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

Murphy TF, Kirkham C - BMC Microbiol. (2002)

Bottom Line: A pilus-deficient variant showed a marked impairment in biofilm formation compared to its isogenic parent.Expression of lipooligosaccharide is altered during growth as a biofilm compared to planktonic growth.Pili are important in biofilm formation.

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Affiliation: Division of Infectious Diseases, Department of Medicine, University at Buffalo, State University of New York, USA. murphyt@acsu.buffalo.edu

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.

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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.
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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.

Mentions: To further assess the expression of epitopes on selected individual outer membrane antigens, purified cell envelopes were assayed in immunoblot assays with well characterized polyclonal and monoclonal antibodies. Polyclonal antibodies to a peptide corresponding to loop 6 of the outer membrane protein P2 [33], which is the only known porin of nontypeable H. influenzae, revealed expression of P2 in the bacteria grown as a biofilm and in planktonically grown bacteria (Figure 4). Similarly, the epitopes recognized by monoclonal antibodies to outer membrane protein P5, an OMP A-like protein, and P6, a highly conserved protein in all strains of nontypeable H. influenzae, were expressed in the outer membranes of cells grown as biofilms and cells grown planktonically (Figure 4).


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

Murphy TF, Kirkham C - BMC Microbiol. (2002)

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.
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Related In: Results  -  Collection

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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.
Mentions: To further assess the expression of epitopes on selected individual outer membrane antigens, purified cell envelopes were assayed in immunoblot assays with well characterized polyclonal and monoclonal antibodies. Polyclonal antibodies to a peptide corresponding to loop 6 of the outer membrane protein P2 [33], which is the only known porin of nontypeable H. influenzae, revealed expression of P2 in the bacteria grown as a biofilm and in planktonically grown bacteria (Figure 4). Similarly, the epitopes recognized by monoclonal antibodies to outer membrane protein P5, an OMP A-like protein, and P6, a highly conserved protein in all strains of nontypeable H. influenzae, were expressed in the outer membranes of cells grown as biofilms and cells grown planktonically (Figure 4).

Bottom Line: A pilus-deficient variant showed a marked impairment in biofilm formation compared to its isogenic parent.Expression of lipooligosaccharide is altered during growth as a biofilm compared to planktonic growth.Pili are important in biofilm formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, University at Buffalo, State University of New York, USA. murphyt@acsu.buffalo.edu

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.

Show MeSH
Related in: MedlinePlus