Neighbour Typing Using Long Read Sequencing Provides Rapid Prediction of Sequence Type and Antimicrobial Susceptibility of Klebsiella pneumoniae

Budia-Silva, M.; Carroll, A. C.; Ghosh, H.; McGeer, A.; Giani, T.; Rossolini, G. M.; Brinda, K.; Hanage, W.; Grundmann, H.; MacFadden, D. R.; Reuter, S.

doi:10.1101/2025.09.03.673989

bioRxiv · 10.1101/2025.09.03.673989

Neighbour Typing Using Long Read Sequencing Provides Rapid Prediction of Sequence Type and Antimicrobial Susceptibility of Klebsiella pneumoniae

Budia-Silva, M.·Carroll, A. C.·Ghosh, H.·McGeer, A.·Giani, T.·Rossolini, G. M.·Brinda, K.·Hanage, W.·Grundmann, H.·MacFadden, D. R.·Reuter, S.

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Abstract

The rapid genome-based diagnostic approach of long read sequencing coupled with neighbor typing offers the potential to improve empiric treatment of infection. However, this approach is still in development, and clinical validation is needed to support its use. In this study, we present an assessment of a neighbour typing method (RASE - resistance associated sequence elements) to predict lineage or sequence type (ST) and antimicrobial susceptibility in real time for Klebsiella pneumoniae sensu lato. We analysed the initial reads generated during the early phase of long read sequencing from pure culture (n=99), mock communities (n=20) and metagenomic samples (n=20). RASE accurately identified 69.7% and 70% of STs in pure culture and metagenomes, respectively, and identified the STs of the isolates representing the highest proportion in mock communities. Regarding antimicrobial susceptibility prediction, the probability of susceptibility increased to 72% (95% CI 63%-80%) across all tested antibiotics, when RASE predicted susceptibility, and decreased probability of susceptibility to 8.9% (95% CI 6.4%-9.6%) when was indicative of a resistant phenotype. Our study confirmed that genomic neighbor typing in K. pneumoniae sensu lato is capable of providing informative predictions of ST and antibiotic susceptibility in less than ten minutes (after the start of sequencing) with 200-500 reads. IMPORTANCEThe growing burden of antimicrobial resistance is leading to high rates of mortality and morbidity worldwide. This situation has made the selection of empirical antibiotic therapy challenging, due to the risk of treatment failure and the overuse of last-resort antibiotics. The development of new sequencing technologies is helping to reduce the waiting time for a microbiological diagnosis, providing information in the early phase of bacterial infections, which could help improve clinical outcomes in a time of rising antimicrobial resistance. In this context, we assessed the performance of RASE (resistance associated sequence elements) in Klebsiella pneumoniae, an opportunistic pathogen frequently associated with nosocomial infections, which can rapidly acquire antibiotic resistance genes. Thus, in our study we provide insights that may aid in the validation of RASE for clinical use.

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Budia-Silva, M., Carroll, A. C., Ghosh, H., McGeer, A., Giani, T., Rossolini, G. M., Brinda, K., Hanage, W., Grundmann, H., MacFadden, D. R., Reuter, S.. 2025-09-09. Neighbour Typing Using Long Read Sequencing Provides Rapid Prediction of Sequence Type and Antimicrobial Susceptibility of Klebsiella pneumoniae. https://doi.org/10.1101/2025.09.03.673989

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