D-084. Sub-Lethal Concentrations of Antimicrobial Peptides Alter Biofilm Formation by Nontypeable Haemophilus influenzae

J. A. Jurcisek, K. M. Mason, L. O. Bakaletz;
The Res. Inst. at Nationwide Children's Hosp., Columbus, OH.

The upper airway has an important role in the first encounter of antigens and pathogens. The epithelial lining of the upper airway has developed several mechanisms for protection against microorganisms. There are nonspecific mechanical factors such as mucociliary clearance and triggering of sneezing which contribute to the prevention of microbial invasion. Antimicrobial peptides (AP), expressed by mucosal surfaces, co-operate with these mechanical factors. Nontypeable Haemophilus influenzae (NTHI) is a commensal which resides in the human nasopharynx despite the presence of the constitutively expressed APs. As a commensal, NTHI has developed multiple mechanisms to counteract the host response during colonization of this niche. One mechanism may be the formation of a biofilm, as biofilm formation has been shown to facilitate increased survivability of bacteria under stressful conditions. This protection is mediated by the extracellular matrix, which can block access to bacteria, by electrostatic interactions, or by the bacteria within the biofilm having an altered metabolic rate. We hypothesized that NTHI would respond to the environmental stresses in the upper airway by formation of biofilm, and that exposure to AP would induce the formation of a more robust biofilm. To that end, we first exposed biofilms formed by NTHI when grown in chamber slides, to either hBD3, LL37, or mellitin and found that compared to planktonic bacteria, bacteria within the biofilm were more resistant to killing by these APs. We then exposed NTHI biofilms to a sub-lethal concentration of mellitin for 24 hours. By microscopy, biofilms exposed to mellitin had an altered morphology. Bacteria harvested from duplicate wells showed that the bacterial counts were similar, indicating that while exposure to a sublethal concentration of AP did not kill the bacteria, it nonetheless influenced the structure of the biofilm formed by NTHI. We are currently investigating the biological consequences of the observed altered biofilm structure as part of our overarching goal of understanding of the role of APs in the upper airway. Work funded by RO1 DC03915 to LOB from NIH/NIDCD