(542) Genomic diversity of nontypeable Haemophilus influenzae impacts colonization and persistence in vivo

Background: Cystic fibrosis (CF) causes reduced mucociliary clearance in the airways, inflammation, and epithelial damage, all of which increase patient susceptibility to respiratory infections. Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative opportunist that is one of the most common early-life pathogens found in children with CF. NTHi strains exhibit a wide range of genetic diversity which potentially impacts colonization and persistence in vivo. This work aims to identify genes of interest in NTHi clinical CF isolates and characterize how they contribute to disease consequences of respiratory infection.

Methods: Using three CF sputum-derived clinical isolates of NTHi compared to a reference genome, we applied comparative genomic analyses to identify core and variable genomic regions. We characterized each of these strains for colonization and persistence, inflammation, and biofilm formation in a murine model of respiratory infection and in CF bronchial epithelial cells (CFBEs) grown at airway liquid interface. Isogenic mutations of several genes of interest, including torZ, dlgD, and hktE, were created to understand the importance of epithelial adherence, biofilm formation, and survival under oxidative stress.

Results: Genomic comparisons among three clinical isolates (NTHi HI-1, HI-3, and HI-8) compared to a well-characterized laboratory strain, NTHi 86-028NP, reveal that NTHi HI-1 is particularly phylogenetically distinct. Infection of CFBE cells shows that all clinical isolates, but particularly strain NTHi HI-1, disrupt tight junction barrier integrity in addition to causing mild cytotoxic effects. In our mouse model, strain NTHi HI-1 resulted in increased persistence, increased histopathological damage, and higher measurements of leukocyte infiltration and cytokine influx in bronchoalveolar lavage fluid (BALF) following infection. NTHi HI-1 also produces more biofilm and has higher catalase activity than other strains, suggesting these factors likely contribute to persistence in respiratory diseases. Isogenic mutants of torZ, dlgD, and hktE in strain NTHi HI-1 were used to verify that biofilm formation and catalase production contribute to increased colonization and persistence in vivo.

Conclusions: Phylogenetic comparison revealed significant diversity between clinical isolates NTHi HI-1, HI-3, HI-8 and reference genome NTHi 86-028NP. This diversity was also seen in phenotypic variations in both cell culture and animal models. Genomic regions of interest that are unique to NTHi HI-1 reveal that several genes, torZ, dlgD, and hktE, likely contribute to chronic respiratory infections in CF.

Acknowledgements: We thank Timothy Starner (University of Iowa Children’s Hospital) for providing bacterial strains and Michael Apicella (University of Iowa) for providing NTHi monoclonal antibody.

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