scholarly journals In-Silico Evaluation and Selection of the Best 16S rRNA Gene Primers for Use in Next-Generation Sequencing to Detect Oral Bacteria and Archaea.

2021 ◽  
Author(s):  
Regueira-Iglesias A ◽  
Vázquez-González L ◽  
Balsa-Castro C ◽  
Vila-Blanco N ◽  
Blanco-Pintos T ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
Oral Bacteria ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Sequencing Studies ◽  
Distinct Individual ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Abstract Background: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. Results: A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral-archaea database . Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7 and 3-7 16S rRNA gene regions, with SC levels of 97.14-98.83%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-5 and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5 and 5-9, and produced SC values of 94.54-95.71% and 96.91-99.48% for bacteria and archaea, respectively. Conclusions: Given the three amplicon length categories (100-300, 301-600 and >600 bps), the primer pairs with the best coverage values for detecting oral bacteria were: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined) and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801) and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature.

scholarly journals Molecular Phylogenetic Analysis of 16S rRNA Sequences Identified Two Lineages of Helicobacter pylori Strains Detected from Different Regions in Sudan Suggestive of Differential Evolution

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Abeer Babiker Idris ◽  
Hadeel Gassim Hassan ◽  
Maryam Atif Salaheldin Ali ◽  
Sulafa Mohamed Eltaher ◽  
Leena Babiker Idris ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Human Migration ◽  
Rrna Gene ◽  
Migration Patterns ◽  
H Pylori ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Background. Helicobacter pylori (H. pylori) is ubiquitous among humans and one of the best-studied examples of an intimate association between bacteria and humans. Phylogeny and Phylogeography of H. pylori strains are known to mirror human migration patterns and reflect significant demographic events in human prehistory. In this study, we analyzed the molecular evolution of H. pylori strains detected from different tribes and regions of Sudan using 16S rRNA gene and the phylogenetic approach. Materials and methods. A total of 75 gastric biopsies were taken from patients who had been referred for endoscopy from different regions of Sudan. The DNA extraction was performed by using the guanidine chloride method. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied, and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Results. Molecular detection of H. pylori has shown that 28 (37.33%) of the patients were positive for H. pylori and no significant differences were found in sociodemographic characteristics, endoscopy series, and H. pylori infection. Nucleotide variations were observed at five nucleotide positions (positions 219, 305, 578, 741, and 763–764), and one insertion mutation (750_InsC_751) was present in sixty-seven percent (7/12) of our strains. These six mutations were detected in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding sites; 66.67% of them were located in the central domain of 16S rRNA. The phylogenetic analysis of 16S rRNA sequences identified two lineages of H. pylori strains detected from different regions in Sudan. The presence of Sudanese H. pylori strains resembling Hungarian H. pylori strains could reflect the migration of Hungarian people to Sudan or vice versa. Conclusion. This finding emphasizes the significance of studying the phylogeny of H. pylori strains as a discriminatory tool to mirror human migration patterns. In addition, the 16S rRNA gene amplification method was found useful for bacterial identification and phylogeny.

scholarly journals Extent and Variation of Phage-Borne Bacterial 16S rRNA Gene Sequences in Wastewater Environments

2011 ◽  
Vol 77 (15) ◽  
pp. 5529-5532 ◽  
Author(s):  
Antonio Del Casale ◽  
Paul V. Flanagan ◽  
Michael J. Larkin ◽  
Christopher C. R. Allen ◽  
Leonid A. Kulakov
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
Total Bacterial Community ◽  
Rrna Sequences ◽  
16S Rrna Sequences ◽  
Bacterial 16S Rrna Gene

ABSTRACTPhage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains ofProteobacteria,Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.

scholarly journals IDENTIFIKASI BAKTERI PENGOKSIDASI BESI DAN SULFUR BERDASARKAN GEN 16S rRNA DARI LAHAN TAMBANG TIMAH DI BELITUNG

2014 ◽  
Vol 1 (1) ◽  
pp. 10
Author(s):  
Dhewanti Puspitasari ◽  
Hendro Pramono ◽  
Oedjijono Oedjijono
Keyword(s):  
Heavy Metals ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Micrococcus Luteus ◽  
Morphological Characterization ◽  
Rrna Gene ◽  
Mining Areas ◽  
Gram Staining ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Heavy metals contamination disturb balance and diversity of microorganism in soil. Microorganisms which can able to survive in those conditions are bacteria capable of oxidizing heavy metals. Identification based on 16S rRNA was used to determine characteristics and phylogenetic relationship of bacteria which can oxidize iron and sulphur in tin mining areas. The aim of this research was able to determine the bacterias characteristics isolated from tin mining areas and determine the phylogenetic relation of iron-sulphur oxidizing bacteria on tin mining soil in Belitung based on 16S rRNA sequences. This research was done using descriptive method, including isolation, morphological characterization, and identification based on 16S rRNA sequences. Morphology characterization includes colony and cell morphology through Gram staining. Molecular characterization includes amplification of 16S rRNA gene (Polymerase Chain Reaction/ PCR), electrophoresis amplicon and sequencing. Bacteria identification was done by comparing the 16S rRNA gene sequence in GenBank. The result showed three bacterias were identified by 16S rRNA have a similarity with Bacillus anthracis strain Ames, Bacillus cereus ATCC 14579, Staphylococcus sciuri subsp. Sciuri strains DSM 20345 and Micrococcus luteus NCTC 2665.

