Rapid Optimization of Antibiotic Therapy for Multidrug-Resistant Gram-Negative Infections Using Nanopore Whole Genome Sequencing

Background: Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. We sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole genome sequencing in predicting AST results. Methods: Using a cohort of 40 clinical Klebsiella pneumoniae isolates, three separate sequencing and analysis pipelines were performed: (1) a real-time Nanopore analysis approach that identified acquired AMR genes, (2) an assembly-based Nanopore approach that identified acquired AMR genes and chromosomal mutations, and (3) a Pilon-corrected Nanopore-Illumina hybrid approach. The Pilon-corrected assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. Findings: With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 hours of subcultured isolates. Assemblies sufficient for full resistance gene and single nucleotide polymorphism annotation were available within 14 hours from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 75% (range 30-95%) and 91% (range 80-98%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could substantially shorten the median time to effective antibiotic therapy by 20 hours and 26 hours, respectively, compared to standard AST. Interpretations: Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms as well as to predict AST results. Bioinformatics improvements enabling real-time alignment coupled with rapid extraction and library preparation will further enhance the accuracy and workflow of the Nanopore real-time approach. Funding Statement: The work was supported by funding from the National Institutes of Health K23-AI127935 awarded to PDT, a Canadian Institutes of Health Research fellowship awarded to YF, an R01-HG009190 awarded to WT, an R01-HG006677 awarded to MCS, and an R21-AI130608 awarded to PJS. Declaration of Interests: PDT has received research funding from Merck, outside of the submitted work. KCC has received research funding from GenMark, Inc. and Singulex outside of the submitted work. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. WT, YF, PJS have received travel funds from Oxford Nanopore Technologies. PJS has received grants and personal fees from Accelerate Diagnostics, grants from BD Diagnostics, Inc., grants from bioMerieux, Inc., grants from Check-Points Diagnostics, BV, grants from Hardy Diagnostics, personal fees from Roche Diagnostics, personal fees from Opgen Inc, outside the submitted work. All other authors declare no competing interests. Ethics Approval Statement: The Johns Hopkins University School of Medicine Institutional Review Board approved this study, with a waiver of informed consent.