Open Access Highly Accessed Research article

Non typable-Haemophilus influenzae biofilm formation and acute otitis media

Assaf Mizrahi1, Robert Cohen2, Emmanuelle Varon3, Stephane Bonacorsi4, Stephane Bechet2, Claire Poyart1, Corinne Levy2 and Josette Raymond1*

Author Affiliations

1 Université Paris Descartes, Hôpital Cochin, Bactériologie, 27 rue du Faubourg Saint Jacques, 75679 Paris cedex 14, France

2 ACTIV, Saint Maur des Fossés, Paris, France

3 Université Paris Descartes, Hôpital Georges Pompidou, Bactériologie, Paris, France

4 Université Diderot, Hôpital Robert Debré, Bactériologie, Paris, France

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BMC Infectious Diseases 2014, 14:400  doi:10.1186/1471-2334-14-400

Published: 19 July 2014



Non-typable Haemophilus influenzae (NT-Hi) infection is frequently associated with acute otitis media (AOM) treatment failure, recurrence or chronic otitis media. Persistence of otopathogens in a biofilm-structured community was implicated in these situations. Here, we compared biofilm production by H. influenzae strains obtained by culture of middle ear fluid (MEF) from children with AOM treatment failure and by strains isolated from nasopharyngeal (NP) samples from healthy children or those with AOM (first episode or recurrence). We aimed to evaluate an association of clinical signs and in vitro biofilm formation and establish risk factors of carrying a biofilm-producing strain.


We used a modification of the microtiter plate assay with crystal violet staining to compare biofilm production by 216 H. influenzae strains: 41 in MEF from children with AOM treatment failure (group MEF), 43 in NP samples from healthy children (NP group 1), 88 in NP samples from children with a first AOM episode (NP group 2, n = 43) or recurrent (NP group 3, n = 45) and 44 in NP samples from children with AOM associated with conjunctivitis (NP group 4).


At all, 106/216 (49%) H. influenzae strains produced biofilm as did 26/43 (60.5%) in NP samples from healthy children. Biofilm production in MEF samples and NP samples did not significantly differ (40.5% vs 60.5%, 55.8%, 56.8% and 31.1% for NP groups 1, 2, 3 and 4, respectively). On multivariate analysis, only presence of conjunctivitis was significantly associated with low biofilm production (OR = 0.3, CI [0.16-0.60], p = 0.001). The ampicillin resistance of H. influenzae produced by penicillin-binding protein modification was significantly associated with low biofilm production (p = 0.029).


We found no association of biofilm production and AOM treatment failure or recurrence. Biofilm production was low from H. influenzae strains associated with conjunctivitis-otitis syndrome and from strains with modified penicillin-binding protein.

Haemophilus influenzae; Biofilm; AOM; Conjunctivitis