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

Abstract

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.

N -Acetyl- l -Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae

“…Biofilm formation was determined with a modified CV assay (26). NT H. influenzae was grown at 37°C under 5% CO 2 in sBHI medium to an optical density at 550 nm (OD 550 ) of 0.5, sedimented by centrifugation, resuspended in an equal volume of s(CϩY) medium (22), and diluted 10-or 100-fold. NE S. pneumoniae P233 was grown at 37°C in CϩY medium to an OD 550 of ϳ0.5, sedimented by centrifugation, resuspended in an equal volume of s(CϩY), and diluted 10-or 100-fold.…”

“…NT H. influenzae isolated from patients with AOM, cystic fibrosis (CF), or chronic obstructive pulmonary disease (COPD) has been proposed to form biofilms in vitro and in vivo (19)(20)(21), although a biofilm-specific extracellular polysaccharide has never been described. Indeed, we have recently shown that NT H. influenzae strains are also capable of forming in vitro a true biofilm matrix composed of a ␤-glucan together with proteins and nucleic acids (22).…”

Autolysin-Independent DNA Release inStreptococcus pneumoniae in vitroBiofilms

“…, staining with a variety of DNA-specific fluorophores) to disclose the presence of eDNA in pneumococcal biofilms (7–9, 11, 26, 27). Immunostaining with α-dsDNA combined with CLSM also revealed the existence of long filaments of eDNA in biofilms formed either by Enterococcus faecalis (45) or Haemophilus influenzae ECM (46). Moreover, the presence of eDNA-containing fibrous structures in the ECM of Staphylococcus aureus and Propionibacterium acnes biofilms has also been reported using α-dsDNA and atmospheric scanning electron microscopy (47, 48).…”

Subversion of host immune responses by otopathogens during otitis media

“…The nonisogenic mutant, however, demonstrated elevated levels of fractal structures, which appears to have a role in enabling biofilm nutrient exchange, and feedback mechanisms. Extracellular RNA also plays an important role in the initial steps of biofilm synthesis, though its overall function is less important . Overall, the biofilm synthesized by NTHi has been found to release extracellular DNA and a β ‐glucan …”

“…Extracellular RNA also plays an important role in the initial steps of biofilm synthesis, though its overall function is less important . Overall, the biofilm synthesized by NTHi has been found to release extracellular DNA and a β ‐glucan …”