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

ABSTRACT Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in clinical bacterial isolates. 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, we compared different approaches to identify best practices for detection of AMR genes, including: total genomic DNA and plasmid DNA extractions, solo assembly of Illumina short-reads and of 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, purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. We also found that Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility corresponded well with genotypes identified by sequencing, but 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 reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS to expedite patient treatment and to track AMR genes within the hospital and community.