The characterization of bacterial communities of oropharynx microbiota in healthy children by combining culture techniques and sequencing of the 16S rRNA gene

2020 ◽  
Vol 143 ◽  
pp. 104115 ◽  
Author(s):  
Abbas Maleki ◽  
Maryam Zamirnasta ◽  
Morovat Taherikalani ◽  
Iraj Pakzad ◽  
Jasem Mohammadi ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Communities ◽  
Rrna Gene ◽  
Healthy Children ◽  
Culture Techniques ◽  
The 16S Rrna Gene

Molecular characterization of the bacterial communities present in sheep's milk and cheese produced in South Brazilian Region via 16S rRNA gene metabarcoding sequencing

LWT ◽  
2021 ◽  
Vol 147 ◽  
pp. 111579
Author(s):  
Creciana M. Endres ◽  
Ícaro Maia S. Castro ◽  
Laura D. Trevisol ◽  
Juliana M. Severo ◽  
Michele B. Mann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Bacterial Communities ◽  
Rrna Gene

scholarly journals Molecular Characterization of anEndozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximusL.)

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Irene Cano ◽  
Ronny van Aerle ◽  
Stuart Ross ◽  
David W. Verner-Jeffreys ◽  
Richard K. Paley ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Mass Mortality ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTOne of the fastest growing fisheries in the UK is the king scallop (Pecten maximusL.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resemblingRickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from “CandidatusEndonucleobacter bathymodioli” and 95% withEndozoicomonasspecies.In situhybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences fromEndozoicomonasspp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCEMolluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of anEndozoicomonas-like organism (ELO) associated with an important commercial scallop species.

scholarly journals MOLECULAR CHARACTERIZATION OF GENE 16S rRNA MICRO SYMBIONTS IN SPONGE AT MELAWAI BEACH, EAST KALIMANTAN

2018 ◽  
Author(s):  
Ismail Marzuki ◽  
Alfian Noor ◽  
Nursiah La Nafie
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Isolation And Purification ◽  
East Kalimantan ◽  
Degree Of Similarity ◽  
The 16S Rrna Gene

Molecular characterization studies have been conducted 16S rRNA gene micro symbiont of sponge origin Melawai Beach, Balikpapan in East Kalimantan. Objective analysis of histo- morphological research, isolation-purification, molecular characterization of micro-symbiont genes in order to search symbiont bacteria that can live in extreme environments contaminated hydrocarbon waste. The research method that morphological identification, isolation-purification and molecular characterization of the 16S rRNA gene with Chain Reaction Polymerization method. The results of histo-morphological analysis concluded sponge samples with species of Callyspongia sp. Isolation and purification mikro symbionts of sponge obtained 2 (two) isolates. Characteristics of Isolates 1; spherical shape, colonize and creamy, while isolates 2; jagged shape, oval and white colonies. Molecular characterization of the 16S rRNA gene by PCR, Bacillus subtilis strain BAB-684 identification for isolates one is the number of nucleotide pairs reached 899 bp and the degree of similarity in GenBank reached 89% homologous, while the second is a Bacillus flexus strain PHCDB20 isolates the number reached 950 bp nucleotide pairs with the degree of similarity in GenBank reached 99% homologous

scholarly journals Seasonal changes in bacterial communities associated with healthy and diseasedPoritescoral in southern Taiwan

2016 ◽  
Vol 62 (12) ◽  
pp. 1021-1033 ◽  
Author(s):  
Chorng-Horng Lin ◽  
Chih-Hsiang Chuang ◽  
Wen-Hung Twan ◽  
Shu-Fen Chiou ◽  
Tit-Yee Wong ◽  
...  
Keyword(s):  
Health Status ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Southern Taiwan ◽  
The 16S Rrna Gene

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

scholarly journals Critical Relevance of Stochastic Effects on Low-Bacterial-Biomass 16S rRNA Gene Analysis

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
John R. Erb-Downward ◽  
Nicole R. Falkowski ◽  
Jennifer C. D’Souza ◽  
Lisa M. McCloskey ◽  
Roderick A. McDonald ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Biomass ◽  
Critical Examination ◽  
Rrna Gene ◽  
Stochastic Noise ◽  
Oral Rinse ◽  
Healthy Humans ◽  
The 16S Rrna Gene

