Nontypeable Haemophilus influenzae (NTHi) commonly causes
otitis media,
chronic bronchitis in
emphysema, and early airway
infections in
cystic fibrosis. Long-term, low-dose
azithromycin has been shown to improve clinical outcomes in chronic
lung diseases, although the mechanism of action remains unclear. The inhibition of bacterial biofilms by
azithromycin has been postulated to be one mechanism mediating these effects. We hypothesized that subinhibitory concentrations of
azithromycin would affect NTHi biofilm formation. Laboratory strains of NTHi expressing
green fluorescent protein and
azithromycin-resistant clinical isolates were grown in flow-cell and static-culture biofilm models. Using a range of concentrations of
azithromycin and
gentamicin, we measured the degree to which these
antibiotics inhibited biofilm formation and persistence. Large biofilms formed over 2 to 4 days in a flow cell, displaying complex structures, including towers and channels. Subinhibitory concentrations of
azithromycin significantly decreased biomass and maximal thickness in both forming and established NTHi biofilms. In contrast, subinhibitory concentrations of
gentamicin had no effect on biofilm formation. Furthermore, established NTHi biofilms became resistant to
gentamicin at concentrations far above the MIC. Biofilm formation of highly resistant clinical NTHi isolates (
azithromycin MIC of > 64 microg/ml) was similarly decreased at subinhibitory
azithromycin concentrations. Clinically obtainable
azithromycin concentrations inhibited biofilms in all but the most highly resistant isolates. These data show that subinhibitory concentrations of
azithromycin have antibiofilm properties, provide mechanistic insights, and supply an additional rationale for the use of
azithromycin in chronic biofilm
infections involving H. influenzae.