ArticleCell reports methods2026
A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing.
Article in Cell reports methods, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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.
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