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Updated: Mar 22, 2026
Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Published on: April 2, 2013
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.
Background:
Free-living amoebae (FLA) and particularly acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms. Within the amoebae, L. pneumophila activates a complex regulatory pathway that enables the bacteria to resist amoebal digestion and to replicate. Moreover, the amoebae provide the bacteria protection against harsh environmental conditions and disinfectants commonly used in engineered water systems. To study this ecological relationship, co-culture and infection models have been used. However, there is a lack of data regarding the effectiveness of the different methods used to release intracellular bacteria from their amoebal hosts. The aim of this study was to evaluate the impact of the methods used to release intracellular L. pneumophila cells on the culturability of the bacteria. Furthermore, the standard method ISO 11731:1998 for the recovery and enumeration of Legionella from water samples was evaluated for its suitability to quantify intracellular bacteria.
Results:
The effectiveness of the eight release treatments applied to L. pneumophila and Acanthamoeba strains in a free-living state varied between bacterial strains. Moreover, the current study provides numerical data on the state of co-culture suspensions at different time points. The release treatments enhanced survival of both microorganisms in co-cultures of L. pneumophila and Acanthamoeba. Passage through a needle (21G, 27G) and centrifugation at 10,000 × g showed the highest bacterial counts when releasing the bacteria from the intracellular state. Regarding the ISO 11731:1998 method, one of the tested strains showed no differences between the recovery rates of associated and free-living L. pneumophila. However, a reduced bacterial recovery rate was observed for the second L. pneumophila strain used, and this difference is likely linked to the survival of the amoebae.
Conclusions:
Mechanical release treatments were the most effective methods for providing bacterial release without the use of chemicals that could compromise further study of the intracellular bacteria. The current results demonstrated that the recovery of L. pneumophila from water systems may be underestimated if protozoal membranes are not disrupted.
Insights
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.
