A novel mechanism of unstable heteroresistance via reversible ompP2 mutations in Haemophilus influenzae

Heteroresistance, the presence of a resistant subpopulation within a predominantly susceptible bacterial isolate, is usually difficult to detect and may cause treatment failure. Mechanisms driving unstable heteroresistant phenotypes, which rapidly revert in absence of antibiotics, remain poorly understood. To investigate the mechanisms underlying heteroresistance in H. influenzae , we isolated four heteroresistant clones and characterized them through experimental evolution, gradient diffusion assays, population analysis profiling, and whole genome sequencing. All clones initially exhibited increased ceftriaxone minimum inhibitory concentrations but rapidly reverted to wild-type susceptibility within four days in drug-free conditions. The main mechanism was reversible structural changes of the ompP2 resistance gene. Three clones exhibiting a 168 bp genomic inversion affecting ompP2 reverted to wild-type sequence. A fourth clone with an ompP2 frameshift insertion reproducibly acquired compensatory deletions restoring the open reading frame. PAP analysis still detected minority subpopulations with 4x-8x increased MICs at day 4. We identified site-specific DNA breakpoints with a palindrome and a homologous sequence facilitating rapid structural genome changes and phenotypic switching. Overall, we discovered novel mechanisms of unstable heteroresistance in H. influenzae , mediated by reversible structural changes in the gene ompP2. Phenotypic switching was induced by ceftriaxone exposure and likely reflects the mutational plasticity of ompP2.