Nontypeable Haemophilus influenzae (NTHI) causes
chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces
lipooligosaccharides containing
sialic acid (NeuAc) and
phosphorylcholine (
PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking
PCho. In this study, we asked if alterations in NTHI
PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked
PCho (NTHI 2019 licD) or were constitutively locked in the
PCho-positive phase (NTHI 2019 lic(ON)). Transformants expressing
green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal
laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters.
PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with
ruthenium red in biofilms formed by NTHI 2019 lic(ON). The biofilms of all three strains were comparable in viability, the presence of extracellular
DNA, and the presence of sialylated moieties on or between bacteria. In vivo
infection studies using the chinchilla model of
otitis media showed a direct correlation between
PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between
PCho and persistence in the planktonic phase in
middle-ear effusions. Collectively, these data show that
PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.