Differential Bacterial Predation by Free-Living Amoebae May Result in Blooms of Legionella in Drinking Water Systems

Abstract

Intracellular growth of pathogenic Legionella in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how Legionella spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species, Willaertia magna, Acanthamoeba polyphaga, and Vermamoeba vermiformis regarding Legionella pneumophila and two Escherichia coli strains. Although all the studied FLA species supported intracellular growth of L. pneumophila, they avoided this bacterium to a certain degree in the presence of E. coli and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once L. pneumophila were intracellular, it inhibited digestion of co-occurring E. coli within the same trophozoites. Altogether, based on FLA-bacteria interactions and the shifts in microbial population dynamics, we propose that FLA's feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of Legionella and creates a "forced-feeding" condition facilitating the internalization of Legionella into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of Legionella.

Keywords: Legionella; Legionnaires’ disease; engineered water systems; free-living amoebae.

Figure 1

Preferential feeding on bacteria by W. magna at RT at different time points (0.5, 24, and 96 h) of co-culture. The four images (from top to bottom) represent the same field of view under different fluorescent light channels, Monocolor transmission light channel, Green fluorescent channel to observe GFP-L. pneumophila, Texas-Red channel for mCherry-E. coli MG1655, and DAPI channel for BFP-E. coli TOP10. The clusters of color dots in images indicate the presence of different intracellular bacteria in the food vacuoles of W. magna trophozoites. The scattered color dots (smaller in size) indicate planktonic bacterial cells in the medium.

Figure 2

The number of W. magna trophozoites containing E. coli TOP10, E. coli MG1655, and L. pneumophila cells at different time points of co-culture. The number of trophozoites were counted from four fields of view of three replicates (total 48 images/ time point), the bars represent standard deviations of the mean and the box represent 25–75 percentiles. The t-test indicates significant differences in prey preference (p < 0.001).

Figure 3

Intracellular L. pneumophila interferes with W. magna’s digestion process. W. magna and L. pneumophila were co-cultured at RT, and after 24 h, E. coli TOP10 and E. coli MG1655 were added to the culture. The images represent overlaid images of the same field of view under four light channels, Monocolor transmission light channel, Green fluorescent channel, Texas-Red channel, and DAPI channel at 200× and 1000× magnification after 26 h of co-culture. L. pneumophila cells are green, E. coli MG1655 cells are red, and E. coli TOP10 cells are blue in color.

Figure 4

Intracellular locations of E. coli MG1655 (red) in A. polyphaga cysts at 96 h of co-culture. Many of the cysts did not contain any E. coli MG1655 cells, which indicate that the digestion may depend on the intracellular load of the bacterial cells and encystation time. Releasing of vesicle during encystation and vesicle-containing E. coli MG1655 after 48 h of co-culture (Right image).