Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that frequently colonizes the human nasopharynx; it is a common cause of chronic and recurrent otitis media in children and of exacerbations of chronic obstructive pulmonary disease. To date, no exopolysaccharide clearly contributing to NTHi biofilms has been identified. Consequently, there is some debate as to whether NTHi forms biofilms during colonization and infection. The present work shows that NTHi can form biofilms in vitro, producing an extracellular matrix composed of proteins, nucleic acids, and a β-glucan. Extracellular DNA, visualized by immunostaining and using fluorochromes, is an important component of this matrix and appears to be essential in biofilm maintenance. Extracellular RNA appears to be required only in the first steps of biofilm formation. Evidence of a matrix polysaccharide was obtained by staining with Calcofluor white M2R and by disaggregating biofilms with cellulase. Using strain 54997, residues of Glcp(1→4) in the NTHi biofilm were confirmed by gas-liquid chromatography-mass spectrometry. Evidence that N-acetyl-L-cysteine shows notable killing activity towards in vitro NTHi biofilm-forming bacteria is also provided.