Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure

Significance Several human pathogens exploit genomic variability to adapt to the host environment. Genome sequencing of collections of isolates and classification of strains according to their genomic content are pivotal to the formulation of vaccines able to elicit broad protection. We sequenced a collection of carriage and disease isolates of nontypeable Haemophilus influenzae, a component of the microbial flora of the upper respiratory tract that can cause a spectrum of diseases, including otitis media and meningitis. We identified distinct evolutionary clades that correlate with the presence of selected surface-associated proteins and virulence determinants. The high-resolution definition of the population structure of nontypeable Haemophilus influenzae allowed by whole-genome sequencing will be key for the development of efficacious containment strategies for this important human pathogen. One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.