Investigation of the effect of free-living amoebas cell free supernatans on Staphylococcus

Free-living amoebae (FLAs) are eukaryotic microorganisms that can live in a variety of environments such as soil, air, sea, lake, river, and man-made water systems (taps, swimming pools, dental units, cooling towers, etc.). These microorganisms attract a lot of attention due to the diseases they cause in humans and their relationships with other microorganisms in nature. The most well-known of these relationships is related to the fact that FLAs increase the spread of bacteria by acting as a host for some bacteria. Contrary to this situation, there is limited information regarding the inhibition of FLAs by secreting antibacterial agents against bacteria with which they share the same environment in nature. Therefore, in this study, it was aimed to investigate whether cell-free supernatants (CFS) of FLA strains have antibacterial effects against Staphylococcus bacteria. In this study, four FLA strains (A10, A14, UG, Acanthamoeba castellanii ATCC 50373) were used. The antibacterial effect of CFS samples obtained from these strains on 12 Staphylococcus bacteria was investigated using colony counting method and agar diffusion method. FLA strains were grown in Petri dishes containing heat-inactivated Escherichia coli ATCC 25922 spread non-nutrient agar (NNA). Pepton yeast extract glucose (PYG) medium was used to obtain monolayer axenic cultures of these strains. Amoeba numbers (cells/mL) in axenic cultures were determined using a Thoma slide and viability was determined using trypan blue dye. Cell-free supernatant of FLA strains (FLA-CFS) were obtained by centrifuging the FLA axenic cultures at 3000 rpm for 5 minutes and then passing them through a sterile filter with a pore diameter of 0.22 µm. Suspensions (108 cfu/mL) of Staphylococcus strains were prepared using the spectrophotometric method (OD=595). In order to determine the pathogenicity of FLA strains, osmotolerance and thermotolerance tests were used. According to the colony count method, it was determined that the inhibitory effect of FLA-CFS samples on Staphylococcus strains varied (0-100%). A. castellanii ATCC 50373-CFS sample showed 100% inhibitory effect against one Staphylococcus strain (MRSA-SA17), and UG-CFS sample obtained from the native amoeba isolate showed a close (88.63%) inhibitory effect against the same strain. On the other hand, one Staphylococcus strain (MSSE-SE1) that the A. castellanii ATCC 50373-CFS sample did not inhibit was 99.97% inhibited by the UG-CFS sample. These variations in results are thought to be due to the FLA strains and Staphylococcus strains used. It was determined that the FLA–CFS samples (A10-CFS, A14-CFS, UG-CFS) isolated from the waters of our country had an average inhibitory effect of 0.34%, 1.85%, and 16.35%, respectively, against Staphylococcus strains. However, it was determined that the inhibitory effect of A. castellanii ATCC 50373-CFS sample obtained from the standard strain on all Staphylococcus strains (average 29.12%) was higher than that of the FLA-CFS samples isolated from our country's waters. It was determined that the FLA-CFS samples, which were found to have an inhibitory effect in the colony counting method, did not show a significant inhibitory effect in the agar diffusion method. As a result of osmotolerance and thermotolerance tests, it was concluded that the tested FLA strains had no/low pathogenic potential. This thesis study shows that cell-free supernatants of FLA strains isolated from the waters of our country have antibacterial-effect potential against Staphylococcus bacteria. In order to detect FLA strains with higher anti- Staphylococcus activity, studies involving more strains should be planned first. Then, by detecting the active substance(s) from the FLA-CFS samples with high antibacterial effect, it will be possible to contribute to the discovery of new anti-Staphylococcus effective substance(s).