Interaction between waterborne pathogenic bacteria and Acanthamoeba castellanii

Waterborne bacteria cause global public health problems. Francisella tularensis causes tularemia, which is a fatal disease in humans. Pseudomonas aeruginosa is an opportunistic and nosocomial pathogen of humans. Vibrio cholerae O1 and V. cholerae O139 infect only humans and cause epidemic and pandemic cholera. The principal natural reservoirs of these pathogens are largely unknown. To find their aquatic reservoirs is an important factor in the epidemiology of the infections. Acanthamoeba is a genus of free-living amoebae, which are found in the aquatic system and include several species and seem to have an increased role as reservoirs to many pathogenic bacteria. Acanthamoeba castellanii was co-cultured with each of the above-mentioned bacteria for more than two weeks in order to study the interaction. Growth of the microorganisms, localisation and survival of intracellular bacteria was estimated by cell count, viable count, flow cytometry, PCR, fluorescence as well as electron microscopy. The results showed that F. tularensis localised in A. castellanii, multiplied within vacuoles and survived in intact trophozoites, excreted vesicles, and cysts. Co-cultivation enhanced growth of F. tularensis, which grew and survived intracellularly for more than 3 weeks. In contrast, growth of singly cultured bacteria decreased significantly to non-detectable level within 2 weeks confirming the intracellular behaviour of the bacterium. The co-cultivation decreased growth of the amoebae in comparision to growth of singly cultured amoebae. Co-cultivation of A. castellanii with different strains of P. aeruginosa PA103 producing different effector proteins secreted by type III secretion system (TTSS) resulted in the death of the amoeba populations. Different analysis disclosed that the number of co-cultured amoebae decreased over time in comparision to the number of singly cultured cells. The TTSS effector proteins ExoU and ExoS induced necrotic cell death to the most of A. castellanii. The interaction between V. cholerae and A. castellanii resulted in growth and survival of V. cholerae O1 as well as O139 in the cytoplasm of trophozoites and in the cysts of A. castellanii. Co-cultivation enhanced growth of V. cholerae, which grew and survived intracellularly for more than two weeks, whereas, singly cultured bacteria decreased significantly to non-detectable level within few days disclosing an intracellular behaviour of V. cholerae. In conclusion, methods used in this project showed predation between A. castellanii and the extracellular P. aeruginosa, symbiosis between A. castellanii and each of the facultative intracellular bacterium F. tularensis as well as V. cholerae.