Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, densitydependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

On the basis of these data, we conclude that pneumococci form biofilms in vivo and that this process may be intertwined with the formation of neutrophil extracellular traps. These findings provide new insights into the potential causes of antibiotic treatment failure and bacterial persistence in chronic pneumococcal otitis media.

Otitis media is one of the most common pediatric public health problems worldwide. It is caused by bacterial opportunists that normally reside in the nasopharynx such as non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Once established, these infections are largely resistant to clearance by host immune effectors. Much remains to be learned regarding how these pathogens persist in the middle ear. Dendritic cells (DC) are the major antigen presenting cell at mucosal surfaces and are the link between the innate and adaptive immune responses. The goal of this study was to determine if NTHi and Mcat activate DC in vitro, and if these activated DC are able to stimulate effector T cell responses. Mouse bone marrow derived DC were stimulated with NTHi strain 86-028NP or 2019, Mcat strain 7169, or lipooligosaccharide (LOS) and the level of co-stimulatory molecule expression (CD86, CD80, CD44 and I-A[b]) was measured using flow cytometry. Bacteria activated DC were then used to stimulate CD8+ and CD4+ T cells, and their proliferation and cytokine production were measured. The results indicate that both NTHi and Mcat stimulate DC maturation in vitro. Based on this data, it appears that the mechanism of persistence of these agents in otitis media does not involve evasion of antigen presenting cells. Ongoing work in our laboratory will focus on defining why immune effectors fail to resolve these infections.