scholarly journals Microbiome of Odontogenic Abscesses

2021 ◽  
Vol 9 (6) ◽  
pp. 1307
Author(s):  
Sebastian Böttger ◽  
Silke Zechel-Gran ◽  
Daniel Schmermund ◽  
Philipp Streckbein ◽  
Jan-Falco Wilbrand ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Oral Microbiome ◽  
Minor Role ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Bacterial Strains ◽  
16S Rrna Gene Analysis ◽  
Odontogenic Infections ◽  
Odontogenic Abscess

Severe odontogenic abscesses are regularly caused by bacteria of the physiological oral microbiome. However, the culture of these bacteria is often prone to errors and sometimes does not result in any bacterial growth. Furthermore, various authors found completely different bacterial spectra in odontogenic abscesses. Experimental 16S rRNA gene next-generation sequencing analysis was used to identify the microbiome of the saliva and the pus in patients with a severe odontogenic infection. The microbiome of the saliva and the pus was determined for 50 patients with a severe odontogenic abscess. Perimandibular and submandibular abscesses were the most commonly observed diseases at 15 (30%) patients each. Polymicrobial infections were observed in 48 (96%) cases, while the picture of a mono-infection only occurred twice (4%). On average, 31.44 (±12.09) bacterial genera were detected in the pus and 41.32 (±9.00) in the saliva. In most cases, a predominantly anaerobic bacterial spectrum was found in the pus, while saliva showed a similar oral microbiome to healthy individuals. In the majority of cases, odontogenic infections are polymicrobial. Our results indicate that these are mainly caused by anaerobic bacterial strains and that aerobic and facultative anaerobe bacteria seem to play a more minor role than previously described by other authors. The 16S rRNA gene analysis detects significantly more bacteria than conventional methods and molecular methods should therefore become a part of routine diagnostics in medical microbiology.

scholarly journals Clinical Relevance of the Microbiome in Odontogenic Abscesses

Biology ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 916
Author(s):  
Sebastian Böttger ◽  
Silke Zechel-Gran ◽  
Daniel Schmermund ◽  
Philipp Streckbein ◽  
Jan-Falco Wilbrand ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Oral Microbiome ◽  
Molecular Methods ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Cultural Methods ◽  
Odontogenic Abscess ◽  
Polymicrobial Infections

Odontogenic abscesses are usually caused by bacteria of the oral microbiome. However, the diagnostic culture of these bacteria is often prone to errors and sometimes fails completely due to the fastidiousness of the relevant bacterial species. The question arises whether additional pathogen diagnostics using molecular methods provide additional benefits for diagnostics and therapy. Experimental 16S rRNA gene analysis with next-generation sequencing (NGS) and bioinformatics was used to identify the microbiome of the pus in patients with severe odontogenic infections and was compared to the result of standard diagnostic culture. The pus microbiome was determined in 48 hospitalized patients with a severe odontogenic abscess in addition to standard cultural pathogen detection. Cultural detection was possible in 41 (85.42%) of 48 patients, while a pus-microbiome could be determined in all cases. The microbiomes showed polymicrobial infections in 46 (95.83%) cases, while the picture of a mono-infection occurred only twice (4.17%). In most cases, a predominantly anaerobic spectrum with an abundance of bacteria was found in the pus-microbiome, while culture detected mainly Streptococcus, Staphylococcus, and Prevotella spp. The determination of the microbiome of odontogenic abscesses clearly shows a higher number of bacteria and a significantly higher proportion of anaerobes than classical cultural methods. The 16S rRNA gene analysis detects considerably more bacteria than conventional cultural methods, even in culture-negative samples. Molecular methods should be implemented as standards in medical microbiology diagnostics, particularly for the detection of polymicrobial infections with a predominance of anaerobic bacteria.

scholarly journals The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

2020 ◽  
Vol 8 (1) ◽  
pp. 131 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Christian Milani ◽  
Gabriele Andrea Lugli ◽  
Federico Fontana ◽  
Francesca Turroni ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
In Silico Analysis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Test Case ◽  
Bacterial Populations ◽  
Taxonomic Marker ◽  
The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

Streptococcus orisasini sp. nov. and Streptococcus dentasini sp. nov., isolated from the oral cavity of donkeys

2013 ◽  
Vol 63 (Pt_8) ◽  
pp. 2782-2786 ◽  
Author(s):  
Kazuko Takada ◽  
Masanori Saito ◽  
Osamu Tsudukibashi ◽  
Takachika Hiroi ◽  
Masatomo Hirasawa
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Type Species ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Content Type ◽  
Link Type ◽  
Sequencing Studies

