# 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.

## Key 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.