A multiplex molecular assay for detection of six penA codons to predict decreased susceptibility to cephalosporins in Neisseria gonorrhoeae

2022 ◽  
Author(s):  
Yamei Li ◽  
Lulu Zhang ◽  
Leshan Xiu ◽  
Di Wang ◽  
Yaling Zeng ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
High Throughput ◽  
High Resolution Melting ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rapid Testing ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
Resistant Strains

The emerging cephalosporin-resistant Neisseria gonorrhoeae poses an urgent threat to the continued efficacy of the last-line monotherapy for gonorrhea. Consequently, high-throughput, accurate, and reasonable molecular assays are urgently needed for strengthening antimicrobial-resistance surveillance in N. gonorrhoeae . In this study, we designed a high-throughput multiplex method that incorporates high-resolution melting technology and is based on a 6-codon assay (among the most parsimonious assays) developed following comprehensive and systematic reviews. The results showed that our method can precisely distinguish specific single-nucleotide polymorphisms in resistance-associated genes with a specificity and sensitivity of 100% and a detection limit as low as 10 copies per reaction. This method can be directly applied to clinical samples without cumbersome culture and successfully predicted all cephalosporin-resistant isolates (sensitivity: 100%). The method presented here represents a technique for rapid testing of antimicrobial resistance and will serve as a valuable tool for tailor-made antimicrobial therapy and for monitoring the transmission of cephalosporin-resistant strains.

A multiplex assay for characterization of antimicrobial resistance in Neisseria gonorrhoeae using multi-PCR coupled with mass spectrometry

2020 ◽  
Vol 75 (10) ◽  
pp. 2817-2825
Author(s):  
Yamei Li ◽  
Leshan Xiu ◽  
Jingwei Liu ◽  
Chi Zhang ◽  
Feng Wang ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
High Throughput ◽  
Antimicrobial Agents ◽  
Multiplex Assay ◽  
Clinical Samples ◽  
High Throughput Method ◽  
Powerful Means ◽  
Resistant Strains

Abstract Background Complicated mechanisms and variable determinants related to drug resistance pose a major challenge to obtain comprehensive antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae. Meanwhile, cephalosporin-resistant mosaic penA alleles have been reported worldwide. Therefore, it is urgent to monitor the expansion of cephalosporin-resistant mosaic penA alleles. Objectives To develop a comprehensive high-throughput method to efficiently screen AMR determinants. Methods We developed a method based on multiplex PCR with MALDI-TOF MS, which can simultaneously screen for 24 mutations associated with multiple antimicrobial agents in 19 gonococcal AMR loci (NG-AMR-MS). The performance of the NG-AMR-MS method was assessed by testing 454 N. gonorrhoeae isolates with known MICs of six antibiotics, eight non-gonococcal Neisseria strains, 214 clinical samples and three plasmids with a confirmed mosaic penA allele. Results The results show that NG-AMR-MS had a specificity of 100% with a sensitivity as low as 10 copies per reaction (except for PorB A121D/N/G, 100 copies per reaction). For clinical samples with gonococcal load >5 copies/μL, the method can accurately identify 20 AMR mutations. In addition, the method successfully detected specific cephalosporin-resistant strains with the A311V mutation in the penA allele. Conclusions Our high-throughput method can provide comprehensive AMR profiles within a multiplex format. Furthermore, the method can be directly applied to screening for AMR among clinical samples, serving as an effective tool for overall monitoring of N. gonorrhoeae AMR and also provides a powerful means to comprehensively improve the level of monitoring.

scholarly journals ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads

2017 ◽  
Author(s):  
Martin Hunt ◽  
Alison E. Mather ◽  
Leonor Sánchez-Busó ◽  
Andrew J. Page ◽  
Julian Parkhill ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Single Nucleotide Polymorphisms ◽  
High Throughput ◽  
Bacterial Isolate ◽  
Animal Health ◽  
Gram Negative Bacteria ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Gram Negative

AbstractAntimicrobial resistance (AMR) is one of the major threats to human and animal health worldwide, yet few high-throughput tools exist to analyse and predict the resistance of a bacterial isolate from sequencing data. Here we present a new tool, ARIBA, that identifies AMR-associated genes and single nucleotide polymorphisms directly from short reads, and generates detailed and customisable output. The accuracy and advantages of ARIBA over other tools are demonstrated on three datasets from Gram-positive and Gram-negative bacteria, with ARIBA outperforming existing methods. ARIBA is available at https://github.com/sanger-pathogens/ariba.

scholarly journals Emergence of Resistance Mutations in Salmonella enterica Serovar Typhi Against Fluoroquinolones

