Comparison of Phenotypic and Whole-Genome Sequencing-Derived Antimicrobial Resistance Profiles of Legionella pneumophila Isolated in England and Wales from 2020 to 2023.

Background: Antimicrobial resistance (AMR) in Legionella pneumophila is emerging as a concern, particularly with resistance to macrolides and fluoroquinolones. Although clinically significant resistance in Legionella pneumophila remains uncommon, systematic genomic surveillance using whole-genome sequencing (WGS) is needed to anticipate treatment failure as metagenomic diagnostics move toward routine use. Objectives: We assessed the UK Health Security Agency AMR pipeline for predicting resistance in L. pneumophila by analysing 522 L. pneumophila isolates from England and Wales (2020-2023) together with nine database sequences that carry confirmed 23S rRNA mutations conferring high-level azithromycin resistance. The objective of the present study was to examine the presence of antimicrobial resistance genes (ARGs) in L. pneumophila isolates and to determine whether they exhibited phenotypic resistance through minimum inhibitory concentration (MIC) testing. Methods: Serogroups (sgs) were determined using an in-house qPCR assay, and L. pneumophila non-sg1 isolates were serogrouped using the Dresden monoclonal antibody (mAb) typing method. Sequence types were determined using the standard sequence-based typing method by Sanger sequencing. WGS reads were screened against standard AMR databases to identify resistance genes and resistance-mediating mutations. Agar dilution measured MICs for azithromycin, erythromycin, ampicillin, levofloxacin, tetracycline and spectinomycin in isolates possessing the blaOXA-29, lpeAB or aph(9)-Ia gene. Results: AMR screening detected lpeAB, two allelic β-lactamase variants (blaOXA-29 and blaLoxA) and aph(9)-Ia in 165 of the 522 L. pneumophila isolates, while all high-azithromycin MIC reference sequences contained the expected 23S mutation. Only lpeAB was associated with a significant twofold elevation in macrolide MICs. Neither β-lactamase variant increased ampicillin MICs, and aph(9)-Ia carriage did not correlate with higher spectinomycin MICs. Conclusions: Advanced genomic analytics can now deliver timely therapeutic guidance, yet database-flagged genes may not translate into phenotypic resistance. Continuous pairing of curated mutation catalogues with confirmatory testing remains essential for distinguishing clinically actionable determinants such as 23S mutations and lpeAB from silent markers like blaOXA-29 and aph (9)-Ia.