Role of biofilm formation and antimicrobial resistance in urogenital Haemophilus influenzae isolates

Haemophilus influenzae (Hi) is increasingly detected in genital infections in humans and linked to septic abortion. We aimed to characterize 123 non-capsulated (non-typeable) Hi isolates from genital sites in women and investigate their bacteriological characteristics, antimicrobial resistance profiles, and genetic relationships. We screened H. influenzae isolates received at the French National Reference Centre for Meningococci and Hi between 2017 and 2022. All isolates from genital sites in women and placenta were selected, in addition to isolates from gastric fluids of neonates less than 4 days old. The selected isolates were characterized by antibiotic susceptibility testing (amoxicillin, ampicillin, third-generation cephalosporins, ciprofloxacin, co-trimoxazole, and rifampicin), serotyping, whole-genome sequencing and biofilm formation. All 123 identified bacterial isolates were non-capsulated. Forty-six isolates (37.4%) were resistant to amoxicillin and ampicillin, including 12 beta-lactamase positive. Resistance to third-generation cephalosporins was observed in 20 isolates (16.2%), to co-trimoxazole in 20 isolates, and to ciprofloxacin in 2 isolates. Multi-drug resistance (0.81%) was observed in one isolates. Whole-genome sequencing revealed high diversity: 26 clonal complexes, with 15 unassigned. Biofilm gene analysis clustered isolates into subgroups, including one enriched with neonatal gastric fluid and placenta isolates. These isolates formed strong biofilms, but biofilm formation was reduced with anti-P5 protein antibodies, which is involved in biofilm formation. Heterogeneous unencapsulated Hi isolates associated with genital infections in women exhibit substantial antimicrobial resistance and enhanced biofilm-forming capacity, which may benefit from anti-biofilm treatments.