Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region
Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 1...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-08-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fcimb.2019.00278/full |
| _version_ | 1819114859421237248 |
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| author | Maja Kosecka-Strojek Maja Kosecka-Strojek Artur J. Sabat Viktoria Akkerboom Karsten Becker Evert van Zanten Guido Wisselink Jacek Miedzobrodzki Anna M. D. (Mirjam) Kooistra-Smid Anna M. D. (Mirjam) Kooistra-Smid Alexander W. Friedrich |
| author_facet | Maja Kosecka-Strojek Maja Kosecka-Strojek Artur J. Sabat Viktoria Akkerboom Karsten Becker Evert van Zanten Guido Wisselink Jacek Miedzobrodzki Anna M. D. (Mirjam) Kooistra-Smid Anna M. D. (Mirjam) Kooistra-Smid Alexander W. Friedrich |
| author_sort | Maja Kosecka-Strojek |
| collection | DOAJ |
| description | Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens. |
| first_indexed | 2024-12-22T04:52:00Z |
| format | Article |
| id | doaj.art-e8a528c71374492c8c326914d4982594 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-22T04:52:00Z |
| publishDate | 2019-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-e8a528c71374492c8c326914d49825942022-12-21T18:38:28ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882019-08-01910.3389/fcimb.2019.00278464039Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA RegionMaja Kosecka-Strojek0Maja Kosecka-Strojek1Artur J. Sabat2Viktoria Akkerboom3Karsten Becker4Evert van Zanten5Guido Wisselink6Jacek Miedzobrodzki7Anna M. D. (Mirjam) Kooistra-Smid8Anna M. D. (Mirjam) Kooistra-Smid9Alexander W. Friedrich10Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyCerte, Department of Medical Microbiology, Groningen, NetherlandsCerte, Department of Medical Microbiology, Groningen, NetherlandsDepartment of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsCerte, Department of Medical Microbiology, Groningen, NetherlandsDepartment of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, NetherlandsMany members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.https://www.frontiersin.org/article/10.3389/fcimb.2019.00278/fullNGSStaphylococcus16S-23S rDNAdiagnosticsreal-time PCR |
| spellingShingle | Maja Kosecka-Strojek Maja Kosecka-Strojek Artur J. Sabat Viktoria Akkerboom Karsten Becker Evert van Zanten Guido Wisselink Jacek Miedzobrodzki Anna M. D. (Mirjam) Kooistra-Smid Anna M. D. (Mirjam) Kooistra-Smid Alexander W. Friedrich Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region Frontiers in Cellular and Infection Microbiology NGS Staphylococcus 16S-23S rDNA diagnostics real-time PCR |
| title | Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region |
| title_full | Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region |
| title_fullStr | Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region |
| title_full_unstemmed | Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region |
| title_short | Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region |
| title_sort | development and validation of a reference data set for assigning staphylococcus species based on next generation sequencing of the 16s 23s rrna region |
| topic | NGS Staphylococcus 16S-23S rDNA diagnostics real-time PCR |
| url | https://www.frontiersin.org/article/10.3389/fcimb.2019.00278/full |
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