NanoMLST: A High‐Throughput Bacterial Multi‐Locus Sequence Typing Workflow Using Oxford Nanopore Next‐Generation Sequencing for ESKAPE + E Pathogens

Multi-Locus Sequence Typing (MLST) is a key method for allocation of Sequence Types (STs) for bacterial isolates. Traditionally, this is performed by the Sanger sequencing method, which can be highly time-consuming and laborious. In this study, we present NanoMLST, a high-throughput MLST workflow using multiplex PCR, Oxford Nanopore Technologies Next-Generation Sequencing, and the Krocus program for typing ESKAPE + E pathogens (Enterococcus faecium [E. faecium], Staphylococcus aureus, Klebsiella pneumoniae [K. pneumoniae], Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli). Bacterial isolates were obtained from the Hospital Universitario La Paz's Microbiology Department and the Centro Nacional de Microbiología. Primers that can be multiplexed in a single PCR reaction were designed for the seven housekeeping genes for each species. DNA was extracted from single colonies by heating at 95°C for 10 min, mechanical lysis at 4.20 m/s for 2 min, and then by the MagCore extraction system. Multiplex PCRs were then performed with the respective primer mixes for each species, and libraries were prepared for sequencing by ONT Flongle cells. The Krocus program was then used to determine the STs from the raw FastQ reads. STs for 221 isolates were obtained through this workflow with an average time of 12 h per 24 isolates. In line with local data, the K. pneumoniae and E. faecium isolates were relatively oligoclonal, while the rest were polyclonal. STs from representative isolates showed 100% concordance between Sanger sequencing and the proposed workflow. NanoMLST offers a fast, cheaper, and less labor-intensive alternative for large-scale MLST applications targeting clinically important pathogens.