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.
Highlights
Chronic respiratory diseases including chronic obstructive pulmonary disease (COPD), bronchiectasis or cystic fibrosis (CF) often progress with lower airways colonization by pathogenic microorganisms and, in many cases, chronic infections are established
nontypable H. influenzae (NTHi) isolates used in this study were recovered from sputum samples from 18 episodes of acute exacerbations in 6 adult patients with chronic lung diseases (4 COPD, 1 bronchiectasis, 1 cystic fibrosis and bronchiectasis)
NTHi isolates were typed by Pulse field gel electrophoresis (PFGE) and divided in two groups (Table 1): (i) persistent isolates had identical PFGE profile to those isolated from previous sputa in the same patient
Summary
Chronic respiratory diseases including chronic obstructive pulmonary disease (COPD), bronchiectasis or cystic fibrosis (CF) often progress with lower airways colonization by pathogenic microorganisms and, in many cases, chronic infections are established. Chronic infection sets up a condition where microbial antigens induce a host inflammatory response and disrupt the innate lung defense. These aspects are thought to play a key role in the irreversible airway damage from which some patients die [1,2,3,4]. Acquisition of a new NTHi strain has been shown to be an important cause of lower respiratory tract infection associated with the occurrence of COPD exacerbations [11]. New strain exacerbations are associated with significantly greater increases from baseline in sputum TNF-a, neutrophil elastase and serum Creactive protein than preexisting strain exacerbations [14]
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