A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing(Fig1_HT-5PSeq)

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 four hours 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 3000 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. Overall design: HT-5PSeq library of exponetially growing (OD600=0.6-0.8)b.subtilis and l.plantarum treated with different concentration of chloramphenicol(untreated, 1.6ug/ml, 8ug/ml, 40ug/ml) or erythromycin (untreated, 0.1ug/ml, 0.5ug/ml, 2.5ug/ml) for 10min.