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 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.

scholarly journals BacillOndex: An Integrated Data Resource for Systems and Synthetic Biology

2013 ◽  
Vol 10 (2) ◽  
pp. 103-116 ◽  
Author(s):  
Goksel Misirli ◽  
Anil Wipat ◽  
Joseph Mullen ◽  
Katherine James ◽  
Matthew Pocock ◽  
...  
Keyword(s):  
Laboratory Strain ◽  
Nucleotide Polymorphisms ◽  
Genetic Circuits ◽  
Positive Bacterium ◽  
Single Nucleotide ◽  
Genetic Traits ◽  
Data Resource ◽  
Data Integration System ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

Summary BacillOndex is an extension of the Ondex data integration system, providing a semantically annotated, integrated knowledge base for the model Gram-positive bacterium Bacillus subtilis. This application allows a user to mine a variety of B. subtilis data sources, and analyse the resulting integrated dataset, which contains data about genes, gene products and their interactions. The data can be analysed either manually, by browsing using Ondex, or computationally via a Web services interface. We describe the process of creating a BacillOndex instance, and describe the use of the system for the analysis of single nucleotide polymorphisms in B. subtilis Marburg. The Marburg strain is the progenitor of the widely-used laboratory strain B. subtilis 168. We identified 27 SNPs with predictable phenotypic effects, including genetic traits for known phenotypes. We conclude that BacillOndex is a valuable tool for the systems-level investigation of, and hypothesis generation about, this important biotechnology workhorse. Such understanding contributes to our ability to construct synthetic genetic circuits in this organism.

scholarly journals Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1164
Author(s):  
Shona C. Moore ◽  
Rebekah Penrice-Randal ◽  
Muhannad Alruwaili ◽  
Nadine Randle ◽  
Stuart Armstrong ◽  
...  
Keyword(s):  
Viral Genome ◽  
False Negative ◽  
Point Mutations ◽  
Read Length ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Number Of Patients ◽  
Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

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 Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

2009 ◽  
Vol 75 (17) ◽  
pp. 5667-5675 ◽  
Author(s):  
Michael K�ser ◽  
Julia Hauser ◽  
Pamela Small ◽  
Gerd Pluschke
Keyword(s):  
Buruli Ulcer ◽  
Etiologic Agent ◽  
Mycobacterium Ulcerans ◽  
Nucleotide Polymorphisms ◽  
Differentiation Markers ◽  
Single Nucleotide ◽  
Virulence Plasmids ◽  
Sequence Polymorphisms ◽  
Genetic Features ◽  
Relationship Of

ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

scholarly journals It Is Not All about Single Nucleotide Polymorphisms: Comparison of Mobile Genetic Elements and Deletions in Listeria monocytogenes Genomes Links Cases of Hospital-Acquired Listeriosis to the Environmental Source

2015 ◽  
Vol 53 (11) ◽  
pp. 3492-3500 ◽  
Author(s):  
Qinning Wang ◽  
Nadine Holmes ◽  
Elena Martinez ◽  
Peter Howard ◽  
Grant Hill-Cawthorne ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Snp Analysis ◽  
Nucleotide Polymorphisms ◽  
Environmental Isolates ◽  
Single Nucleotide ◽  
Content Type ◽  
Serotype 1 ◽  
Hospital Acquired

The control of food-borne outbreaks caused byListeria monocytogenesin humans relies on the timely identification of food or environmental sources and the differentiation of outbreak-related isolates from unrelated ones. This study illustrates the utility of whole-genome sequencing for examining the link between clinical and environmental isolates ofL. monocytogenesassociated with an outbreak of hospital-acquired listeriosis in Sydney, Australia. Comparative genomic analysis confirmed an epidemiological link between the three clinical and two environmental isolates. Single nucleotide polymorphism (SNP) analysis showed that only two SNPs separated the three human outbreak isolates, which differed by 19 to 20 SNPs from the environmental isolates and 71 to >10,000 SNPs from sporadicL. monocytogenesisolates. The chromosomes of all human outbreak isolates and the two suspected environmental isolates were syntenic. In contrast to the genomes of background sporadic isolates, all epidemiologically linked isolates contained two novel prophages and a previously unreported clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) locus subtype sequence. The mobile genetic element (MGE) profile of these isolates was distinct from that of the other serotype 1/2b reference strains and sporadic isolates. The identification of SNPs and clonally distinctive MGEs strengthened evidence to distinguish outbreak-related isolates ofL. monocytogenesfrom cocirculating endemic strains.

scholarly journals Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3261-3269 ◽  
Author(s):  
Alessandra Bragonzi ◽  
Lutz Wiehlmann ◽  
Jens Klockgether ◽  
Nina Cramer ◽  
Dieter Worlitzsch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
De Novo ◽  
Nucleotide Polymorphisms ◽  
Aquatic Habitats ◽  
Environmental Isolates ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Airway Colonization

The mucA gene of the muc operon, which is instrumental in the control of the biosynthesis of the exopolysaccharide alginate, is a hotspot of mutation in Pseudomonas aeruginosa, a micro-organism that chronically colonizes the airways of individuals with cystic fibrosis (CF). The mucA, mucB and mucD genes were sequenced in nine environmental isolates from aquatic habitats, and in 37 P. aeruginosa strains isolated from 10 patients with CF, at onset or at a late stage of chronic airway colonization, in order to elucidate whether there was any association between mutation and background genotype. The 61 identified single nucleotide polymorphisms (SNPs) segregated into 18 mucABD genotypes. Acquired and de novo stop mucA mutations were present in 14 isolates (38 %) of five mucABD genotypes. ΔG430 was the most frequent and recurrent mucA mutation detected in four genotypes. The classification of strains by mucABD genotype was generally concordant with that by genome-wide SpeI fragment pattern or multilocus SNP genotypes. The exceptions point to intragenic mosaicism and interclonal recombination as major forces for intraclonal evolution at the mucABD locus.

scholarly journals Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay

2021 ◽  
Vol 70 (3) ◽  
Author(s):  
Kaveesha Bodiyabadu ◽  
Jennifer Danielewski ◽  
Suzanne M. Garland ◽  
Dorothy A. Machalek ◽  
Catriona S. Bradshaw ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Predictive Value ◽  
Sanger Sequencing ◽  
Mycoplasma Genitalium ◽  
Amino Acid Position ◽  
Fluoroquinolone Resistance ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type

Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated. Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H). Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated. Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance. Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

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