Identification of two novel ß-lactamases in H. influenzae

The spread of multidrug resistance through integrative and conjugative elements (ICEs) among Haemophilus spp., following the emergence of blaCTX-M-15 in H. parainfluenzae, was a risk of horizontal transfer to H. influenzae, with potential implications for reduced cephalosporin susceptibility. In this study, two clinical isolates, PTHi-14525 (Portugal) and HUB-HI042681 (Spain), were characterized by antimicrobial susceptibility testing and hybrid whole-genome sequencing (Illumina and Oxford Nanopore) to investigate their resistance profiles and genomic context. PTHi-14525, isolated from a 67-year-old male with severe community-acquired pneumonia requiring ECMO, displayed a multidrug-resistant phenotype, being resistant to ampicillin, cephalosporins, trimethoprim–sulfamethoxazole, and azithromycin. It carried blaCTX-M-15 and blaTEM-1, together with mef(A) and msr(D) within a Tn7471 transposon located in an ICEHpaHUB5-like element inserted between metB and thrC. A second copy of the blaCTX-M-15 gene was integrated into a prophage. PBP3 substitutions (D350N, G490N, N526K, A530S) were consistent with ß-lactam resistance. HUB-HI042681, recovered from a 68-year-old female with bacteremic pneumonia, was resistant to ß-lactams and aminoglycosides and carried a novel ICEHinHUB1, harbouring blaOXA-9, blaTEM-1, aadA1, aac(6')-Ib, and aph(3')-Ia integrated between pgsA and ppa. This element resembled ICEHpaHUB1 but contained rearrangements within Tn7077. PBP3 substitutions (D350N, M377I, A502V, N526K) were also detected. These findings represent the first report of blaCTX-M-15 and blaOXA-9 in H. influenzae, underscoring the genomic plasticity of this species. The acquisition of these clinically relevant ß-lactamases broadens the resistome of H. influenzae and raises concern about potential therapeutic limitations, reinforcing the need for sustained genomic surveillance of antimicrobial resistance.