2017 ◽  
Vol 4 (4) ◽  
Author(s):  
Takashi Matono ◽  
Masatomo Morita ◽  
Koji Yahara ◽  
Ken-ichi Lee ◽  
Hidemasa Izumiya ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Evolutionary Process ◽  
Resistance Mechanisms ◽  
Single Mutation ◽  
Nucleotide Polymorphisms ◽  
Resistance Mutations ◽  
Single Nucleotide ◽  
Salmonella Enterica Serovar Typhi ◽  
Resistant Strains ◽  
Emergence Of Resistance

Abstract Background Little is known about the evolutionary process and emergence time of resistance mutations to fluoroquinolone in Salmonella enterica serovar Typhi. Methods We analyzed S. Typhi isolates collected from returned travelers between 2001 and 2016. Based on ciprofloxacin susceptibility, isolates were categorized as highly resistant (minimum inhibitory concentration [MIC] ≥ 4 μg/mL [CIPHR]), resistant (MIC = 1–2 μg/mL [CIPR]), intermediate susceptible (MIC = 0.12–0.5 μg/mL [CIPI]), and susceptible (MIC ≤ 0.06 μg/mL [CIPS]). Results A total of 107 isolates (33 CIPHR, 14 CIPR, 30 CIPI, and 30 CIPS) were analyzed by whole-genome sequencing; 2461 single nucleotide polymorphisms (SNPs) were identified. CIPS had no mutations in the gyrA or parC genes, while each CIPI had 1 of 3 single mutations in gyrA (encoding Ser83Phe [63.3%], Ser83Tyr [33.3%], or Asp87Asn [3.3%]). CIPHR had the same 3 mutations: 2 SNPs in gyrA (encoding Ser83Phe and Asp87Asn) and a third in parC (encoding Ser80Ile). CIPHR shared a common ancestor with CIPR and CIPI isolates harboring a single mutation in gyrA encoding Ser83Phe, suggesting that CIPHR emerged 16 to 23 years ago. Conclusions Three SNPs—2 in gyrA and 1 in parC—are present in S. Typhi strains highly resistant to fluoroquinolone, which were found to have evolved in 1993–2000, approximately 10 years after the beginning of the ciprofloxacin era. Highly resistant strains with survival advantages arose from strains harboring a single mutation in gyrA encoding Ser83Phe. Judicious use of fluoroquinolones is warranted to prevent acceleration of such resistance mechanisms in the future.

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 Investigation of Genetic Relationships within Miscanthus using SNP Markers Identified using SLAF-Seq

2021 ◽  
Author(s):  
ZHIYONG Chen ◽  
Yancen He ◽  
Yasir Iqbal ◽  
Yanlan Shi ◽  
Hongmei Huang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Genetic Relationships ◽  
Female Parent ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Breeding Programs ◽  
Sequencing Method ◽  
Specific Locus

Abstract Background: Miscanthus, which is a leading dedicated-energy grass in Europe and in parts of Asia, is expected to play a key role in the development of the future bioeconomy. However, due to its complex genetic background, it is difficult to investigate phylogenetic relationships and the evolution of gene function in this genus. Here, we investigated 50 Miscanthus germplasms: 1 female parent (M. lutarioriparius), 30 candidate male parents (M. lutarioriparius, M. sinensis, and M. sacchariflorus), and 19 offspring. We used high-throughput Specific-Locus Amplified Fragment sequencing (SLAF-seq) to identify informative single nucleotide polymorphisms (SNPs) in all germplasms.Results: We identified 800,081 SLAF tags, of which 160,368 were polymorphic. Each tag was 264–364 bp long. The obtained SNPs were used to investigate genetic relationships within Miscanthus. We constructed a phylogenetic tree of the 50 germplasms using the obtained SNPs, and found that the germplasms fell into two clades: one clade of M. sinensis only and one clade that included the offspring, M. lutarioriparius, and M. sacchariflorus. Genetic cluster analysis indicated that M. lutarioriparius germplasm C3 was the most likely male parent of the offspring.Conclusions: As a high-throughput sequencing method, SLAF-seq can be used to identify informative SNPs in Miscanthus germplasms and to rapidly characterize genetic relationships within this genus. Our results will support the development of breeding programs utilizing Miscanthus cultivars with elite biomass- or fiber-production potential.

scholarly journals Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection

2021 ◽  
Author(s):  
Leland B Hyman ◽  
Clare R Christopher ◽  
Philip A Romero
Keyword(s):  
Point Of Care ◽  
High Specificity ◽  
Common Source ◽  
Lamp Method ◽  
Universal Method ◽  
Nucleotide Polymorphisms ◽  
One Pot ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
And Shifts

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive "sink" strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.

