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

TL;DR

This study compares antibiotic susceptibility testing methods for less common Legionella species, finding significant differences compared to the more common L. pneumophila.

Contribution

The study provides new insights into antimicrobial susceptibility patterns and testing protocols for non-pneumophila Legionella species.

Findings

Non-pneumophila Legionella species showed broader MIC ranges for azithromycin and levofloxacin compared to L. pneumophila.

BYE-BMD was the most effective method for MIC determination, though some species required BCYE-α due to poor growth.

Rifampicin susceptibility varied among species, with L. longbeachae showing a higher MIC50 despite overlapping ranges.

Abstract

Background/Objectives: Infections caused by Legionella species are primarily associated with Legionella pneumophila, but non-pneumophila species are increasingly implicated in human disease. Despite this, antimicrobial susceptibility testing (AST) data for non-pneumophila 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-pneumophila Legionella species relative to L. pneumophila using three methodologies. Methods: AST was conducted on 89 Legionella 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. Results: MIC profiles were obtained using LASARUS and BYE-BMD for 53.9% and 93.3% of isolates, respectively. While L. pneumophila reached sufficient turbidity in BYE-BMD after a 48 h incubation, non-pneumophila species required an extended incubation (72–96 h). Non-pneumophila 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 L. pneumophila. L. longbeachae exhibited a higher MIC50 for rifampicin despite overlapping susceptibility ranges across all species (0.001–0.016 mg/L). Conclusions: This study demonstrates species-specific differences in Legionella susceptibility and highlights the limitations in extrapolating L. pneumophila-based AST data. Azithromycin MICs in non-pneumophila species exceeded those of L. pneumophila, 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-pneumophila infections.

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