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

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

doi:10.1099/mgen.0.000312

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.

Highlights

Bacteroides fragilis is a Gram-negative anaerobic bacterium that is commensal to the human gut but can act as an opportunistic pathogen; it is the most commonly isolated anaerobic bacteria from non-faecal clinical samples (1)
Based on DNA-DNA hybridisation studies, B. fragilis can be divided into two DNA homology groups, whose ribosomal contents are so different that the two divisions can be distinguished by mass spectrometry routinely used to identify isolates in clinical laboratories (11)
Based on initial test assemblies using Unicycler without filtering or Canu correction it was concluded that data from the first Oxford Nanopore Technologies (ONT) sequencing runs were to be supplemented by additional runs to increase the chance of complete assembly of all isolates

Summary

Introduction

Bacteroides fragilis is a Gram-negative anaerobic bacterium that is commensal to the human gut but can act as an opportunistic pathogen; it is the most commonly isolated anaerobic bacteria from non-faecal clinical samples (1). Antimicrobial susceptibility testing of anaerobes using agar dilution or gradient strip methods can be costly and labour intensive and despite efforts to validate disk diffusion as a less expensive option, turn-around time will still be least 18 hours and validation for individual species will be required (4). Antimicrobial resistance prediction from bacterial whole genome sequences, from cultured isolates as well as metagenomes, could be implemented in clinical microbiology in the near future, with the potential for improved sample-to-report turnover time and possibly eliminating the need for phenotypical testing for individual species (5–8). Based on DNA-DNA hybridisation studies, B. fragilis can be divided into two DNA homology groups (division I and II), whose ribosomal contents are so different that the two divisions can be distinguished by mass spectrometry routinely used to identify isolates in clinical laboratories (11)

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