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J. Bacteriol. doi:10.1128/JB.00532-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Phosphorylcholine expression by nontypeable Haemophilus influenzae correlates with maturation of biofilm communities in vitro and in vivo

Department of Microbiology and Immunology, Wake Forest University Health Sciences

^* To whom correspondence should be addressed. Email: wswords{at}wfubmc.edu.

	Abstract

Nontypeable Haemophilus influenzae (NTHi) causes chronic infections that feature the formation of biofilms communities. NTHi variants within biofilms have on their surfaces lipooligosaccharides (LOS) containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in 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 lacking PCho- (NTHi 2019 licD) or constitutively in-phase PCho+ (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 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. Biofilms of all three strains had comparable viability, presence of extracellular DNA, and sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model for 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 planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, maturation of NTHi biofilms. Further, this structure may be disadvantageous in the planktonic phase.

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