scholarly journals Molecular Characterization Based on MLST and ECDC Typing Schemes and Antibiotic Resistance Analyses of Treponema pallidum subsp. pallidum in Xiamen, China

2021 ◽  
Vol 10 ◽  
Author(s):  
Dan Liu ◽  
Shu-Min He ◽  
Xiao-Zhen Zhu ◽  
Li-Li Liu ◽  
Li-Rong Lin ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Treponema Pallidum ◽  
23S Rrna ◽  
Clinical Samples ◽  
Molecular Characteristics ◽  
Nucleotide Polymorphisms ◽  
Penicillin Binding Protein ◽  
Allelic Profile ◽  
Single Nucleotide ◽  
Control And Prevention

In total, 49 clinical samples were analyzed using two typing schemes, Enhanced Centers for Disease Control and Prevention (ECDC) and multilocus sequence typing (MLST), to describe the molecular characteristics of circulating Treponema pallidum isolates in Xiamen between 2016 and 2017. In addition, genetic mutations potentially related to antibiotic resistance of T. pallidum were also analyzed. Forty five samples were fully typed by ECDC, and 14 different subtypes were detected. The most common subtype was 16d/f (24.4%), followed by 14d/f (20.0%). All forty nine samples were successfully typed by MLST, while only four allelic profiles were identified, including three SS14-like profiles and one Nichols-like profile. Among them, the major allelic profile was 1.1.8 (85.7%). Interestingly, the allelic profile 1.3.1 widespread in Europe and North America was not detected in this region. Additionally, A2058G mutation in 23S rRNA was found in all detectable samples (38/38), and no mutation in 16S rRNA was observed (36/36). Four non-synonymous single-nucleotide polymorphisms in penicillin-binding protein genes were found in the 35 samples eligible for Sanger sequencing. Among them, the variant in tp0500 (P564I) can only be found in the SS14-like isolates. Homoplastic changes in tp0760 (I415F/I415M) and tp0705 (A506V/A506T) were found. Moreover, the variant tp0705 A506V and the variant tp0705 A506T separately appeared in the SS14-like isolates and Nichols-like isolates, respectively. This study showed that the genotypes of T. pallidum isolates in Xiamen between 2016 and 2017 were different from those in other geographic areas. The resistance-related variants of T. pallidum isolates identified in this study could provide awareness for clinicians in the treatment of syphilis.

scholarly journals Discriminatory Power, Typability, and Accuracy of Single Nucleotide Extension Microarrays

2007 ◽  
Vol 90 (3) ◽  
pp. 802-809 ◽  
Author(s):  
Ingunn Berget ◽  
Even Heir ◽  
Jelena Petcovic ◽  
Knut Rudi
Keyword(s):  
Binary Data ◽  
Discriminatory Power ◽  
Classification Error ◽  
Bayesian Posterior Probability ◽  
Weighted Sum ◽  
Nucleotide Polymorphisms ◽  
Allelic Profile ◽  
Single Nucleotide ◽  
Parameter Estimations ◽  
Wide Acceptance

Abstract Despite great conceptual promise, the use of microarrays in typing approaches has not yet gained wide acceptance. The establishment of proper criteria for determining discriminatory power as well as typability and the accuracy of microarray data remains to be solved. Purely experimental estimations of these parameters would far exceed what is experimentally practical. We therefore used simulations in combination with experimental results in parameter estimations. Our assay was based on 26 single nucleotide polymorphisms (SNPs) identified in the Campylobacter jejuni Multi Locus Sequence Typing (MLST) database (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://pubmlst.org/campylobacter/">http://pubmlst.org/campylobacter/</ext-link>). The SNPs were detected using a single nucleotide extension (SNE) typing microarray. Unknown isolates were assigned to the known sequence type(s) by calculating weighted sum of matches minus a weighted sum of mismatches between predicted and candidate genotype. The weights were set according to the Bayesian posterior probability of the SNP classification. These studies showed that any typing or profiling method based on binary data requires an accuracy of <23% error for each datapoint (in our case SNPs) to classify the isolates to the correct allelic profile in 90% of the cases. The classification error for our experimental data was 3.2% (after removing 5 high error SNPs). We therefore conclude that SNE microarrays are promising for future high-throughput typing of bacteria.

