Multiple combination antibiotic susceptibility testing of nontypeable Haemophilus influenzae biofilms

Haemophilus influenzae is a cause of otitis media with effusion (OME). Animal models demonstrate growth of H. influenzae biofilms in OME, which may explain why OME does not respond well to conventional antibiotic therapy. Using a previously developed in vitro model, we performed H. influenzae susceptibility studies to see if H. influenzae biofilm cultures were more resistant to antibiotics than planktonic (broth) cultures, and to determine which antibiotics were most effective against H. influenzae biofilms. H. influenzae isolates were grown as biofilms on polystyrene pins. Biofilm and planktonic minimum inhibitory concentrations (MICs) were measured for 8 antibiotics, and multiple combination testing was performed with 66 groupings of 1, 2, or 3 antibiotics. We found that biofilm cultures were more resistant to antibiotics than planktonic ones. Antibiotic combinations containing rifampin and ciprofloxacin were most effective against biofilms. Biofilm testing reveals differences in effectiveness among antibiotics not apparent from conventional susceptibility testing, and suggests novel antibiotic regimens that could be studied for treatment of OME.