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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Biofilm formation by clinical isolates of nontypeable H. influenzae. Bars represent the means of three independent experiments performed in quadruplicate. Error bars represent standard deviation. Y axis is optical density at 570 nm. la: middle ear isolates: a. 83; b. 955; c. 1128; d. 1749; e. 1826; f. 2536; g. 2846; h. C4556; i. 8131; j. 8183; k. 8981; 1. 9289; m. C1425; n. DL200; o. 4505. 1b: sputum isolates: a. 5P10H1; b. 5P11H1; c. 5P30H; d. 6P5H; e. 6P8H1; f. 13P26H1; g. 14P13H5; h. 31P13H1; i. 56P40Hl; j. 74P10H1; k. 7P49H1; l. 6P32H1; m. 12P37H1; n. 13P33H1; o.18P16H1.

Figure 2

Time course of biofilm formation by four strains of nontypeable H. influenzae as noted on the right. X axis: time in hours. Y axis: optical density at 570 nm.

Figure 3

Coomassie blue stained sodium dodecyl sulfate polyacrylamide gel of cell envelopes of nontypeable H. influenzae 74P10H1 grown on agar plates (lane a) and as a biofilm (lane b). Molecular mass standards are noted in kDa on the left.

Figure 4

Immunoblot assays of cell envelopes of nontypeable H. influenzae 74P10H1 grown on agar plates (lanes a) and as a biofilm (lanes b). Panels were probed with antibodies to outer membrane antigens as follows: P2: rabbit polyclonal antiserum raised to loop 6 of P2; P5: monoclonal antibody 2C7; P6: monoclonal antibody 7F3; LOS (lipooligosaccharide): monoclonal antibody 6E4. Molecular mass standards are noted in kDa on the left.