Otitis media is an extremely common pediatric ailment caused by opportunists that reside within the nasopharynx. Inflammation within the upper airway can promote ascension of these opportunists into the middle ear chamber. Otitis media can be chronic/recurrent in nature, and a wealth of data indicates that in these cases the bacteria persist within biofilms. Epidemiological data demonstrates most cases of otitis media are polymicrobial, which may have significant impact on antibiotic resistance. In this study, we used in vitro biofilm assays and rodent infection models to examine the impact of polymicrobial infection with Moraxella catarrhalis and Streptococcus pneumoniae (pneumococcus) on biofilm resistance to antibiotic treatment and persistence in vivo. Consistent with prior work, M. catarrhalis conferred beta-lactamase dependent passive protection from beta-lactam killing to pneumococci within polymicrobial biofilms. Moreover, pneumococci increased resistance of M. catarrhalis to macrolide killing in polymicrobial biofilms. However, pneumococci increased colonization in vivo by M. catarrhalis in a quorum signal-dependent manner. We also found that co-infection with M. catarrhalis affects middle ear ascension of pneumococci in both mice and chinchillas. Therefore, we conclude that residence of M. catarrhalis and pneumococci within the same biofilm community significantly impacts resistance to antibiotic treatment and bacterial persistence in vivo.

Acinetobacter baumannii has few known virulence factors and yet causes a variety of opportunistic infections. Many gram-negative bacteria are directly killed by complement, but we hypothesized that A. baumannii would be resistant to serum killing. A serum bactericidal assay assessed the resistance of seven A. baumannii isolates to serum killing, and C2-deficient serum was used to examine its activation of the alternative pathway. Flow cytometry was utilized to determine whether complement regulator factor H (FH) was bound by A. baumannii, and to assay C3 deposition on cells. A microtiter biofilm assay compared biofilm production among isolates. Of seven isolates, four were serum sensitive and three were serum resistant. The C2-deficient serum demonstrated that A. baumannii can activate the alternative pathway. None of the isolates bound FH. Serum-resistant strains accumulated little C3 when exposed to human serum, while sensitive strains had a high amount of surface C3 deposition. Biofilm production varied extensively among strains. Most serum-resistant isolates formed a substantial amount of biofilm, while sensitive isolates produced negligible amounts of biofilm. Our data indicate that some strains of A. baumannii are resistant to serum killing and produce biofilms and by understanding the resistance mechanisms used by this bacterium, we can further elucidate its complex pathogenicity.

Acinetobacter baumannii is an important nosocomial pathogen. Infections are often preceded by intubation or catheter use, promoting the formation of biofilm, and some strains are able to cause severe cases of bacteremia because of their ability to resist killing by complement. We identified a secreted serine protease, termed "PKF," that provided resistance to complement killing and suppressed biofilm formation. Serum resistance was abrogated in A. baumannii treated with protease inhibitors, as well as in a PKF-negative mutant. Serum resistance could be restored by recombinant PKF, which was shown to reduce the complement activity of normal human serum by almost 50%. PKF was shown to inhibit biofilm formation, because the PKF-negative mutant and wild-type A. baumannii treated with protease inhibitors produced biofilm that could be inhibited by addition of recombinant PKF. Our data indicate that PKF is required for serum resistance and that it suppresses biofilm formation in A. baumannii.

Streptococcus pneumoniae (pneumococcus) is both a widespread nasal colonizer and a leading cause of otitis media, one of the most common diseases of childhood. Pneumococcal phase variation influences both colonization and disease and thus has been linked to the bacteria's transition from colonizer to otopathogen. Further contributing to this transition, coinfection with influenza A virus has been strongly associated epidemiologically with the dissemination of pneumococci from the nasopharynx to the middle ear. Using a mouse infection model, we demonstrated that coinfection with influenza virus and pneumococci enhanced both colonization and inflammatory responses within the nasopharynx and middle ear chamber. Coinfection studies were also performed using pneumococcal populations enriched for opaque or transparent phase variants. As shown previously, opaque variants were less able to colonize the nasopharynx. In vitro, this phase also demonstrated diminished biofilm viability and epithelial adherence. However, coinfection with influenza virus ameliorated this colonization defect in vivo. Further, viral coinfection ultimately induced a similar magnitude of middle ear infection by both phase variants. These data indicate that despite inherent differences in colonization, the influenza A virus exacerbation of experimental middle ear infection is independent of the pneumococcal phase. These findings provide new insights into the synergistic link between pneumococcus and influenza virus in the context of otitis media.