A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing (Fig3_Illumina)
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: s5PSeq library were prepared using 200ng extracted total RNA from exponetially growing (OD600=0.6-0.8) C.difficile clinical isolates that are resistant or sensitive to erythromycin. Bacteria cells are treated with erythromycin(1ug/ml) for 10min or without treatment.