Otitis media (OM) is an infection of the middle ear (ME) and one of the most prevalent diseases in young children. It can lead to hearing loss, cholesteatoma, and meningitis and represents a substantial financial burden as it is a main cause of pediatric antibiotic prescription and surgery; the rapidly rising numbers of AMR bacteria, however, mean alternative therapies are urgently needed. NTHi, one of the most common otopathogens in acute OM, is an opportunistic pathogen of the human respiratory tract, commonly associated with respiratory infections. Interactions with airway epithelial cells have been investigated, however little is known about its contribution to disease in the ME. Interactions of NTHi with cell receptors, such as platelet-activating factor receptor (PAFr), through lipooligosaccharide (LOS) phosphorylcholine (PCho), have been linked to the invasion of airway epithelial cells. Still, to date, only the adhesion of NTHi in ME has been investigated. This study investigates the mechanisms of NTHi infection and invasion by examining the effects of NTHi PCho on human ME epithelial cell invasion using gentamicin protection assays. Invasion of strains differentially expressing the protein, H457 (wt), H446 (PCho-), H491 (PCho+) will be assessed. The role of epithelial PAFr in the invasion of bacteria will also be determined by blocking bacterial access to the receptor via competitive binding with a PAFr antagonist, ABT-491. A better understanding of how NTHi evades the immune system and promotes persistence will allow us to elucidate novel therapeutic strategies, avoiding repetitive use of antibiotics.