B-246. Extracellular DNA Mediates Intercellular Adhesion in Non-Typeable Haemophilus influenzae Biofilms

E. A. Izano, D. Rupani, J. B. Kaplan;
UMDNJ, Newark, NJ.

Background: Non-typeable Haemophilus influenzae (NTHi) causes numerous chronic infections including otitis media, sinusitis, bronchitis and chronic obstructive pulmonary disease (COPD). Evidence suggests that the ability to form matrix-encased biofilms contributes to NTHi pathogenesis. Type IV pili have been shown to be a major intercellular adhesin in NTHi biofilms in vitro. Extracellular DNA (eDNA) has also been shown to be a component of the NTHi biofilm matrix. The role of eDNA in biofilm cohesion has not been determined. We hypothesized that eDNA functions as a matrix adhesin in NTHi biofilms. Methods: Eight different NTHi strains isolated from COPD patients were tested. The intercellular adhesion phenotype of each strain was tested using a visual test tube autoaggregation assay. Biofilm formation was assayed in 17 x 100 mm polystyrene tubes. Biofilms were grown in the presence or absence of bovine DNase I or proteinase K. In addition, pre-formed biofilms were treated with DNase I, proteinase K, the carbohydrate-modifying agent sodium metaperiodate (NaMPer), or various detergents including sodium dodecyl sulfate (SDS), cetylpyridinium chloride (CPC), and chlorhexidine (CHX), in order to assess the ability of these agents to induce biofilm detachment. Control biofilms were treated with the appropriate buffer alone. Biofilm biomass was measured by crystal violet staining and CFU counts. Results: All eight clinical isolates exhibited a strong autoaggregation phenotype which was inhibited by proteinase K but not by DNase I or NaMPer. Six out of the eight clinical isolates exhibited a strong biofilm phenotype. Biofilm formation by all biofilm-forming isolates was inhibited by DNase I and proteinase K. Pre-formed biofilms were detached at the early stages by DNase I, and at all stages of biofilm formation by proteinase K, SDS and CPC. CHX and NaMPer did not detach NTHi biofilms at any stage of growth. Conclusions: eDNA function as biofilm matrix adhesins in the early stages of NTHi biofilm development. Unlike most other biofilms, polysaccharides do not appear to play a major role in intercellular adhesion or biofilm formation in NTHi.