Haemophilus influenzaelocalized in epithelial cell layers is shielded from antibiotics and antibody-mediated bactericidal activity

Abstract

NonencapsulatedHaemophilus influenzaefrequently persists in the lungs of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) patients for prolonged periods of time. The bacteria are not eradicated by antibiotic treatment of the patients or by specific antibodies that are found in the sputum and sera of these patients. We investigated whetherH. influenzae, when localized in lung epithelial cell layers, is shielded from antibiotics and from antibody-mediated bactericidal activity of specific antibodies. Anin vitromodel system consisting of lung epithelial NCI-H292 cells on permeable supports was developed to allow long term association ofH. influenzaewith the cells. Microscopic examination showed increasing numbers ofH. influenzaebacteria between the epithelial cells up to 24 h of incubation. Coinciding with the microscopic observations the maximum number of cell-associated bacteria surviving gentamicin treatment of the cell layers was obtained after 24 h of incubation. AllH. influenzaestrains, and oneHaemophilus parainfluenzaestrain tested penetrated into the cell layer as determined by gentamicin killing. Cell-associated bacteria were shielded from the bactericidal activity of several antibiotics and from antibody-mediated bactericidal activity. After prolonged incubation in the cell system in the presence of a specific bactericidal antibody against major outer membrane protein (MOMP) P2, antigenic variation occurred due to a point mutation in the MOMP P2 gene, similar to point mutations observedin vivo. We conclude that penetration ofH. influenzaebetween lung epithelial cells results in shielding the bacteria from killing by antibody dependent defense mechanisms and by antibiotics. Therefore, penetration ofH. influenzaebetween epithelial cells may contribute to the persistence of this microorganism in COPD and CF patients.