scholarly journals From raw reads to trees: Whole genome SNP phylogenetics across the tree of life

2015 ◽  
Author(s):  
Sanaa Afroz Ahmed ◽  
Chien-Chi Lo ◽  
Po-E Li ◽  
Karen W Davenport ◽  
Patrick S.G. Chain
Keyword(s):  
Ad Hoc ◽  
Phylogenetic Reconstruction ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Complex Samples ◽  
Phylogenetic Characterization ◽  
Genome Wide ◽  
Genome Assemblies

Next-generation sequencing is increasingly being used to examine closely related organisms. However, while genome-wide single nucleotide polymorphisms (SNPs) provide an excellent resource for phylogenetic reconstruction, to date evolutionary analyses have been performed using different ad hoc methods that are not often widely applicable across different projects. To facilitate the construction of robust phylogenies, we have developed a method for genome-wide identification/characterization of SNPs from sequencing reads and genome assemblies. Our phylogenetic and molecular evolutionary (PhaME) analysis software is unique in its ability to take reads and draft/complete genome(s) as input, derive core genome alignments, identify SNPs, construct phylogenies and perform evolutionary analyses. Several examples using genomes and read datasets for bacterial, eukaryotic and viral linages demonstrate the broad and robust functionality of PhaME. Furthermore, the ability to incorporate raw metagenomic reads from clinical samples with suspected infectious agents shows promise for the rapid phylogenetic characterization of pathogens within complex samples.

Multiplexed genotyping using a novel digitally inscribed bead-based system

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21089-21089
Author(s):  
S. M. Lipkin ◽  
J. Yeakley ◽  
E. Chao ◽  
J. Velasquez ◽  
M. Lopez ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Sample Material ◽  
Msh2 Gene ◽  
Goldengate Assay ◽  
Single Nucleotide ◽  
System A ◽  
Qpcr Analysis ◽  
Low Levels ◽  
Reference Samples

21089 Background: Genotyping of clinical samples has been limited to low levels of multiplexing, ranging from one to a few dozen single nucleotide polymorphisms (SNPs) per sample. By increasing multiplexing levels, a clinical lab can increase information content per sample, decreasing costs and sample material requirements. Methods: We have adapted the GoldenGate® Assay for simultaneously genotyping 96 to 1,536 SNPs to the BeadXpress™ System, a new high-throughput platform that utilizes digitally inscribed VeraCode™ beads in a compact fluidic instrument. Genotyping on this platform ranges from 96 to 384 multiplexing, using the same GoldenGate Assay that has proven highly robust for millions of genotypes. In preliminary tests, we have observed greater than 99% call rates, and greater than 99.5% rates for reproducibility and heritability. In a test of 96 SNP genotypes chosen for a study of colorectal cancer, a point mutation in the MSH2 gene, previously implicated in predisposition to several cancers, was correctly genotyped when compared to qPCR analysis of the same samples. Conclusion: Together with genotyping data from reference samples, the GoldenGate Assay on the BeadXpress System has yielded highly reproducible and accurate genotypes, suggesting that this approach will prove useful for rapid refinement of SNPs for development of clinical genotyping tests. No significant financial relationships to disclose.

scholarly journals Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

scholarly journals Phylogeographical Analyses and Antibiotic Resistance Genes of Acinetobacter johnsonii Highlight Its Clinical Relevance

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Santiago Castillo-Ramírez ◽  
Valeria Mateo-Estrada ◽  
Gerardo Gonzalez-Rocha ◽  
Andrés Opazo-Capurro
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Clinical Importance ◽  
Antibiotic Resistance Genes ◽  
Genomic Diversity ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Acinetobacter Johnsonii ◽  
Well Differentiated

