Abstract
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.
Highlights
Since the identification of biofilms within the middle ear of children with chronic otitis media with effusion (OME), OME has been considered a biofilm driven disease (Hall-Stoodley et al, 2006; Thornton et al, 2011)
There were a greater number of viable cells in BHIb than in BHIs at 12 h (BHIb, Mean single colony forming units per mL (SCFU/mL) [SD] = 7.7 × 106, [3.1 × 106] vs. BHIs, 1.0 × 106 [7.5 × 105], p = 0.02), 24 h (BHIb, 6.7 × 107 [1.2 × 107] vs. BHIs, 9.0 × 106 [1.0 × 106], p = 0.001), and 48 h (BHIb, 3.2 × 107 [1.6 × 107] vs. BHIs, 5.6 × 106 [5.7 × 105], p = 0.046) (Supplementary Figure 1)
confocal laser scanning microscopy (CLSM) with LIVE/DEAD Baclight bacterial viability stains indicated that when grown in BHIb, A. otitidis formed dense, satellite-aggregations of cells with extracellular matrix, consistent with biofilm (Figure 1A)
Summary
Since the identification of biofilms within the middle ear of children with chronic otitis media with effusion (OME), OME has been considered a biofilm driven disease (Hall-Stoodley et al, 2006; Thornton et al, 2011). The formation of biofilms is significant, as bacteria within biofilms are conferred protection from environmental, host and chemical stressors, and have an increased antibiotic resistance (Donlan and Costerton, 2002; Fergie et al, 2004). There remain biofilm forming bacteria within the middle ear that have yet to be identified (Thornton et al, 2011). One such bacterial species that may potentially be forming biofilms within the middle ear is A. otitidis. The potential of A. otitidis to form biofilms has yet to be investigated
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