ABSTRACT The bacterial microbiome of human body sites, previously considered sterile, remains highly controversial because it can be challenging to isolate signal from noise when low-biomass samples are being analyzed. We tested the hypothesis that stochastic sequencing noise, separable from reagent contamination, is generated during sequencing on the Illumina MiSeq platform when DNA input is below a critical threshold. We first purified DNA from serial dilutions of Pseudomonas aeruginosa and from negative controls using three DNA purification kits, quantified input using droplet digital PCR, and then sequenced the 16S rRNA gene in four technical replicates. This process identified reproducible contaminant signal that was separable from an irreproducible stochastic noise, which occurred as bacterial biomass of samples decreased. This approach was then applied to authentic respiratory samples from healthy individuals (n = 22) that ranged from high to ultralow bacterial biomass. Using oral rinse, bronchoalveolar lavage (BAL) fluid, and exhaled breath condensate (EBC) samples and matched controls, we were able to demonstrate (i) that stochastic noise dominates sequencing in real-world low-bacterial-biomass samples that contain fewer than 104 copies of the 16S rRNA gene per sample, (ii) that critical examination of the community composition of technical replicates can be used to separate signal from noise, and (iii) that EBC is an irreproducible sampling modality for sampling the microbiome of the lower airways. We anticipate that these results combined with suggested methods for identifying and dealing with noisy communities will facilitate increased reproducibility while simultaneously permitting characterization of potentially important low-biomass communities. IMPORTANCE DNA contamination from external sources (reagents, environment, operator, etc.) has long been assumed to be the main cause of spurious signals that appear under low-bacterial-biomass conditions. Here, we demonstrate that contamination can be separated from another, random signal generated during low-biomass-sample sequencing. This stochastic noise is not reproduced between technical replicates; however, results for any one replicate taken alone could look like a microbial community different from the controls. Using this information, we investigated respiratory samples from healthy humans and determined the narrow range of bacterial biomass where samples transition from producing reproducible microbial sequences to ones dominated by noise. We present a rigorous approach to studies involving low-bacterial-biomass samples to detect this source of noise and provide a framework for deciding if a sample is likely to be dominated by noise. We anticipate that this work will facilitate increased reproducibility in the characterization of potentially important low-biomass communities.

scholarly journals The impact of persistent bacterial bronchitis on the pulmonary microbiome of children

2017 ◽  
Author(s):  
Leah Cuthbertson ◽  
Vanessa Craven ◽  
Lynne Bingle ◽  
William O.C.M. Cookson ◽  
Mark L. Everard ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Diversity ◽  
Community Composition ◽  
Bacterial Communities ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Healthy Children ◽  
Significant Difference ◽  
The Impact

AbstractPersistent bacterial bronchitis is a leading cause of chronic wet cough in young children. This study aimed to characterise the respiratory bacterial microbiota of healthy children and to assess the impact of the changes associated with the development of persistent bacterial bronchitis.Blind, protected brushings were obtained from 20 healthy controls and 24 children with persistent bacterial bronchitis, with an additional directed sample obtained from persistent bacterial bronchitis patients. DNA was extracted, quantified using a 16S rRNA gene quantitative PCR assay prior to microbial community analysis by 16S rRNA gene sequencing.No significant difference in bacterial diversity or community composition (R2 = 0.01, P = 0.36) was observed between paired blind and non-blind brushes, showing that blind brushings are a valid means of accessing the airway microbiota. This has important implications for collecting lower respiratory samples from healthy children. A significant decrease in bacterial diversity (P < 0.001) and change in community composition (R2 = 0.08, P = 0.004) was observed between controls and patients. Bacterial communities within patients with PBB were dominated by Proteobacteria, and indicator species analysis showed that Haemophilus and Neisseria were significantly associated with the patient group. In 15 (52.9%) cases the dominant organism by sequencing was not identified by standard routine clinical culture.The bacteria present in the lungs of patients with persistent bacterial bronchitis were less diverse in terms of richness and evenness. The results validate the clinical diagnosis, and suggest that more attention to bacterial communities in children with chronic cough may lead to more rapid recognition of this condition with earlier treatment and reduction in disease burden.

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