Long-term experimental evolution of β-lactam resistance in Haemophilus influenzae

Introduction: Haemophilus influenzae is an opportunistic bacterial pathogen that can cause pneumonia, meningitis, and bacteremia. The increased prevalence of ampicillin resistant strains led to the endorsement of third-generation cephalosporins which overcome resistance mediated by certain β-lactamases. However, mutational resistance, and long-term bacterial adaptation under β-lactam exposure is only poorly understood, thus we established a long-term evolution experiment.

Materials/Methods: H. influenzae DSM 11121 was cultivated over several weeks with stepwise increasing concentrations of ampicillin, cefotaxime, and ceftriaxone. For over 300 clones we determined minimum inhibitory concentrations (MICs) and analyzed genomic variations with next generation sequencing. In addition, we measured the bacterial fitness of evolved mutants with a competitive growth assay.

Results: Presence of ampicillin and cefotaxime led to stepwise evolution of mutations in ftsI (encoding the main target of β-lactam antibiotics) which were associated with increased MICs to all three β-lactams. Exposure to ceftriaxone, however, independently selected for different mutations in ompP2, coding for an outer membrane protein. In addition, a genomic inversion affecting ompP2 was associated with reproducible but unstable heteroresistance. Selected clones, showed variable bacterial fitness effects, and acquired possible fitness compensating mutations.

Discussion: Our data provide new insights into the evolutionary pathways of β-lactam resistance of H. influenzae. We highlight the role of ompP2 as resistance determinant, and possible mechanism to induce a heteroresistant phenotype.