Effect of pH and oxygen on biofilm formation in acute otitis media associated NTHi clinical isolates.

OBJECTIVES/HYPOTHESIS: Biofilms occur in animal models of acute otitis media (AOM) and in children with recurrent AOM (rAOM) and chronic otitis media with effusion (OME). We therefore studied the ability of nontypeable Haemophilus influenzae (NTHi) strains from children to form biofilms in vitro under conditions we presumed occurred in the middle ear during AOM, rAOM, and OME. STUDY DESIGN: Evaluate NTHi isolates for biofilm formation across a pH range under aerobic, microaerophilic, and anaerobic conditions. METHODS: Using a crystal violet biofilm assay we studied 12 NTHi pediatric clinical isolates to investigate biofilm formation over a pH range of 4.5 to 10 under aerobic, microaerophilic, and anaerobic conditions. RESULTS: Our findings included: 1) not all clinical NTHi strains form biofilms (75% did); 2) the pH of middle ear fluid collected from AOM (n = 170; age range, 4-36 months), rAOM (n = 54; age range, 7-36 months), and OME (n = 30; age range, 9-60 months) subjects tested immediately after withdrawal was similar (mean = 8.0;range 7.0-9.0); 3) biofilms formed optimally at pH 8.0, a finding that is consistent with previous studies by other investigators; 4) biofilms did not form under aerobic conditions as likely occurs in AOM, whereas under microaerophilic and anaerobic conditions biofilm formation was observed as likely occurs during rAOM and OME. CONCLUSIONS: We concluded that biofilm formation by NTHi does not occur in all strains, occurs best where the pH = 8.0 and in anaerobic conditions as likely occurs in children during rAOM and OME. However, biofilm formation is limited or absent under aerobic conditions as likely occurs during AOM. LEVEL OF EVIDENCE: NA.