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.

Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection

“…A mouse model of NTHI pulmonary infection was used as described previously (54,55). CD1 female mice (18 to 20 g) aged 4 to 5 weeks were purchased from Charles River Laboratories (France), housed under pathogen-free conditions at the Institute of Agrobiotechnology facilities (registration number ES/31-2016-000002-CR-SU-US), and used at 22 to 25 g. Animal handling and procedures were performed in accordance with the current European (directive 86/609/EEC) and national (Real Decreto 53/2013) legislations, following the FELASA and ARRIVE guidelines, and with the approval of the Universidad Pública de Navarra (UPNa) Animal Experimentation Committee (Comité de Ética, Experimentación Animal y Bioseguridad) and the local government.…”

“…We showed previously that NTHI infection triggers IL-8 secretion by A549 cells (54,55). We sought to determine the effect of pretreatment with 50 g/ml AZM on IL-8 secretion by infected A549 cells.…”

Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

“…These include type IV pili (PilA) overexpression (14), the presence of fimbriae (13), quorum sensing (15), the presence of outer membrane proteins (OMPs) P2 and P5 (16), and the presence of phosphorylcholine (PCho) and sialic acid in the lipooligosaccharide (LOS) molecule (17,18). Despite the previously shown role of PCho in biofilm growth (19), a longitudinal study on NT H. influenzae isolates from patients with chronic respiratory disease found no clear correlation between biofilm growth and the presence of PCho in the LOS molecule (20). We previously showed the absence of a clear correlation between in vitro biofilm formation and the presence of PCho in the LOS of NT H. influenzae using a collection of 111 clinical isolates from different clinical sources (21).…”

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

“…Infections frequently persist and recur despite the host's development of bactericidal antibodies and the use of antibiotics. Our current understanding of the molecular mechanisms involved in NTHi infection remains limited, but identical strains have been repeatedly isolated from the lungs of COPD patients in serial clinic visits, suggesting that NTHi presents features that promote chronic infection (7,8). NTHi is a facultative intracellular patho-gen (9)(10)(11)(12)(13)(14)(15)(16), and epithelial invasion may allow bacterial cells to temporarily evade the immune system and/or therapeutic interventions.…”