scholarly journals Inheritance Pattern and Molecular Markers for Resistance to Blackleg Disease in Cabbage

Plants ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 583
Author(s):  
Mostari Jahan Ferdous ◽  
Mohammad Rashed Hossain ◽  
Jong-In Park ◽  
Arif Hasan Khan Robin ◽  
Denison Michael Immanuel Jesse ◽  
...  
Keyword(s):  
Molecular Markers ◽  
High Resolution Melting ◽  
Leptosphaeria Maculans ◽  
Inbred Lines ◽  
Indel Marker ◽  
Detection Accuracy ◽  
Nucleotide Polymorphisms ◽  
Blackleg Disease ◽  
Marker Assisted Breeding ◽  
Single Nucleotide

The inheritance and causal loci for resistance to blackleg, a devastating disease of Brassicaceous crops, are yet to be known in cabbage (Brassica oleracea L.). Here, we report the pattern of inheritance and linked molecular marker for this trait. A segregating BC1 population consisting of 253 plants was raised from resistant and susceptible parents, L29 (♀) and L16 (♂), respectively. Cotyledon resistance bioassay of BC1 population, measured based on a scale of 0–9 at 12 days after inoculation with Leptosphaeria maculans isolate 03–02 s, revealed the segregation of resistance and ratio, indicative of dominant monogenic control of the trait. Investigation of potential polymorphism in the previously identified differentially expressed genes within the collinear region of ‘B. napus blackleg resistant loci Rlm1′ in B. oleracea identified two insertion/deletion (InDel) mutations in the intron and numerous single nucleotide polymorphisms (SNPs) throughout the LRR-RLK gene Bol040029, of which six SNPs in the first exon caused the loss of two LRR domains in the susceptible line. An InDel marker, BLR-C-InDel based on the InDel mutations, and a high resolution melting (HRM) marker, BLR-C-2808 based on the SNP C2808T in the second exon were developed, which predicated the resistance status of the BC1 population with 80.24%, and of 24 commercial inbred lines with 100% detection accuracy. This is the first report of inheritance and molecular markers linked with blackleg resistance in cabbage. This study will enhance our understanding of the trait, and will be helpful in marker assisted breeding aiming at developing resistant cabbage varieties.

scholarly journals Genomic analysis and antimicrobial resistance of Neisseria gonorrhoeae isolates from Vietnam in 2011 and 2015–16

2020 ◽  
Vol 75 (6) ◽  
pp. 1432-1438 ◽  
Author(s):  
Pham Thi Lan ◽  
Daniel Golparian ◽  
Johan Ringlander ◽  
Le Van Hung ◽  
Nguyen Van Thuong ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Reproductive Health ◽  
Neisseria Gonorrhoeae ◽  
Molecular Epidemiology ◽  
Genomic Analysis ◽  
Illumina Miseq ◽  
Phylogenomic Analysis ◽  
Asian Countries ◽  
Resistant Strains ◽  
A Minor

Abstract Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae, compromising gonorrhoea treatment, is a threat to reproductive health globally. South-East and East Asia have been major sources of emergence and subsequent international spread of AMR gonococcal strains during recent decades. We investigated gonococcal isolates from 2011 and 2015–16 in Vietnam using AMR testing, WGS and detection of AMR determinants. Methods Two hundred and twenty-nine gonococcal isolates cultured in 2015–16 (n = 121) and 2011 (n = 108) in Vietnam were examined. AMR testing was performed using Etest and WGS with Illumina MiSeq. Results Resistance among the 2015–16 isolates was as follows: ciprofloxacin, 100%; tetracycline, 79%; benzylpenicillin, 50%; cefixime, 15%; ceftriaxone, 1%; spectinomycin, 0%; and 5% were non-WT to azithromycin. Eighteen (15%) isolates were MDR. The MIC range for gentamicin was 2–8 mg/L. Among the 2015–16 isolates, 27% (n = 33) contained a mosaic penA allele, while no isolates had a mosaic penA allele in 2011. Phylogenomic analysis revealed introduction after 2011 of two mosaic penA-containing clones (penA-10.001 and penA-34.001), which were related to cefixime-resistant strains spreading in Japan and Europe, and a minor clade (eight isolates) relatively similar to the XDR strain WHO Q. Conclusions From 2011 to 2015–16, resistance in gonococci from Vietnam increased to all currently and previously used antimicrobials except ceftriaxone, spectinomycin and tetracycline. Two mosaic penA-containing clones were introduced after 2011, explaining the increased cefixime resistance. Significantly increased AMR surveillance, antimicrobial stewardship and use of WGS for molecular epidemiology and AMR prediction for gonococcal isolates in Vietnam and other Asian countries are crucial.

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