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Open Access Open Badges Research article

Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili

Timothy F Murphy12* and Charmaine Kirkham12

Author Affiliations

1 Division of Infectious Diseases, Department of Medicine, and Department of Microbiology, University at Buffalo, State University of New York

2 Department of Veterans Affairs Western New York Healthcare System (151) 3495 Bailey Avenue, Buffalo, NY 14215, USA

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BMC Microbiology 2002, 2:7  doi:10.1186/1471-2180-2-7

Published: 15 April 2002



Nontypeable Haemophilus influenzae is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Several lines of evidence suggest that the bacterium grows as a biofilm in the human respiratory tract.


Fifteen clinical isolates from middle ear fluid of children with otitis media and 15 isolates from sputum of adults with COPD were studied in an in vitro assay of biofilm formation. Striking variability among isolates was observed in their ability to form biofilms. Analysis of cell envelopes revealed minimal differences in banding patterns in polyacrylamide gels, alteration of expression of an epitope on lipooligosaccharide, and preservation of expression of selected epitopes on outer membrane proteins P2, P5 and P6 in biofilms compared to planktonically grown cells. A pilus-deficient variant showed a marked impairment in biofilm formation compared to its isogenic parent.


Nontypeable H. influenzae forms biofilms in vitro. Clinical isolates show substantial variability in their ability to grow as biofilms. Three major outer membrane proteins (P2, P5 and P6) are expressed during growth as a biofilm. Expression of lipooligosaccharide is altered during growth as a biofilm compared to planktonic growth. Pili are important in biofilm formation. As the role of biofilms in human infection becomes better defined, characterization of biofilms may be important in understanding the pathogenesis of infection and immune response to nontypeable H. influenzae in children with otitis media and adults with COPD.