Whole Genome Sequencing of Haemophilus influenzae. Multiple interspecies recombination events documented by whole genome sequencing in multidrug resistant Haemophilus influenzae clinical isolates

Introduction: Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium but the rise of resistance against beta-lactams and other critically important antibiotics is now an increasing concern. We describe here the whole genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018 – 2019 by the Belgian National Reference Centre (NRC) for Hi as they presented an unusual multi-resistant profile. Results: The three isolates were resistant to ampicillin, cefuroxime, cefotaxime, ceftriaxone, ciprofloxacin, macrolides and trimethoprim-sulfamethoxazole. Two of them were also resistant to co-amoxiclav and showed a reduced susceptibility to meropenem. WGS analysis revealed that the three isolates were very similar. They belonged to the MLST Clonal Complex (CC) 422, thus closely related to a multidrug resistant clone circulating in Japan. Their Antimicrobial Resistance (AMR) genotype was studied. They harbored mobile genetic elements (plasmids and transposons carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodeling events in ompP2 that might be related to lower penems susceptibility or heteroresistance profile. The isolates also harbored interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. Conclusion: The Hi evolution towards AMR is a complex and poorly understood phenomenon. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping AMR in Hi, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.