In VitroProbiotic Potential of Hemophilin-producing Strains ofHaemophilus haemolyticus

ABSTRACT Non-typeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections, including otitis media and acute exacerbations of chronic obstructive pulmonary disease. Despite the enormous disease burden associated with NTHi infections, there are currently no effective prevention strategies, and the rapid development of antibiotic resistance is compromising treatment. We previously discovered Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we have assessed the feasibility of this approach by in vitro competition assays between NTHi and Hh strains with varying capacity to produce HPL. HPL-producing strains of Hh exhibited enhanced growth and consistently outcompeted NTHi compared to Hh strains unable to produce the protein. This competitive advantage was maintained over a period of six days, culminating in the complete eradication of NTHi. Expression analysis of HPL during competition coincided with the NTHi-inhibitory capacity of HPL-producers, confirming that inhibition was mediated by the presence of HPL. Together, results suggest that natural levels of HPL production by Hh are sufficient to limit NTHi’s access to heme, even under excess heme conditions unlikely to be encountered in vivo. Further investigation is required to determine the protective capacity of HPL-producers in vivo and their ability to interrupt NTHi colonization of host cells.