Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids

Thomas V Sydenham*, Søren Overballe-Petersen, Henrik Hasman, Hannah Wexler, Michael Kemp, Ulrik S Justesen

*Kontaktforfatter for dette arbejde

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Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.

OriginalsprogEngelsk
Artikelnummer000312
TidsskriftMicrobial Genomics
Vol/bind5
Udgave nummer11
Antal sider18
ISSN2057-5858
DOI
StatusUdgivet - nov. 2019

Fingeraftryk

Metagenomics
Microbiota
Databases
Research
Gastrointestinal Microbiome
Lead

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title = "Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids",
abstract = "Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.",
keywords = "Antimicrobial resistance, Bacteroides fragilis, Genome sequencing, Hybrid assembly, Insertion sequences, Oxford Nanopore, Plasmid",
author = "Sydenham, {Thomas V} and S{\o}ren Overballe-Petersen and Henrik Hasman and Hannah Wexler and Michael Kemp and Justesen, {Ulrik S}",
year = "2019",
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doi = "10.1099/mgen.0.000312",
language = "English",
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Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids. / Sydenham, Thomas V; Overballe-Petersen, Søren; Hasman, Henrik; Wexler, Hannah; Kemp, Michael; Justesen, Ulrik S.

I: Microbial Genomics, Bind 5, Nr. 11, 000312, 11.2019.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids

AU - Sydenham, Thomas V

AU - Overballe-Petersen, Søren

AU - Hasman, Henrik

AU - Wexler, Hannah

AU - Kemp, Michael

AU - Justesen, Ulrik S

PY - 2019/11

Y1 - 2019/11

N2 - Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.

AB - Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.

KW - Antimicrobial resistance

KW - Bacteroides fragilis

KW - Genome sequencing

KW - Hybrid assembly

KW - Insertion sequences

KW - Oxford Nanopore

KW - Plasmid

U2 - 10.1099/mgen.0.000312

DO - 10.1099/mgen.0.000312

M3 - Journal article

C2 - 31697231

VL - 5

JO - Microbial Genomics

JF - Microbial Genomics

SN - 2057-5858

IS - 11

M1 - 000312

ER -