ABSTRACT Acinetobacter johnsonii has been severely understudied and its population structure and the presence of antibiotic resistance genes (ARGs) are very much uncertain. Our phylogeographical analysis shows that intercontinental transmission has occurred frequently and that different lineages are circulating within single countries; notably, clinical and nonclinical strains are not well differentiated from one another. Importantly, in this species recombination is a significant source of single nucleotide polymorphisms. Furthermore, our results show this species could be an important reservoir of ARGs since it has a significant amount of ARGs, and many of them show signals of horizontal gene transfer. Thus, this study clearly points out the clinical importance of A. johnsonii and the urgent need to better appreciate its genomic diversity.

scholarly journals Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years

2021 ◽  
Author(s):  
Peter Braun ◽  
Wolfgang Beyer ◽  
Matthias Hanczaruk ◽  
Julia Riehm ◽  
Markus Antwerpen ◽  
...  
Keyword(s):  
Soil Samples ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Environmental Isolates ◽  
Single Nucleotide ◽  
Virulence Plasmids ◽  
Bacterium Bacillus ◽  
Phage Receptor ◽  
Contaminated Area

The zoonotic disease anthrax caused by the endospore-forming bacterium Bacillus anthracis is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009 resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture and the presence of both virulence plasmids (pXO1 and pXO2) were confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA-sequenced and phylogenetically grouped within the B.Br.CNEVA clade that is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate of 2009, separated only by three single nucleotide polymorphisms on the chromosome, one on plasmid pXO2 and three indel-regions. Further, B. anthracis DNA was detected by PCR from soil-samples taken from spots, where the cow had fallen onto the pasture. New tools based on phage receptor binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil-samples. These environmental isolates were genotyped and found to be SNP-identical to BF-1. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The contaminated area was subsequently disinfected with formaldehyde.

scholarly journals Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

2006 ◽  
Vol 72 (2) ◽  
pp. 1295-1301 ◽  
Author(s):  
Daniele Daffonchio ◽  
Noura Raddadi ◽  
Maya Merabishvili ◽  
Ameur Cherif ◽  
Lorenzo Carmagnola ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Restriction Site ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Trna Genes ◽  
Nucleotide Polymorphisms ◽  
Efficient Tool ◽  
Single Nucleotide ◽  
The Relationship ◽  
Different Origin

ABSTRACT Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.

scholarly journals The Arg753Gln Polymorphism of Toll-Like Receptor 2 Has a Lower Occurrence in Patients with Syphilis, Suggesting Its Protective Effect in Czech and Slovak Individuals

2020 ◽  
Vol 89 (1) ◽  
pp. e00503-20
Author(s):  
Linda Grillová ◽  
Jana Musilová ◽  
Klára Janečková ◽  
Petra Pospíšilová ◽  
Ivana Kuklová ◽  
...  
Keyword(s):  
Treponema Pallidum ◽  
Signaling Cascade ◽  
Nucleotide Polymorphisms ◽  
Toll Like Receptor ◽  
Healthy Individuals ◽  
Single Nucleotide ◽  
Content Type ◽  
Tlr2 Polymorphism ◽  
History Of ◽  
Toll Like Receptor 2

ABSTRACTSyphilis is a bacterial infection caused by Treponema pallidum subsp. pallidum. Infection with T. pallidum subsp. pallidum and its dissemination lead to the synthesis of proinflammatory cytokines triggered by the interaction of bacterial lipoproteins with Toll-like receptor 2 (TLR2). TLR2 contains several nonsynonymous single-nucleotide polymorphisms that may impact the activation of its signaling cascade and alter the responsiveness to, or the course of, various infectious diseases, including those caused by pathogenic spirochetes. To investigate whether TLR2 polymorphism may influence susceptibility to syphilis, 221 healthy individuals with no history of syphilis (controls) and 137 patients diagnosed with syphilis (cases) were screened for the presence of the Arg753Gln polymorphism in the TLR2 gene (2258G→A; rs5743708). The Arg753Gln variant occurs at a significantly lower frequency in syphilis patients (4 of 137 [3%]) than in controls (24 of 221 [10.9%]). These data suggest that TLR2 Arg753Gln may protect from the development of syphilis due to reduced signaling.

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