Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Abstract

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.

Figure 1. Infection of epithelial cells by NTHi isolates recovered from chronic respiratory patients.

(A) NTHi adhesion to A549 epithelial cells. NTHi cells were incubated with A549 cells for 30 min. Bacterial adhesion was quantified by lysis, serial dilution and viable counting on sBHI-agar plates. Mean numbers for newly acquired strains (grey bar) were significantly higher (p<0.0001) than those obtained for strains recovered from patients 1, 2 and 3 (white bar). (B) Stimulation of IL-8 secretion by infection for NTHi clinical isolates. Quantification by ELISA of IL-8 secreted to the supernatant by A549 cells upon infection with NTHi1a, 1b, 1c, 2a, 2b, 3a, 3b, 3c, 5a, 5b, 5c 5d and 6a. [IL-8] was quantified at 6 (white bar) and 20 (grey bar) h post-infection.

Figure 2. H. influenzae clinical isolates self-aggregation.

Tube-settling experiment of stationary phase cultures of isolates NTHi1a (open circles), NTHi1b (open squares), NTHi1c (open triangles), NTHi2a (black inverted triangle), NTHi2b (black diamond), NTHi3a (grey circle), NTHi3b (grey square), NTHi3c (grey triangle), NTHi5b (open inverted triangle) and NTHi6a (open diamond), after incubation at room temperature for 7 h. E. coli C600 (asterisk) was used as a negative control. Bacterial aggregation was quantified by measuring the decrease of absorbance at OD600nm. NTHi1a-1c self-aggregated significantly slower than NTHi2a-2b (p<0.0001 at 1 h; p<0.001 at 1.5 h). NTHi6a aggregated slower than strains from patients 1 and 2 (p<0.0001 at 1 and 1.5 h), and faster than isolates from patient 3 (p<0.0001, all time points tested).