Four Gram-positive, catalase-negative, coccoid isolates that were obtained from donkey oral cavities formed two distinct clonal groups when characterized by phenotypic and phylogenetic studies. From the results of biochemical tests, the organisms were tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed the organisms to be members of the genus Streptococcus . Two of the isolates were related most closely to Streptococcus ursoris with 95.6 % similarity based on the 16S rRNA gene and to Streptococcus ratti with 92.0 % similarity based on the 60 kDa heat-shock protein gene (groEL). The other two isolates, however, were related to Streptococcus criceti with 95.0 and 89.0 % similarities based on the 16S rRNA and groEL genes, respectively. From both phylogenetic and phenotypic evidence, the four isolates formed two distinct clonal groups and are suggested to represent novel species of the genus Streptococcus . The names proposed for these organisms are Streptococcus orisasini sp. nov. (type strain NUM 1801T = JCM 17942T = DSM 25193T) and Streptococcus dentasini sp. nov. (type strain NUM 1808T = JCM 17943T = DSM 25137T).

scholarly journals Evidence and molecular characterization ofBartonellaspp. and hemoplasmas in neotropical bats in Brazil

2017 ◽  
Vol 145 (10) ◽  
pp. 2038-2052 ◽  
Author(s):  
P. IKEDA ◽  
M. C. SEKI ◽  
A. O. T. CARRASCO ◽  
L. V. RUDIAK ◽  
J. M. D. MIRANDA ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Clinical Manifestations ◽  
Rrna Gene ◽  
Zoonotic Virus ◽  
The World ◽  
Neotropical Bats ◽  
Latin America Countries ◽  
Rrna Sequences ◽  
16S Rrna Sequences

SUMMARYThe order Chiroptera is considered the second largest group of mammals in the world, hosting important zoonotic virus and bacteria.Bartonellaand hemotropic mycoplasmas are bacteria that parasite different mammals’ species, including humans, causing different clinical manifestations. The present work aimed investigating the occurrence and assessing the phylogenetic positioning ofBartonellaspp. andMycoplasmaspp. in neotropical bats sampled from Brazil. Between December 2015 and April 2016, 325 blood and/or tissues samples were collected from 162 bats comprising 19 different species sampled in five states of Brazil. Out of 322 bat samples collected, while 17 (5·28%) were positive to quantitative PCR forBartonellaspp. based onnuoGgene, 45 samples (13·97%) were positive to cPCR assays for hemoplasmas based on 16S rRNA gene. While seven sequences were obtained forBartonella(nuoG) (n= 3),gltA(n= 2),rpoB(n= 1),ftsZ(n= 1), five 16S rRNA sequences were obtained for hemoplasmas. In the phylogenetic analysis, theBartonellasequences clustered withBartonellagenotypes detected in bats sampled in Latin America countries. All five hemoplasmas sequences clustered together as a monophyletic group by Maximum Likelihood and Bayesian Inference analyses. The present work showed the first evidence of circulation ofBartonellaspp. and hemoplasmas among bats in Brazil.

scholarly journals Streptococcus loxodontisalivarius sp. nov. and Streptococcus saliviloxodontae sp. nov., isolated from oral cavities of elephants

2014 ◽  
Vol 64 (Pt_9) ◽  
pp. 3288-3292 ◽  
Author(s):  
Masanori Saito ◽  
Noriko Shinozaki-Kuwahara ◽  
Masatomo Hirasawa ◽  
Kazuko Takada
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Type Species ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Content Type ◽  
Link Type ◽  
Sequencing Studies

Four Gram-stain-positive, catalase-negative, coccoid-shaped organisms were isolated from elephant oral cavities. The isolates were tentatively identified as streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed the organisms to be members of the genus Streptococcus . Two isolates (NUM 6304T and NUM 6312) were related most closely to Streptococcus salivarius with 96.8 % and 93.1 % similarity based on the 16S rRNA gene and the RNA polymerase β subunit encoding gene (rpoB), respectively, and to Streptococcus vestibularis with 83.7 % similarity based on the 60 kDa heat-shock protein gene (groEL). The other two isolates (NUM 6306T and NUM 6318) were related most closely to S. vestibularis with 97.0 % and 82.9 % similarity based on the 16S rRNA and groEL genes, respectively, and to S. salivarius with 93.5 % similarity based on the rpoB gene. Based on phylogenetic and phenotypic evidence, these isolates are suggested to represent novel species of the genus Streptococcus , for which the names Streptococcus loxodontisalivarius sp. nov. (type strain NUM 6304T = JCM 19287T = DSM 27382T) and Streptococcus saliviloxodontae sp. nov. (type strain NUM 6306T = JCM 19288T = DSM 27513T) are proposed.

scholarly journals Empirical Testing of 16S rRNA Gene PCR Primer Pairs Reveals Variance in Target Specificity and Efficacy Not Suggested by In Silico Analysis

2009 ◽  
Vol 75 (9) ◽  
pp. 2677-2683 ◽  
Author(s):  
Sergio E. Morales ◽  
William E. Holben
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
In Silico ◽  
In Silico Analysis ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Empirical Testing ◽  
Specific Target ◽  
Silico Analysis

ABSTRACT Phylogenetic and “fingerprinting” analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing ∼5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

Sign in / Sign up

Export Citation Format

Share Document