RAMP-Seq: a single-tube nanopore workflow for co-amplifying full-length 16S rRNA and antimicrobial resistance genes for integrated taxonomic and resistance profiling

Antimicrobial resistance (AMR) surveillance needs workflows that both identify bacteria and detect resistance genes from a single sample, but current targeted nanopore assays typically split these steps into separate reactions or library preparations. We introduce RAMP-Seq (Rapid AMR Profiling by Sequencing), a workflow that simultaneously amplifies the full-length 16S rRNA gene (~1,500 bp) together with eight resistance determinants (~600 - 700 bp) in a single multiplex PCR and then sequences the resulting mixed-length amplicon pool as a single nanopore library. The eight-primer pair set targets dominant alleles of {beta}-lactamase, carbapenemase, quinolone-resistance and silver-resistance families. Read-length distributions confirmed that both size classes were co-amplified evenly using a single, non-equimolar primer pool. From extracted DNA to the first taxonomically classified reads took 8.2 {+/-} 0.6 h. Full-length 16S sequencing of an eight-member mock community identified all anticipated taxa, and the observed abundances closely followed the expected log-scaled composition (Pearson's r = 0.879). The full nineplex amplicon assay was subsequently tested on three distinct matrices: pooled clinical isolates, spiked human blood, and environmental water samples. Using an equal-mass pool of genomic DNA from eleven multidrug-resistant clinical and repository isolates, the full assay produced genus-level and resistance-gene profiles that were consistent across biological replicates (r = 0.998 and r = 0.824), and it specifically identified all seven panel targets present, confirmed by Illumina whole-genome sequencing. After carbonate-based host DNA depletion, Klebsiella pneumoniae was recovered from spiked human blood at every titration down to 102 CFU/mL, as were all five panel targets carried by that strain. Across five water-sampling sites from a sewage treatment plant (STP) to a connected lake system, microbial communities and resistance-gene profiles were site-specific: influent and effluent communities overlapped little, while the lake inlet and outlet were similar. Target resistance genes were most numerous and abundant in STP influent, fewer in effluent, and rare or absent in lake and well samples. Because bacterial identification relies on amplification of the conserved, near-universal 16S rRNA gene, rather than on species-specific primer sets, RAMP-Seq can detect and taxonomically classify bacteria in a design-agnostic manner. The same single-tube chemistry therefore serves sample types as different as clinical specimens and environmental water and is extendable to longer ribosomal targets and further resistance-gene families.