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 CCUG4856^T (=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: Bacteroides fragilis; Oxford Nanopore; antimicrobial resistance; genome sequencing; hybrid assembly; insertion sequences; plasmid.

Fig. 1.

Dot plot matrix of the alignment of the reference assembly and the hybrid Unicycler assembly using Gepard v1.40 [81]. The B. fragilis NCTC9343 (RefSeq accession number GCF_000025985.1) reference assembly derived from Sanger sequencing is on the x-axis and the hybrid Unicycler assembly on the y-axis. On this otherwise near-perfect alignment with high similarity, an 88 045 bp inversion with 100 % ID is observed at nucleotide positions 2 941 962…3 030 006 on the Unicycler assembly (2 005 742…2 093 786 on the reference sequence) (indicated by the blue arrow).

Fig. 2.

Evolution of genome assemblies with added data and manual finishing. The best SPAdes assembly graphs by Unicycler with short reads only are shown on the far left. Supplying ONT reads improved the assemblies overall, but only three were circularized with singular chromosome contigs with data from the initial MinION sequencing runs. Adding additional ONT data and correcting reads with Canu did not improve assemblies for all isolates. Manual finishing was necessary to finish assemblies for three isolates. Assembly graph images generated with Bandage. Read information can be found in Table S1.

Fig. 3.

Linear representation of an alignment of putative circular plasmid sequences pBFO17_1 and pBFS01_1 (reverse complement for better visualization) using EasyFig [82]. EasyFig uses blast to identify sequences of similarity. Sequence similarities of >98 % are indicated by full colouring, a darker colour indicates a higher % ID. Products of annotated CDSs are shown. CDSs annotated as hypothetical or domain of unknown function are coloured white. The two sequences show a very high degree of similarity. pBFO17_1 is 7586 bp longer than pBFS01_1. This is mainly due to the insertion of a reverse transcriptase (pBFO17_1, 11367…13034) (disrupting a DNA methylase), the insertion of prophage (from position 56125 to 61162) (identified as an incomplete prophage using phaster [83] and an IS1380 family-like transposase (67933…69237). The regions pBFO17_1 50711…52501 and pBFS01_1 32248…30304 are not similar. Possibly, the insertion of two transposases in pBFO17_1 have excised most of the ParB-family DNA partitioning protein in the corresponding sequence range in pBFS01_1.