Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer

Ayumu Ohno,Satomi Asai,Kirill Kryukov,Hayato Miyachi,Kazuo Umezawa,Tadashi Imanishi,So Nakagawa

doi:10.1038/s41598-021-82903-z

Abstract

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

Highlights

Since the discovery of penicillin by Alexander Fleming in 1928, numerous infectious diseases have been cured by various antibiotics
Exhibits drug resistance to ampicillin and MDRP exhibits drug resistance to ampicillin and gentamicin[14], and Escherichia coli (E. coli) exhibits sensitivity to ampicillin and gentamicin, but we confirmed the reactivity of antibiotics in actual bacteria
We proposed a new method for rapid identification of drug-resistant bacteria by a combination of 16S metagenome sequencing and the use of propidium monoazide (PMA) and antibiotics, and examined its feasibility

Summary

Introduction

Since the discovery of penicillin by Alexander Fleming in 1928, numerous infectious diseases have been cured by various antibiotics. A rapid method for the identification of pathogens and their antimicrobial spectra is necessary in addition to antimicrobial stewardship, in order to treat individual infected patients properly and to prevent the spread of drug-resistant bacteria, as a control measure of nosocomial infection. To achieve this purpose, we incorporated two kinds of technologies. We previously developed a portable system for rapid 16S rRNA metagenome analysis using the nanopore DNA sequencer MinION and laptop computers[11,12,13] By combining these two technologies, we aimed at establishing a rapid and accurate diagnostic technology that can detect drug-resistant bacteria

Methods

Results

Conclusion