Microb Ecol (2017) 73: 699. doi:10.1007/s00248-016-0905-7

Effects of Bacillus cereus Endospores on Free-Living Protist Growth

Susana S. Santos1, Niels Bohse Hendriksen1, Hans Henrik Jakobsen2, Anne Winding1#

1Department of Environmental Science, Aarhus University, Denmark

2Department of Bioscience, Aarhus University, Denmark



We studied the predator – prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on the survival and dispersal of endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists after antibiotic treatment of the protist surface. This was notably observed in the amoeboid protist. These observations support that protists can act as a potential breeding ground for B. cereus.



The Bacillus cereus group comprises a versatile spectrum of species and strains which range from being probiotics to being pathogens – even with human lethal abilities [1]. Genomic analysis has confirmed that B. cereus group bacteria found in soil are adapted to proliferate under nutrient rich conditions and revert to resting endospores when nutrients are scarce [2, 3]. Therefore, B. cereus mainly exists in soil as endospores, and germinates when in contact with easily degradable organic matter or animal (eukaryotic) hosts [3]. In this study, we refine our understanding of predator-prey interactions between diverse protists (Acanthamoeba castellanii, Cercomonas sp. and Tetrahymena pyriformis) and the two B. cereus stages (vegetative cells and endospores). Different outcomes can derive from a predator – prey contact interaction [4], with consequent growth or no growth of the predator (ingestion/digestion; ingestion/no digestion; no interaction). The ability of certain bacteria to resist digestion, indicates potential for long-term intracellular survival and emphasises the potential for survival within the ecosystems [5].


B. cereus Cells as Food for the Different Protist Predators

Epifluorescence microscopy and qPCR techniques allowed us to confirm that the diverse protists grazers consumed the endospores as well as the vegetative cells (Figure 1, A-C). Protist growth as a function of ingestion displayed two stages for T. pyriformis and A. castellanii strains, which corresponds to their maintenance requirements. This has been previously described as an effective mechanism of evasion from phagocytic predation [6] and could be a result of B. cereus vegetative cells being a relatively poor-quality food source for soil protists. Nevertheless, the positive relationship between active and inactivated endospore ingestion versus protist growth observed in our study (Figure 1, D-G), suggests that above a specific ingestion threshold of approximately 2% of predator body volume, positive growth of protist was always accompanied by predation.





Figure 1: A-C: Growth dynamics of protist cultures when feeding on both B. cereus life stages. A – Tetrahymena pyriformisB – Acanthamoeba castellanii and C – Cercomonas sp. Starving corresponds to control protist monocultures (without bacteria). D-G: Protist growth rates versus ingestion rates when fed with B. cereus. The straight lines represent a linear regression with significant slopes (P < 0.05). Data indicate the average and standard errors for three independent experiments. Significant differences (Tukey; P < 0.05) among the different stages are indicate with different letters within the same graph. Notice the different scales on y-axes.


Uptake and Intracellular Growth of B. cereus

The results of our experiments reveal that after 24 hours post-antibiotic treatment bacteria inside food vacuoles had significantly increased (P < 0.05). Cercomonas sp. showed a higher ratio of intracellular growth of vegetative cells than endospores, while A. castellanii showed higher capacity to take-up endospores. T. pyriformis showed a similar low ratio of intracellular growth after 24 h for both bacterial life forms (Figure 2). This indicates that bacterial cells survived the ingestion and digestion process, and could even proliferate inside protists food vacuoles. A strong reciprocal link between Cercomonas sp. predation/ingestion and growth on B. cereus endospores was observed (Figure 1). This demonstrates that bacterial spores can be a relevant food source for protists. In addition, depending on the protist, the outcome of the intracellular Bacillus-protist interaction may be different and result in bacterial growth, survival or death. The study highlights the dependency of life stage on the outcome of a bacteria-protist interaction and shows that B. cereus benefits from diverse eukaryotic predators assemblages in their survival and proliferation.



Figure 2: Intracellular survival and growth of B. cereus vegetative cells and endospores within each protist species. Data are means and standard errors of three to four independent experiments. Different superscripts indicate significant difference (P < 0.05; Tukey).




This work was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. (289949) and by the Danish Centre for Environment and Energy at Aarhus University through the eDNA center. HHJ received support from the VELUX foundation to procure microscopes and tools from epi-fluorescence microscopy grant no. (VKR022608).



Anne Winding

Department of Environmental Science

University of Aarhus Frederiksborgvej 399,

DK-4000 Roskilde, Denmark Phone: +45 87158615

e-mail: aw@envs.au.dk



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