A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing
Ontology highlight
ABSTRACT:
ORGANISM(S): Enterococcus faecalis Clostridioides difficile Lactiplantibacillus plantarum Staphylococcus aureus Bacillus subtilis
PROVIDER: GSE293532 | GEO | 2025/12/23
REPOSITORIES: GEO
Similar Datasets
Project description: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.
2025-12-23 | GSE293523 | GEO
Project description: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.
2025-12-23 | GSE293530 | GEO
Project description: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.
2025-12-23 | GSE293525 | GEO
Project description: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.
2025-12-23 | GSE293524 | GEO
Project description: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.
2025-12-23 | GSE293528 | GEO
Project description: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.
2025-12-23 | GSE312901 | GEO
Project description: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.
2025-12-23 | GSE312902 | GEO
Project description:Slow progress towards implementation of conventional clinical bacteriology in low resource settings and strong interest in greater speed for antimicrobial susceptibility testing (AST) more generally has focused attention on next-generation rapid AST technologies. In this Review, we systematically synthesize publications and submissions to regulatory agencies describing technologies that provide phenotypic AST faster than conventional methods. We characterize over ninety technologies in terms of underlying technical innovations, technology readiness level, extent of clinical validation, and time-to-results. This work provides a guide for technology developers and clinical microbiologists to understand the rapid phenotypic AST technology landscape, current development pipeline, and AST-specific validation milestones.
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Project description:Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.
| S-EPMC5530678 | biostudies-literature
Project description:Growing antimicrobial resistance (AMR) is a serious global threat to human health. Current methods to detect resistance include phenotypic antibiotic sensitivity testing (AST), which measures bacterial growth and is therefore hampered by a slow time to obtain results (∼12-24 h). Therefore, new rapid phenotypic methods for AST are urgently needed. Nanomechanical cantilever sensors have recently shown promise for rapid AST but challenges of bacterial immobilization can lead to variable results. Herein, a novel cantilever-based method is described for detecting phenotypic antibiotic resistance within ∼45 min, capable of detecting single bacteria. This method does not require complex, variable bacterial immobilization and instead uses a laser and detector system to detect single bacterial cells in media as they pass through the laser focus. This provides a simple readout of bacterial antibiotic resistance by detecting growth (resistant) or death (sensitive), much faster than the current methods. The potential of this technique is demonstrated by determining the resistance in both laboratory and clinical strains of Escherichia coli (E. coli), a key species responsible for clinically burdensome urinary tract infections. This work provides the basis for a simple and fast diagnostic tool to detect antibiotic resistance in bacteria, reducing the health and economic burdens of AMR.
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