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Article: Rational design of an acidic erythritol (ACER) medium for the enhanced isolation of the environmental pathogen

Assig, Karoline / Lichtenegger, Sabine / Bui, Linh N H / Mosbacher, Bettina / Vu, Anh T N / Erhart, Daniel / Trinh, Trung T / Steinmetz, Ivo

Frontiers in microbiology

2023 Volume 14, Page(s) 1213818

Abstract: ... The soil ... ...

Abstract	The soil bacterium
Language	English
Publishing date	2023-06-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2023.1213818
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Article ; Online: Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance.

Wagner, Gabriel E / Dabernig-Heinz, Johanna / Lipp, Michaela / Cabal, Adriana / Simantzik, Jonathan / Kohl, Matthias / Scheiber, Martina / Lichtenegger, Sabine / Ehricht, Ralf / Leitner, Eva / Ruppitsch, Werner / Steinmetz, Ivo

Journal of clinical microbiology

2023 Volume 61, Issue 4, Page(s) e0163122

Abstract: Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and ... ...

Abstract	Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.
MeSH term(s)	Antigens, Bacterial/genetics ; Bacteria/genetics ; Bacteria/isolation & purification ; Bacteria/pathogenicity ; Bacterial Vaccines/genetics ; Bordetella pertussis/genetics ; Bordetella pertussis/isolation & purification ; Bordetella pertussis/pathogenicity ; Drug Resistance, Bacterial/genetics ; Environmental Monitoring ; Genome, Bacterial ; Genotyping Techniques ; High-Throughput Nucleotide Sequencing/methods ; Multilocus Sequence Typing/methods ; Nanopore Sequencing/methods ; Phylogeny ; Reproducibility of Results ; Virulence Factors/genetics
Chemical Substances	Antigens, Bacterial ; Bacterial Vaccines ; Virulence Factors
Language	English
Publishing date	2023-03-29
Publishing country	United States
Document type	Evaluation Study ; Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/jcm.01631-22
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Article ; Online: Newly detected paediatric melioidosis cases in a single referral children's hospital in Ho Chi Minh City indicate the probable underrecognition of melioidosis in South Vietnam.

Pham, Thai Son / König, Elisabeth / Bui, The Trung / Vu, Thi Ngoc Anh / Nguyen, Tran Nam / Do, Chau Viet / Lichtenegger, Sabine / Bui, Nguyen Hai Linh / Trinh, Huu Tung / Steinmetz, Ivo / Trinh, Thanh Trung

Transactions of the Royal Society of Tropical Medicine and Hygiene

2023 Volume 118, Issue 3, Page(s) 190–198

Abstract: Background: The epidemiology of melioidosis in Vietnam, a disease caused by the soil bacterium Burkholderia pseudomallei, remains unclear. This study aimed to detect paediatric melioidosis in South Vietnam and describe clinical features and the ... ...

Abstract	Background: The epidemiology of melioidosis in Vietnam, a disease caused by the soil bacterium Burkholderia pseudomallei, remains unclear. This study aimed to detect paediatric melioidosis in South Vietnam and describe clinical features and the geographic distribution. Methods: We introduced a simple laboratory algorithm for detecting B. pseudomallei from clinical samples at Children's Hospital 2 in Ho Chi Minh City in July 2015. A retrospective observational study of children <16 y of age with culture-confirmed melioidosis between July 2015 and August 2019 was undertaken. Results: Thirty-five paediatric cases of melioidosis were detected, with cases originating from 13 of 32 provinces and cities in South Vietnam. The number of paediatric melioidosis cases detected from a certain region correlated with the overall number of inpatients originating from the respective geographic area. Suppurative parotitis (n=15 [42.8%]) was the most common clinical presentation, followed by lung infection (n=10 [28.6%]) and septicaemia (n=7 [20%]). Fourteen (40%) children had disseminated disease, including all cases of lung infection, four cases with central nervous system symptoms and four (11.4%) deaths. Conclusions: The patients' origin indicates a wide distribution of melioidosis in South Vietnam. It seems probable that cases not only in children, but also in adults, remain grossly undiagnosed. Further awareness raising and laboratory capacity strengthening are needed in this part of the country.
MeSH term(s)	Adult ; Child ; Humans ; Burkholderia pseudomallei ; Cities ; Hospitals ; Melioidosis/diagnosis ; Melioidosis/epidemiology ; Melioidosis/microbiology ; Referral and Consultation ; Vietnam/epidemiology ; Retrospective Studies
Language	English
Publishing date	2023-11-23
Publishing country	England
Document type	Observational Study ; Journal Article
ZDB-ID	441375-1
ISSN	1878-3503 ; 0035-9203
ISSN (online)	1878-3503
ISSN	0035-9203
DOI	10.1093/trstmh/trad080
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Article: A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants

