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

Nonencapsulated Haemophilus influenzae frequently persists in the lungs of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) patie nts for prolonged periods of time. The bacteria are not eradicated by antib iotic treatment of the patients or by specific antibodies that are found in the sputum and sera of these patients. We investigated whether H. influenz ae, when localized in lung epithelial cell layers, is shielded from antibio tics and from antibody-mediated bactericidal activity of specific antibodie s. An in vifro model system consisting of lung epithelial NCI-H292 cells on permeable supports was developed to allow long term association of H. infl uenzae with the cells. Microscopic examination showed increasing numbers of H. influenzae bacteria between the epithelial cells up to 24 h of incubati on. 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. All H. influenzae strains, and one Haemo philus parainfluenzae strain tested penetrated into the cell layer as deter mined by gentamicin killing. Cell-associated bacteria were shielded from th e bactericidal activity of several antibiotics and from antibody-mediated b actericidal activity. After prolonged incubation in the cell system in the presence of a specific bactericidal antibody against major outer membrane p rotein (MOMP) P2, antigenic variation occurred due to a point mutation in t he MOMP P2 gene, similar to point mutations observed in vivo. We conclude t hat penetration of H. influenzae between lung epithelial cells results in s hielding the bacteria from killing by antibody dependent defense mechanisms and by antibiotics. Therefore, penetration of H. influenzae between epithe lial cells may contribute to the persistence of this microorganism in COPD and CF patients. (C) 1999 Academic Press.