Evolution of beta-lactam resistance causes fitness reductions and several cases of collateral sensitivities in the human pathogen <i>Haemophilus influenzae</i>

The evolution of antimicrobial-resistant pathogens poses a global health threat, and it is unclear if evolutionary trajectories to resistance lead to predictable phenotypes. We analyzed antimicrobial resistance (AMR) evolution in the human pathogen <i>Haemophilus influenzae</i> in controlled evolution experiments with increasing concentrations of either ampicillin, cefotaxime, or ceftriaxone. We isolated 315 clones from different time points of six independent experiments and characterized changes in genome sequences, bacterial fitness, and minimum inhibitory concentrations (MICs) to 14 antibiotics. Resistance evolution under ampicillin and cefotaxime was mainly driven by mutations in the <i>ftsI</i> gene, encoding the penicillin-binding protein 3. However, ceftriaxone exposure repeatedly selected for amino acid substitutions in the outer membrane protein P2 (OmpP2). Some OmpP2 mutants reproducibly showed phenotypic heterogeneity not only for ceftriaxone but also for fluoroquinolones, rifampicin, and tetracycline. Bacterial fitness assessments revealed trade-offs between resistance-associated mutations and growth, though no systematic correlation of MIC increase and growth deficit was detected. Over 50% of the selected clones became more susceptible to aminoglycosides, clarithromycin, and colistin, while some mutation patterns resulted in cross-resistance to meropenem and fluoroquinolones. Overall, beta-lactam antibiotics reproducibly selected mutants with increased MICs, but evolutionary pathways and resulting phenotypes remain unpredictable. These findings highlight the complexity of resistance evolution and suggest that future AMR treatment strategies may need to consider strain-specific and collateral effects more closely.