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Journal of Bacteriology, November 2007, p. 8300-8307, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00532-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Phosphorylcholine Expression by Nontypeable Haemophilus influenzae Correlates with Maturation of Biofilm Communities In Vitro and In Vivo{triangledown}

Wenzhou Hong, Bing Pang, Shayla West-Barnette, and W. Edward Swords*

Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157

Received 6 April 2007/ Accepted 6 June 2007

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 licON). 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 licON. 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Wake Forest University Health Sciences, 5101A Gray Building, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336) 713-5049. Fax: (336) 716-9928. E-mail: wswords{at}wfubmc.edu

{triangledown} Published ahead of print on 15 June 2007.

Journal of Bacteriology, November 2007, p. 8300-8307, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00532-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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