Nontypable Haemophilus influenzae
(NTHi) has emerged as an important opportunistic pathogen causing infection in
adults suffering obstructive lung diseases. Existing evidence associates
chronic infection by NTHi to the progression of the chronic respiratory
disease, but specific features of NTHi associated with persistence
have not been comprehensively addressed. To
provide clues about adaptive strategies adopted by NTHi during persistent
infection, we compared sequential persistent isolates with newly
acquired isolates in sputa from six patients with chronic obstructive lung
disease. Pulse
field gel electrophoresis (PFGE) identified three patients with consecutive persistent
strains and three with new strains. Phenotypic
characterisation included infection of respiratory
epithelial cells, bacterial self-aggregation, biofilm formation and resistance
to antimicrobial peptides (AMP). Persistent isolates differed from new strains
in showing low epithelial adhesion and inability to form biofilms when grown
under continuous-flow culture conditions in microfermenters.
Self-aggregation clustered the strains by patient, not by persistence.
Increasing resistance to AMPs was observed for each series of persistent
isolates; this was not associated with lipooligosaccharide decoration with
phosphorylcholine or with lipid A acylation. Variation was further analyzed for
the series of three persistent isolates recovered from patient 1. These
isolates displayed comparable growth rate, natural transformation frequency and
murine pulmonary infection. Genome sequencing of these three isolates revealed
sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic
acid kinase kdkA, the long-chain
fatty acid transporter ompP1,
and the phosphoribosylamine glycine ligase purD. Collectively,
we frame a range of pathogenic traits and a
repertoire of genetic variants in
the context of persistent infection by NTHi.