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Otitis media with effusion (OME) is a biofilm driven disease and commonly accepted otopathogens, such as  Haemophilus influenzae, Streptococcus pneumonia, and Moraxella catarrhalis , have been demonstrated to form polymicrobial biofilms within the middle ear cleft. However,  Alloiococcus otitidis  ( A. otitidis ), which is one of the most commonly found bacteria within middle ear aspirates of children with OME, has not been described to form biofilms. The aim of this study was to investigate whether  A. otitidis  can form biofilms and investigate the impact on antibiotic susceptibility and survivability in polymicrobial biofilms with  H. influenzae in vitro . The ability of  A. otitidis  to form single-species and polymicrobial biofilms with  H. influenzae  was explored. Clinical and commercial strains of  A. otitidis  and  H. influenzae  were incubated in brain heart infusion with and without supplementation. Biofilm was imaged using confocal laser scanning microscopy and scanning electron microscopy. Quantification of biofilm biomass and viable bacterial number was assessed using crystal violet assays and viable cell counting in both optimal growth conditions and in adverse growth conditions (depleted media and sub-optimal growth temperature). Antimicrobial susceptibility and changes in antibiotic resistance of single-species and multi-species co-culture were assessed using a microdilution method to assess minimal bactericidal concentration and E-test for amoxicillin and ciprofloxacin.  A. otitidis  formed single-species and polymicrobial biofilms with  H. influenzae . Additionally, whilst strain dependent, combinations of polymicrobial biofilms decreased antimicrobial susceptibility, albeit a small magnitude, in both planktonic and polymicrobial biofilms. Moreover,  A. otitidis  promoted  H. influenzae  survival by increasing biofilm production in depleted media and at suboptimal growth temperature. Our findings suggest that  A. otitidis  may play an indirect pathogenic role in otitis media by altering  H. influenzae  antibiotic susceptibility and enhancing growth under adverse conditions.

Alloiococcus otitidis Forms Multispecies Biofilm with Haemophilus influenzae: Effects on Antibiotic Susceptibility and Growth in Adverse Conditions