Wagner, Gabriel E. / Totaro, Massimo G. / Volland, André / Lipp, Michaela / Saiger, Sabine / Lichtenegger, Sabine / Forstner, Patrick / von Laer, Dorothee / Oberdorfer, Gustav / Steinmetz, Ivo

Viruses. 2021 Dec. 19, v. 13, no. 12

2021

Abstract: Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics ... ...

Abstract	Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated “S-Protein-Typer” tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad Cₜ range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12–13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.
Keywords	Severe acute respiratory syndrome coronavirus 2 ; amino acids ; automation ; bioinformatics ; monitoring ; nanopores ; pathogenicity
Language	English
Dates of publication	2021-1219
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v13122548
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Wagner, Gabriel E / Berner, Andreas / Lipp, Michaela / Kohler, Christian / Assig, Karoline / Lichtenegger, Sabine / Saqib, Muhammad / Müller, Elke / Trinh, Trung T / Gad, Anne-Marie / Söffing, Hans Hermann / Ehricht, Ralf / Laroucau, Karine / Steinmetz, Ivo

Journal of clinical microbiology

2022 Volume 61, Issue 1, Page(s) e0123422

Abstract: Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of ... ...

Abstract	Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (
MeSH term(s)	Humans ; Horses ; Animals ; Glanders/diagnosis ; Melioidosis/diagnosis ; Melioidosis/veterinary ; Protein Array Analysis ; Burkholderia mallei/genetics ; Burkholderia pseudomallei
Language	English
Publishing date	2022-12-21
Publishing country	United States
Document type	Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/jcm.01234-22
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Article ; Online: Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout.

Lichtenegger, Sabine / Saiger, Sabine / Hardt, Melina / Kulnik, Susanne / Wagner, Gabriel E / Kleinhappl, Barbara / Assig, Karoline / Zauner, Andrea / Ober, Michelle / Kimpel, Janine / von Laer, Dorothee / Zatloukal, Kurt / Steinmetz, Ivo

Journal of clinical microbiology

2022 Volume 60, Issue 7, Page(s) e0037622

Abstract: Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated ... ...

Abstract	Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (r
MeSH term(s)	Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19/diagnosis ; Humans ; Neutralization Tests/methods ; Pandemics ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2022-06-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/jcm.00376-22
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Article ; Online: Serum resistance and phase variation of a nasopharyngeal non-typeable Haemophilus influenzae isolate.

Lichtenegger, Sabine / Bina, Isabelle / Durakovic, Sanel / Glaser, Philippe / Tutz, Sarah / Schild, Stefan / Reidl, Joachim

International journal of medical microbiology : IJMM

2017 Volume 307, Issue 2, Page(s) 139–146

Abstract: Haemophilus influenzae harbours a complex array of factors to resist human complement attack. As non-typeable H. influenzae (NTHi) strains do not possess a capsule, their serum resistance mainly depends on other mechanisms including LOS decoration. In ... ...

