Haemophilus spp in COPD patients with long-term azithromycin treatment

Patients with severe chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter respiratory colonising bacteria. The goal of this study was to identify genetic changes in Haemophilus influenzae and Haemophilus parainfluenzae colonising strains during prolonged azithromycin treatment. Azithromycin susceptibility was studied in respiratory H. influenzae and H. parainfluenzae isolates from COPD patients with long-term azithromycin treatment at Hospital Universitari de Bellvitge (Spain). Strains were phylogenetically characterised by pulse-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). Antimicrobial resistance determinants and the genetic adaptation traits were studied in the persistent strains. Four patients were persistently colonised by H. influenzae (P02 by ST139-ST2111; P08 by ST107; P11 by ST147 and ST2480; and P13 by ST165), and two patients by H. parainfluenzae (P04 by HPAR04.05; and P11 by HPAR11.02). All strains isolated before treatment were azithromycin susceptible, but developed resistance within the first months, with the exception of patient 2 strains (P02), who discontinued the treatment due to adverse effects: P04-HPAR04.05 acquired changes in RlmC; P08-ST107 gradually acquired 23S rRNA mutations; P11-ST2480 and P13-ST165 showed alterations in ribosomal proteins L4 and L22; and P11-HPAR11.02 carried the MefE/MsrD efflux pumps in an integrative conjugative element with tet(M)-MEGA element, which was later identified in H. influenzae P11-ST147. Most genetic changes occurred in genes associated with cell wall (22.2%) and inorganic ion transport and metabolism (13.5%). All persistent H. influenzae strains had phase variation changes in licA and hgpB, linked to lipooligosaccharide synthesis and heme uptake, respectively. Other genes (lex1, lic3A, hgpC, and fadL) also presented recurrent alterations. In addition, persistent strains showed loss or acquisition of prophages and an accumulation of genetic changes in prophage-associated regions. In conclusion, long-term azithromycin therapy results in macrolide resistance through mutations and horizontal gene transfer, as well as changes related to bacterial adaptation and survival.