Continuous microevolution accelerates disease progression during sequential episodes of infection

Nontypeable Haemophilus influenzae (NTHI), the causative agent of otitis media (OM), can cause chronic episodes of disease. Hypothesizing that during chronic infection bacterial adaptation would promote fitness and facilitate long term survival within the host, we used a chinchilla model of sequential episodes of OM to determine the role of microevolution in NTHI adaptation during disease. After successive episodes of disease, we observed a significant increase in biofilm development and the number of bacteria recovered from biofilms, suggesting in vivo strain adaptation by passage through animals. We also observed a significant increase in the number of intracellular bacterial communities of NTHI within middle ear mucosae, as well as increased fibrosis and inflammation. Whole genome sequencing of genomic DNA isolated from NTHI that had undergone four episodes of disease identified mutations in 12 genes. Confirmatory sequencing showed a subset of these mutations arose within the first seven days of infection.