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Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA
Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. De...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598756/ https://www.ncbi.nlm.nih.gov/pubmed/36290058 http://dx.doi.org/10.3390/antibiotics11101400 |
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author | Maboni, Grazieli Baptista, Rodrigo de Paula Wireman, Joy Framst, Isaac Summers, Anne O. Sanchez, Susan |
author_facet | Maboni, Grazieli Baptista, Rodrigo de Paula Wireman, Joy Framst, Isaac Summers, Anne O. Sanchez, Susan |
author_sort | Maboni, Grazieli |
collection | PubMed |
description | Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community. |
format | Online Article Text |
id | pubmed-9598756 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95987562022-10-27 Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA Maboni, Grazieli Baptista, Rodrigo de Paula Wireman, Joy Framst, Isaac Summers, Anne O. Sanchez, Susan Antibiotics (Basel) Article Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community. MDPI 2022-10-12 /pmc/articles/PMC9598756/ /pubmed/36290058 http://dx.doi.org/10.3390/antibiotics11101400 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Maboni, Grazieli Baptista, Rodrigo de Paula Wireman, Joy Framst, Isaac Summers, Anne O. Sanchez, Susan Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title | Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title_full | Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title_fullStr | Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title_full_unstemmed | Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title_short | Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA |
title_sort | three distinct annotation platforms differ in detection of antimicrobial resistance genes in long-read, short-read, and hybrid sequences derived from total genomic dna or from purified plasmid dna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598756/ https://www.ncbi.nlm.nih.gov/pubmed/36290058 http://dx.doi.org/10.3390/antibiotics11101400 |
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