Dataset Information

Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

ABSTRACT: Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10?minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and blaCTX-M subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes.

SUBMITTER: Taxt AM

PROVIDER: S-EPMC7203151 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

Taxt Arne M AM Avershina Ekaterina E Frye Stephan A SA Naseer Umaer U Ahmad Rafi R

Scientific reports 20200506 1

Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnosti ...[more]

PMID: 32376847

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Project description:Infections with multidrug-resistant bacteria often leave limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we demonstrate the application of Nanopore sequencing in a hospital setting for monitoring transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species. In 2009, we experienced an outbreak with extensively multidrug-resistant Pseudomonas aeruginosa harboring the carbapenemase-encoding bla IMP-8 gene. In 2012, the first Citrobacter freundii and Citrobacter cronae strains harboring the same gene were detected. Using Nanopore and Illumina sequencing, we conducted comparative analysis of all bla IMP-8 bacteria isolated in our hospital over a 6-year period (n?=?54). We developed the computational platform plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de novo assembly of genomes and plasmids, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates, and visualization of results. Using plasmIDent, we identified a 40-kb plasmid carrying bla IMP-8 in P. aeruginosa and C. freundii, verifying the plasmid transfer. Within C. freundii, the plasmid underwent further evolution and plasmid fusion, resulting in a 164-kb megaplasmid, which was transferred to C. cronae Multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs. In summary, plasmid transfer, plasmid fusion, and rearrangement of the ARG cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of near-real-time monitoring of plasmid evolution and ARG transfer in clinical settings, enabling successful countermeasures to contain plasmid-mediated outbreaks.IMPORTANCE Infections with multidrug-resistant bacteria represent a major threat to global health. While the spread of multidrug-resistant bacterial clones is frequently studied in the hospital setting, surveillance of the transfer of mobile genetic elements between different bacterial species was difficult until recent advances in sequencing technologies. Nanopore sequencing technology was applied to track antimicrobial gene transfer in a long-term outbreak of multidrug-resistant Pseudomonas aeruginosa, Citrobacter freundii, and Citrobacter cronae in a German hospital over 6?years. We developed a novel computational pipeline, pathoLogic, which enables de novo assembly of genomes and plasmids, antimicrobial resistance gene annotation and visualization, and comparative analysis. Applying this approach, we detected plasmid transfer between different bacterial species as well as plasmid fusion and frequent rearrangements of the antimicrobial resistance gene cassette. This study demonstrated the feasibility of near-real-time tracking of plasmid-based antimicrobial resistance gene transfer in hospitals, enabling countermeasures to contain plasmid-mediated outbreaks.

Dataset Information

Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

Publications

Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing.

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