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 Haemophilus influenzae as they presented an unusual multi-resistant profile. Methods: All three isolates were sequenced by WGS and mapped to the Reference isolate Hi Rd KW20. Shorten Uptake signal sequence (USS) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared to the database of Blast nucleotide to look for similarity with other Haemophilus species. Their Antimicrobial Resistance (AMR) genotype was studied. Core-genome MLST was performed on NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. Results:  The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI regioninvolved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST Clonal Complex (CC) 422, and WGS revealed that the three were very similar. They harbored mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility.  Conclusion: The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked in majority to the presence of USS and exchange within the Pasteurellaceae family. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR , both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infectionsshould be conducted.