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Optimized methods for Legionella pneumophila release from its Acanthamoeba hosts
Elisabeth Dietersdorfer1, Sílvia Cervero-Aragó2,3, Regina Sommer4,5
1Institute of Specific Prophylaxis and Tropical Medicine, Department of Medical Parasitology, Medical University of Vienna, Kinderspitalgasse 15, A-1090, Vienna, Austria.
BMC Microbiology
|April 27, 2016
Summary
Mechanical release methods, like needle passage and centrifugation, are most effective for recovering intracellular Legionella pneumophila from Acanthamoeba. Standard methods may underestimate bacterial counts in water systems if amoebal membranes are not disrupted.
Area of Science:
- Microbiology
- Environmental Science
- Water Quality
Background:
- Free-living amoebae (FLA), particularly Acanthamoeba, host and protect pathogenic bacteria like Legionella pneumophila.
- Engineered water systems create environments where amoebae shield L. pneumophila from disinfectants.
- Current methods for studying intracellular bacteria lack standardized protocols for releasing bacteria from amoebae.
Purpose of the Study:
- To evaluate the impact of different release methods on the culturability of intracellular L. pneumophila.
- To assess the suitability of the ISO 11731:1998 standard for quantifying intracellular bacteria.
Main Methods:
- Co-culture of L. pneumophila and Acanthamoeba strains.
- Application of eight different release treatments, including mechanical methods (needle passage, centrifugation) and chemical treatments.
- Evaluation of bacterial recovery rates using standard plating techniques and the ISO 11731:1998 method.
Main Results:
- Mechanical release methods, specifically needle passage (21G, 27G) and high-speed centrifugation (10,000 × g), yielded the highest bacterial counts.
- Release treatments generally enhanced the survival of both L. pneumophila and Acanthamoeba in co-cultures.
- The ISO 11731:1998 method showed variable results, with one L. pneumophila strain exhibiting reduced recovery rates, likely due to amoebal survival.
Conclusions:
- Mechanical disruption of protozoal membranes is crucial for effective intracellular bacterial release.
- Chemical-free release methods preserve bacterial viability for further analysis.
- Standard Legionella enumeration methods may underestimate bacterial loads in water if amoebal hosts are not disrupted.
