Holistic understanding of trimethoprim resistance in using an integrative approach of genome-wide association study, resistance reconstruction, and machine learning.

Unlabelled: Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen . We explored a collection of 662 . genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the and loci are responsible for TMP non-susceptibility in and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic.

Importance: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the locus produces trimethoprim (TMP) resistance in . These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.