COPD Exacerbation Associated Strains Of Nontypeable Haemophilus Influenzae Form Greater Flow Cell Biofilms Than Colonizing Strains



Shaun Bowman, Jingjing Yin, Lori Grove, Catherine Wrona and Sanjay Sethi

INTRODUCTION: COPD sputum isolates of nontypeable Haemophilus influenzae (NTHi) form biofilms to varying degrees in vitro. We wanted to determine if the ability of NTHi to form biofilms in vitro in flow cells that emulate airway conditions is correlated with the duration of carriage and the propensity to cause exacerbations in patients with COPD.


METHODS: NTHi isolates collected from a cohort of COPD patients at the Buffalo VAMC during monthly visits and at exacerbations during a 10-year prospective study were studied. Each isolate was designated as an “exacerbation” (E) strain if its acquisition was associated with a clinical exacerbation, otherwise it was designated as a “colonizing” (C) strain. Biofilm formation of 43 NTHi strains (27 C and 16 E) was analyzed using the BioFlux 200 system (Fluxion Biosciences), which utilizes pneumatic pressure to create a constant flow of medium through glass, microfluidic

viewing channels. For this study, biofilms were formed for 24hrs at 37°C with a constant flow rate of 0.5 dynes/cm2, consistent with the normal breathing induced-shear of the airways. All biofilms were stained with BacLight Green (Molecular Probes) and imaged by confocal microscopy. Biofilms were quantitated by summation of the fluorescence intensity of successive Z-stacks covering the entire area of the viewing channel. The biofilm forming ability of each strain was expressed as the average fluorescence intensity of duplicate channels, and was compared between C and E strains and with duration of airway carriage.


RESULTS: Biofilm forming abilities of the NTHi strains demonstrated measurable differences, with fluorescence intensity values ranging 7 fold from 14.4 to 102.1 units. A significant difference in the biofilm forming abilities of C and E strains was seen, with E strains having higher overall average fluorescence intensity (log) values, 3.65±0.16 and 3.26±0.10 for E and C strains respectively; p=0.03. Biofilm forming ability did not correlate with the duration of carriage.


CONCLUSIONS: Exacerbation strains formed better biofilms than colonizing strains. Biofilm formation may allow NTHi strains to resist innate and adaptive immune host responses, proliferate in the airway, and cause exacerbations. Although the formation of biofilms by NTHi does not seem to be related to the duration of colonization, that relationship may be confounded by antibiotic exposure.