# A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

## TL;DR

A new method called s5PSeq quickly detects antibiotic resistance by measuring how antibiotics affect bacterial ribosomes, without needing to grow bacteria in the lab.

## Contribution

s5PSeq is a streamlined, nanopore-compatible protocol that enables rapid phenotypic antimicrobial sensitivity testing by profiling 5′P mRNA degradation intermediates.

## Key findings

- s5PSeq reduces library preparation time to under 4 hours and uses a novel rRNA blocking strategy.
- It identifies antibiotic resistance in Clostridioides difficile isolates using as few as 3,000 nanopore sequencing reads.
- The method provides a molecular phenotypic readout of antimicrobial susceptibility without culturing bacteria.

## Abstract

Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present “simplified 5PSeq” (s5PSeq), a streamlined protocol for profiling 5′ monophosphorylated (5′P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition—offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under 4 h and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3,000 reads. In addition to simplifying the study of 5′P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics.

•s5PSeq is a streamlined protocol for profiling 5′P mRNA degradation intermediates•Context-specific ribosome stalls can assess phenotypic antimicrobial sensitivity•Blocking rRNA sequencing at the ligation step streamlines library preparation•Nanopore sequencing allows same-day AST in species with 5′-3′ exonuclease

s5PSeq is a streamlined protocol for profiling 5′P mRNA degradation intermediates

Context-specific ribosome stalls can assess phenotypic antimicrobial sensitivity

Blocking rRNA sequencing at the ligation step streamlines library preparation

Nanopore sequencing allows same-day AST in species with 5′-3′ exonuclease

Current phenotypic antimicrobial susceptibility testing typically requires many hours of culture to detect growth inhibition, delaying timely and effective treatment decisions. Although molecular diagnostics can be faster, they often depend on known resistance genes and may not accurately reflect true phenotypic response to antibiotics. A method that rapidly captures the functional effect of antibiotic exposure would address a major gap in current clinical diagnostics. s5PSeq was developed to meet this challenge by measuring antibiotic-induced ribosome stalling as an immediate molecular indicator of susceptibility, enabling a rapid phenotypic susceptibility readout within a streamlined 4-h workflow.

Liu et al. present s5PSeq, a rapid 4-h sequencing workflow that detects antibiotic-induced ribosome stalling within minutes to provide a molecular readout of phenotypic antimicrobial susceptibility. Its compatibility with nanopore sequencing enables fast and affordable phenotypic AMR testing, including in complex mixed-species samples.