demonstrate applicability to microbial 16S rDNA sequencing data from the Human Vaginal Microbiome Project.

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Profiling of Bacterial Species Associated with Haddock Larviculture by PCR Amplification of 16S rDNA and Denaturing Gradient Gel Electrophoresis

Journal of Aquatic Animal Health ◽

10.1577/1548-8667(2001)013<0355:pobsaw>2.0.co;2 ◽

2001 ◽

Vol 13 (4) ◽

pp. 355-363 ◽

Cited By ~ 41

Author(s):

Steve Griffiths ◽

Krista Melville ◽

Marcia Cook ◽

Sherry Vincent ◽

Maurice Pierre ◽

...

Keyword(s):

16S Rdna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Pcr Amplification ◽

Gradient Gel Electrophoresis

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Phylogeny of the family Pasteurellaceae based on rpoB sequences

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.03043-0 ◽

2004 ◽

Vol 54 (4) ◽

pp. 1393-1399 ◽

Cited By ~ 153

Author(s):

Bożena Korczak ◽

Henrik Christensen ◽

Stefan Emler ◽

Joachim Frey ◽

Peter Kuhnert

Keyword(s):

16S Rdna ◽

Dna Hybridization ◽

Gene Encoding ◽

Phylogenetic Studies ◽

Study Selection ◽

Gene Sequence Analysis ◽

Rdna Sequencing ◽

The Family ◽

Type Strains ◽

Selection Of

Sequences of the gene encoding the β-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA–DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

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