Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates.

Abdolrahman Khezri,Ekaterina Avershina,Rafi Ahmad

doi:10.3390/microorganisms9122560

Abdolrahman Khezri, Ekaterina Avershina + Show 1 more

Open Access

https://doi.org/10.3390/microorganisms9122560

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Abstract

Emerging new sequencing technologies have provided researchers with a unique opportunity to study factors related to microbial pathogenicity, such as antimicrobial resistance (AMR) genes and virulence factors. However, the use of whole-genome sequence (WGS) data requires good knowledge of the bioinformatics involved, as well as the necessary techniques. In this study, a total of nine Escherichia coli and Klebsiella pneumoniae isolates from Norwegian clinical samples were sequenced using both MinION and Illumina platforms. Three out of nine samples were sequenced directly from blood culture, and one sample was sequenced from a mixed-blood culture. For genome assembly, several long-read, (Canu, Flye, Unicycler, and Miniasm), short-read (ABySS, Unicycler and SPAdes) and hybrid assemblers (Unicycler, hybridSPAdes, and MaSurCa) were tested. Assembled genomes from the best-performing assemblers (according to quality checks using QUAST and BUSCO) were subjected to downstream analyses. Flye and Unicycler assemblers performed best for the assembly of long and short reads, respectively. For hybrid assembly, Unicycler was the top-performing assembler and produced more circularized and complete genome assemblies. Hybrid assembled genomes performed substantially better in downstream analyses to predict putative plasmids, AMR genes and β-lactamase gene variants, compared to MinION and Illumina assemblies. Thus, hybrid assembly has the potential to reveal factors related to microbial pathogenicity in clinical and mixed samples.

Highlights

The pathogenicity of bacteria is often associated with antimicrobial resistance genes and/or virulence factors
Illumina reads clearly had higher coverage compared to MinION reads for E. coli isolates, whereas for K. pneumoniae, an opposite trend was observed
Coverage of E. coli isolates was calculated by dividing the number of bp in each read over the number of bp in reference genome (E. coli NCTC 13441)

Summary

Introduction

The pathogenicity of bacteria is often associated with antimicrobial resistance genes and/or virulence factors. Antimicrobial resistance (AMR) is the ability of microorganisms to defy antimicrobials, such as antibiotics. Infections due to AMR bacteria are increasing and considered a threat to modern health care [1,2]. During the last two decades, scientific communities have seen a growing trend towards using next-generation sequencing (NGS) technology such as Illumina sequencing to identify AMR genes and virulence factors. NGS provides high depth coverage data, the output reads from. NGS platforms such as Illumina are only about a few hundred base pairs long. Constructing a genome assembly based on the short-reads often results in an incomplete and fragmented assembly, which makes downstream analyses challenging [3]

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