Increased biofilm formation by nontypeable Haemophilus influenzae isolates from patients with invasive disease or otitis media versus strains recovered from cases of respiratory infections

Biofilm formation by nontypeable (NT) Haemophilus influenzae remains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.This study was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (PI 0901904) and MINECO (SAF2012- 31166) and by CIBER de Enfermedades Respiratorias, CIBERES (CB06/ 06/0037), run by the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The work with the BioFilm ring test was performed in the framework of a collaboration with Biofilm Control. C.P. was supported by FPU grant (Formación de Profesorado Universitario, Ministerio de Educación, Spain). S.M. was supported by Sara Borrell postdoctoral contract CD10/ 00298 from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain