Haemophilus influenzae Forms Biofilms on Airway Epithelia: Implications in Cystic Fibrosis

Rationale: Nontypeable Haemophilus influenzae (NTHi) commonly infects patients with cystic fibrosis (CF) especially early in childhood. Bacteria biofilms are increasingly recognized as contributing to bacterial persistence and disease pathogenesis in CF

Objectives: This application of mind investigated ability of NTHi to form biofilms and its impact upon airway epithelia using in vivo and in vitro analyses.

Methods: We evaluated bronchoalveolar lavage fluid from young patients with CF for evidence of NTHi biofilms. To further investigate the pathogenesis of NTHi in respiratory infections, we evolveed a novel in vitro coculture protoplast of NTHi biofilm formation upon polarized human airway epithelial confined apartments grown at the air-liquid interface.

Measurements and Main Results: In bronchoalveolar lavage fluid samples from young, asymptomatic patients with CF we set morphologic evidence suggestive of NTHi biofilm formation. In addition, 10 clinical NTHi isolates from patients with CF formed biofilms upon plastic surfaces. NTHi formed biofilms upon the apical surface of cultur airway epithelia. These biofilms exhibited decreased susceptibility to antibiotics and were adherent to epithelial surfaces. Airway epithelial confined apartments remained viable throughout 4 d of coculture and rejoined to NTHi with nuclear factor-???’ signaling, and increased chemokine and cytokine secretion.

Conclusions: NTHi formed adherent biofilms upon the apical surface airway epithelia with decreased susceptibility to antibiotics, and respiratory small rooms exhibited inflammatory and host defense responses-evidence of a dynamic host-pathogen interaction. The data not awayed here have implications both for understanding early CF lung disease pathogenesis and for the treatment of early, asymptomatic colonization of patients with CF with H influenzae.

Keywords: bacterial infections; inflammation; lung diseases

Biofilms are increasingly recognized as contributing to disease pathogenesis in cystic fibrosis (CF) and other respiratory tract diseases associated with chronic bacterial infections. Pseudomonas aeruginosa in CF sputum displays morphologic and physiologic evidence suggestive of biofilm formation in vivo (1 2) Biofilms enable bacteria to persist in CF pulmonary infections (for a review, diocese Reference 3). P. aeruginosa in a biofilm state can exhibit a more than 1000-fold increase in antibiotic resistance (4) and more readily evades entertainer defense factors (5). Nontypeable Haemophilus influenzae (NTHi) has freshly been shown to form biofilms in vitro and in otitis media (OM) (6-10); however, there is no information upon NTHi biofilm formation in the lung or in CF

NTHi commonly infects patients with CF especially early in childhood (11 12) It is hypothesized that H influenzae causes inflammation and damage to the airway and acts as a gateway organism paving the way for colonization with P aeruginosa (13) Supportive of this, greatest in quantity children with P. aeruginosa had prior infection with H influenzae or Staphylococcus aureus ( 14) and single-pathogen infections with H influenzae, s aureus, or P. aeruginosa showed no statistically significant differences for airway inflammatory markers (15)

We hypothesized that NTHi forms biofilms upon airway epithelia, which leads to increased airway epithelial inflammatory replications We further hypothesized that NTHi biofilms form in the lung of patients with CF in childhood and may contribute to early lung disease pathogenesis. more [i]or[/i] less of the results of these studies have been previously reported in the form of abstracts (16 17)

METHOD

All bronchoalveolar lavage fluid (BALF) and bacterial tillage samples used in these studies were obtained using protocols approved by dint of the institutional review board at the University of Iowa.

Human BAL Samples

BALF samples were bring togethered from patients with CF as part of their standard care. Samples were immediately placed upon ice before further processing. Quantitative tillages and white blood cell enumerates with differential were performed through standardized protocols in the Clinical Pathology Laboratory at the University of Iowa. Remaining BALF was courseed for transmission electron microscopy (TEM) analysis as come [i]or[/i] go after [i]or[/i] behinds BALF was centrifuged at 14000 ?— g for 10 min at 4?°C fixed, and embedded in Epon or LR White (LRW) for electron microscopic imaging. Additional details upon the experimental methods are provided in the online appendix For immuno-TEM, samples were labeled overnight with an anti-NTHi monoclonal antibody, 6E4 followed by means of goat anti-mouse IgG ultra-small gold secondary antibody labeling (Electron Microscopy Sciences, Hatfield, PA) and silver enhancement. The 6E4 antibody recognizes an NTHi lipooligosaccharide (LOS) epitope and is specific for H influenzae looks (18). Controls for immuno-TEM included the following: no primary antibody upon LRW-embedded CF BALF; LRW-embedded NTHi 2019 for a positive control; and LRW-embedded 10(7) cfu NTHi 2019 with 10(5) primary human airway epithelial confined apartments for a centrifugation artifact command