Abstract
Mutation acquisition is a major mechanism of bacterial antibiotic resistance that remains insufficiently characterised. Here we present RM-seq, a new amplicon-based deep sequencing workflow based on a molecular barcoding technique adapted from Low Error Amplicon sequencing (LEA-seq). RM-seq allows detection and functional assessment of mutational resistance at high throughput from mixed bacterial populations. The sensitive detection of very low-frequency resistant sub-populations permits characterisation of antibiotic-linked mutational repertoires in vitro and detection of rare resistant populations during infections. Accurate quantification of resistance mutations enables phenotypic screening of mutations conferring pleiotropic phenotypes such as in vivo persistence, collateral sensitivity or cross-resistance. RM-seq will facilitate comprehensive detection, characterisation and surveillance of resistant bacterial populations (https://github.com/rguerillot/RM-seq).
Highlights
Antimicrobial resistance is on the rise and is responsible for millions of deaths every year [1]
We developed an innovative workflow called resistance mutation sequencing (RM-seq) that enables the unbiased quantification of resistance alleles from complex in vitro-derived resistant clone libraries, selectable under any experimental condition, allowing identification and characterisation of mutational resistance and its consequences
The RM-seq workflow RM-seq is an amplicon-based, deep sequencing technique founded on the single molecule barcoding method [42]
Summary
Antimicrobial resistance is on the rise and is responsible for millions of deaths every year [1]. Bacterial populations consistently and rapidly overcome the challenge imposed by the use of a new antibiotic. Their remarkable ability to quickly develop resistance is due to their capacity to exchange genes and to their high mutation supply rate. Genomics has become a powerful tool to understand, combat and control the rise of resistance [2, 3]. A precise definition of resistance at the genomic level is crucial to enable fast, culture-independent DST by high-throughput sequencing in the clinical context and to track and fight the spread and persistence of resistant clones globally [3, 4]
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