Antimicrobial Susceptibility Determination of Less Frequently Isolated Legionella Species by Broth and Agar Dilution

<b>Background/Objectives</b>: Infections caused by <i>Legionella</i> species are primarily associated with <i>Legionella pneumophila</i>, but non-<i>pneumophila</i> species are increasingly implicated in human disease. Despite this, antimicrobial susceptibility testing (AST) data for non-<i>pneumophila</i> species remain scarce, and standardised testing protocols or resistance thresholds have not been established. This study aimed to address this gap by evaluating and comparing AST performance for non-<i>pneumophila Legionella</i> species relative to <i>L. pneumophila</i> using three methodologies. <b>Methods</b>: AST was conducted on 89 <i>Legionella</i> isolates using LASARUS agar dilution, buffered yeast extract broth microdilution (BYE-BMD), and BCYE-α agar dilution, against ampicillin, azithromycin, chloramphenicol, doxycycline, levofloxacin, and rifampicin. Growth performance and minimum inhibitory concentrations (MICs) were assessed after a 96 h incubation. <b>Results</b>: MIC profiles were obtained using LASARUS and BYE-BMD for 53.9% and 93.3% of isolates, respectively. While <i>L. pneumophila</i> reached sufficient turbidity in BYE-BMD after a 48 h incubation, non-<i>pneumophila</i> species required an extended incubation (72-96 h). Non-<i>pneumophila</i> species displayed broader MIC ranges against azithromycin (0.016-1 mg/L) and levofloxacin (0.016-0.25 mg/L), but a narrower rifampicin range (≤0.0005-0.032 mg/L) relative to <i>L. pneumophila</i>. <i>L. longbeachae</i> exhibited a higher MIC₅₀ for rifampicin despite overlapping susceptibility ranges across all species (0.001-0.016 mg/L). <b>Conclusions</b>: This study demonstrates species-specific differences in <i>Legionella</i> susceptibility and highlights the limitations in extrapolating <i>L. pneumophila</i>-based AST data. Azithromycin MICs in non-<i>pneumophila</i> species exceeded those of <i>L. pneumophila</i>, raising clinical concern. While BYE-BMD was the most effective method for MIC determination, three species required BCYE-α due to poor growth. These findings support developing standardised, species-specific AST protocols and thresholds amid rising macrolide resistance and the increasing detection of non-<i>pneumophila</i> infections.