Abstract	Haemophilus influenzae harbours a complex array of factors to resist human complement attack. As non-typeable H. influenzae (NTHi) strains do not possess a capsule, their serum resistance mainly depends on other mechanisms including LOS decoration. In this report, we describe the identification of a highly serum resistant, nasopharyngeal isolate (NTHi23) by screening a collection of 77 clinical isolates. For NTHi23, we defined the MLST sequence type 1133, which matches the profile of a previously published invasive NTHi isolate. A detailed genetic analysis revealed that NTHi23 shares several complement evading mechanisms with invasive disease isolates. These mechanisms include the functional expression of a retrograde phospholipid trafficking system and the presumable decoration of the LOS structure with sialic acid. By screening the NTHi23 population for spontaneous decreased serum resistance, we identified a clone, which was about 10
MeSH term(s)	Adult ; Alveolar Epithelial Cells/microbiology ; Antigenic Variation ; Blood Bactericidal Activity ; Cell Line ; Child ; Endocytosis ; Genotype ; Glycosyltransferases/genetics ; Glycosyltransferases/metabolism ; Haemophilus influenzae/genetics ; Haemophilus influenzae/immunology ; Haemophilus influenzae/isolation & purification ; Haemophilus influenzae/physiology ; Humans ; Immune Evasion ; Multilocus Sequence Typing ; Nasopharynx/microbiology
Chemical Substances	Glycosyltransferases (EC 2.4.-)
Language	English
Publishing date	2017-01-30
Publishing country	Germany
Document type	Journal Article
ZDB-ID	2006518-8
ISSN	1618-0607 ; 1438-4221
ISSN (online)	1618-0607
ISSN	1438-4221
DOI	10.1016/j.ijmm.2017.01.005
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Article ; Online: A Novel High-Throughput Nanopore-Sequencing-Based Strategy for Rapid and Automated S-Protein Typing of SARS-CoV-2 Variants.

Wagner, Gabriel E / Totaro, Massimo G / Volland, André / Lipp, Michaela / Saiger, Sabine / Lichtenegger, Sabine / Forstner, Patrick / von Laer, Dorothee / Oberdorfer, Gustav / Steinmetz, Ivo

Viruses

2021 Volume 13, Issue 12

Abstract	Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated "S-Protein-Typer" tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad C
MeSH term(s)	COVID-19/epidemiology ; COVID-19/virology ; Epidemiological Monitoring ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Immune Evasion/genetics ; Mutation ; Nanopore Sequencing/methods ; SARS-CoV-2/genetics ; SARS-CoV-2/immunology ; Spike Glycoprotein, Coronavirus/genetics
Chemical Substances	Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2021-12-19
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v13122548
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Article ; Online: RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence.

Phenn, Julia / Pané-Farré, Jan / Meukow, Nikolai / Klein, Annelie / Troitzsch, Anne / Tan, Patrick / Fuchs, Stephan / Wagner, Gabriel E / Lichtenegger, Sabine / Steinmetz, Ivo / Kohler, Christian

PLoS pathogens

2021 Volume 17, Issue 5, Page(s) e1009604

Abstract: Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain ... ...

Abstract	Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.
MeSH term(s)	Adaptation, Physiological ; Anaerobiosis ; Animals ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Burkholderia pseudomallei/genetics ; Burkholderia pseudomallei/pathogenicity ; Burkholderia pseudomallei/physiology ; Female ; Gene Expression Regulation, Bacterial ; Hypoxia ; Melioidosis/microbiology ; Mice ; Mice, Inbred BALB C ; Mutation ; Nitrates/metabolism ; Oxidation-Reduction ; Transcriptome ; Virulence
Chemical Substances	Bacterial Proteins ; Nitrates ; RegA protein, Bacteria
Language	English
Publishing date	2021-05-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1009604
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Article ; Online: Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Lichtenegger, Sabine / Trinh, Trung T / Assig, Karoline / Prior, Karola / Harmsen, Dag / Pesl, Julian / Zauner, Andrea / Lipp, Michaela / Que, Tram A / Mutsam, Beatrice / Kleinhappl, Barbara / Steinmetz, Ivo / Wagner, Gabriel E

Journal of clinical microbiology

2021 Volume 59, Issue 8, Page(s) e0009321

Abstract: Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application ... ...

Abstract	Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.
MeSH term(s)	Burkholderia pseudomallei/genetics ; Genome, Bacterial/genetics ; Humans ; Molecular Epidemiology ; Multilocus Sequence Typing ; Whole Genome Sequencing
Language	English
Publishing date	2021-07-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.00093-21
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