Abstract
Otitis media (OM), inflammation of the middle ear, is an upper respiratory tract infection (URTI) caused by viral and bacterial infections. OM is the most common pediatric disease and the leading cause of pediatric health care visits, antibiotic usage and surgery. A subset of OM affected children develops chronic otitis media with effusion (COME) and/or recurrent otitis media (ROM), with significant consequences on hearing and development. The aim of this research was to find novel risk factors that increase OM susceptibility by looking at both bacterial communities during URTI and host genetics.
To explore how viral infection affects bacterial composition of the nasopharynx in the setting of URTI, two complementary approaches were used in a longitudinal examination following experimental viral challenge. Ten healthy adults were infected with a rhinovirus, and nasal lavage fluid (NLF) samples were collected before, during, and after inoculation. Microarray technology tested samples for a panel of species, and sequencing of the 16S rRNA bacterial gene was used to survey the relative abundance of bacteria down to the genus level. Pathogenic bacteria were detected in NLF samples from healthy adults, including species known to cause OM (Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae). Sequencing results demonstrated that relative abundance of two genera Neisseria (P=0.02) and Propionibacterium (P=0.04) were significantly different between infected and not infected individuals. No significant shifts in the microbiome were detected between time points before, during, or after inoculation. This understanding of the complexity of the nasopharyngeal microbiome is important for future study design and appropriate treatment of URTIs.
To discover novel OM host risk factors, a multi-stage approach was used. First, a genome-wide association study (GWAS) of COME/ROM was conducted using a family-based population. The most significantly associated SNP, rs1110060, is located on chromosome 15 in an intron of kinesin family member 7 (KIF7). Association at SNP rs10497394, an intergenic SNP on chromosome 2, was replicated in an independent family-based population of OM. Fine-mapping of associated regions of chromosomes 2 and 15 was carried out using targeted resequencing and subsequent genotyping. Six SNPs were significantly associated (P<2.6x10-4), two in the chromosome 2 region and four in the chromosome 15 region. Explorations of the functional potential of these two loci were carried out. Investigation of chromatin marks revealed an enhancer region on chromosome 2 in the human middle ear epithelial cell line, HMEEC-1. The “G” risk allele of the most significantly-associated chromosome 15 SNP, rs1110060, was found to impact expression of IQGAP1 in adenoids (P=0.026). Also, both lipopolysaccharide (LPS) and TGFβ treatment were found to regulate expression of KIF7 in HMEEC-1.
The data presented in this dissertation show the complex nature of the microbiome of the nasopharynx during URTI, and we have identified novel regions and genes involved in susceptibility to OM. Moreover, functional assays support the participation of previously unsuspected mechanisms in OM pathogenesis. In summary, this research has provided insights into OM susceptibility from both host and bacterial perspectives.
