Genomic constraints shape the evolution of alternative routes to drug resistance in prokaryotes.

Abstract

Antimicrobial resistance (AMR) is often modelled as the accumulation of resistance genes leading to multidrug resistance (MDR). We show that gene co-occurrence patterns in two opportunistic pathogens are consistent with fitness trade-offs that constrain which combinations of resistance mechanisms coexist. We applied a combined pangenomic and machine-learning analysis to 9,584 Escherichia coli genomes (99.2% phylogroup B2) and 7,057 Pseudomonas aeruginosa genomes. In E. coli, we identified eight cases of mutually exclusive gene pairs that independently predicted the same MDR phenotype, suggesting alternative routes to resistance whose components are typically not co-inherited. In a separate dataset of 352 strains with paired minimum inhibitory concentration (MIC) data, these dissociated combinations co-occurred more often in resistant than susceptible strains, consistent with the constraints being conditional on antibiotic selection. 33 gene pairs showed opposing association patterns between the two species, with combinations significantly associated in one species and significantly dissociated in the other (e.g. associated in E. coli and dissociated in P. aeruginosa, or vice versa). This indicates that genomic context modifies the contribution of individual genes to resistance phenotypes, and offers one explanation for the observation that 106 ARGs are present in >95% of strains yet do not predict resistance phenotype on their own. The findings are consistent with resistance evolution being shaped by fitness trade-offs and suggest that the dissociation patterns we identify could be targets for follow-up experimental work on resistance-associated fitness costs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/671315v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7a717corg.highwire.dtl.DTLVardef@b08e64org.highwire.dtl.DTLVardef@154820forg.highwire.dtl.DTLVardef@126b9ee_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPACT STATEMENTMultidrug resistance threatens global health; however, resistance acquisition remains poorly understood. We show that genomes cannot simply accumulate all available resistance mechanisms. Instead, species-specific grammatical rules render certain gene combinations typically incompatible. Analysing 9,584 Escherichia coli genomes, we identify eight mutually exclusive routes to resistance, demonstrating that resistance is more constrained than previously thought. Strikingly, 33 gene pairs that cooperate in E. coli actively disassociate in Pseudomonas aeruginosa, revealing that genomic context determines resistance outcomes. This explains why 106 resistance genes present in all strains fail to confer universal resistance, challenging current diagnostics. Our findings transform our understanding of resistance evolution from an unlimited accumulation model to a grammatically constrained pathways model, presenting new therapeutic strategies that exploit evolutionary dead ends.