Genetic organization and mobile-element basis of β-lactam resistance in a polyclonal respiratory Haemophilus influenzae population
Abstract
Haemophilus influenzae is an important respiratory colonizer and opportunistic pathogen. In the post-Hib vaccine era, non-type b and nontypeable strains have become increasingly associated with clinical disease, while β-lactam resistance complicates treatment and surveillance. To investigate the genetic basis and dissemination of β-lactam resistance, we performed comparative whole-genome analysis of 65 respiratory H. influenzae isolates from sputum and bronchial lavage fluid. The collection showed a highly polyclonal structure, including 28 known sequence types and 12 unassigned isolates. Phylogenetic analysis demonstrated that β-lactamase-positive isolates were distributed across multiple lineages. Genotype-phenotype association analysis identified blaTEM-1 as strongly associated with ampicillin resistance, whereas PBP3 substitutions including ftsI_S357N , ftsI_S385T , and ftsI_L389F were significantly linked to cefuroxime resistance. Several blaTEM -negative but ampicillin-resistant isolates carried similar compound ftsI mutation profiles, indicating that β-lactam susceptibility is shaped by both enzymatic resistance and PBP3 target modification. Pangenome analysis identified 4,223 gene clusters, comprising 1,224 core, 1,913 dispensable, and 1,086 private clusters. Presence/absence association analysis revealed that significant signals were concentrated in ampicillin resistance and formed a co-occurring dispensable-genome module containing bla , tnpA , tnpR , and multiple hypothetical proteins. This module mapped to a putative ICEHinR2866/ICEHin1056-like mobile genetic region carrying blaTEM-1B , conjugation-associated components, and macrolide resistance determinants. These findings suggest that β-lactam resistance in respiratory H. influenzae is driven by combined effects of β-lactamase production, composite PBP3 substitutions, and ICE-like mobile resistance platforms.