A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing.
JSON
Liu H, Huch S, Hull R, Garcia FR, Nersisyan L, Yin X, Chen WH, Du J, Pelechano V
Cell Reports Methods 6 (3) 101327 [2026-03-23; online 2026-03-12]
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.
NGI Stockholm (Genomics Applications) [Service]
National Genomics Infrastructure [Service]
PubMed 41825450
DOI 10.1016/j.crmeth.2026.101327
Crossref 10.1016/j.crmeth.2026.101327
pmc: PMC13030957
pii: S2667-2375(26)00027-5