[Investigation of the Inhibition Effect of Acanthamoeba Cell-Free Supernatants Against Pseudomonas aeruginosa]. / Acanthamoeba Hücresiz Sivilarinin Pseudomonas aeruginosa Karsisinda Inhibe Edici Etkisinin Arastirilmasi.

Free-living amoebae belonging to the genus Acanthamoeba are microorganisms that live in air, soil and aquatic environments. In humans, they cause infections such as amoebic keratitis, graulamotous amoebic encephalitis that are difficult to treat and can be fatal. In addition, it is known that they contribute to the replication of bacteria and increase their pathogenicity by being a host for various bacteria. However, information on its inhibitory properties against bacteria and its production of antimicrobial agents is very limited. In this context, in this study, it was aimed to investigate whether cell-free supernatants of Acanthamoeba strains have antibacterial effects against Pseudomonas aeruginosa isolates. Four different Acanthamoeba strains (A10, A13, A14, U.GÖL) isolated from aquatic environments in our country were selected and used in the study, P.aeruginosa isolates (PA2, PA3, PA4, PA5) were selected from clinical strains belonging to patients in our country. Acanthamoeba castellanii ATCC 50373 and P.aeruginosa ATCC 27853 were used as standard strains. P.aeruginosa isolates were grown on nutrient agar at 37 °C and Acanthamoeba strains were grown on E.coli spread non-nutrient agar at 30 °C under aerobic conditions. Pepton yeast extract glucose (PYG) medium supplemented with penicillin and streptomycin was used to obtain axenic cultures of Acanthamoeba strains. After the centrifugation of axenic cultures at 3000 rpm for five minutes, Acanthamoeba-cell-free supernatants were obtained by filtering the supernatant part through a sterile filter with a pore diameter of 0.22 µm. The antibacterial activities of these supernatants against P.aeruginosa isolates were determined using the colony counting method. Analysis of each Acanthamoeba-cell-free supernatants was performed according to the GC-MS method. Acanthamoeba-cell-free supernatants were found to have varying degrees of inhibitory effects (3.9-91.5%) against tested P.aeruginosa isolates. It was determined that the cell-free supernatant of A.castellanii ATCC 50373 strain showed the highest antibacterial effect (91.5%) against PA5 isolate. A14 strain showed similar inhibitory effects (89.4%) against the same Pseudomonas isolate. The average inhibitory effect of most of the Acanthamoeba strains of our country was found to be higher than that of the reference strain A.castellanii ATCC 50492. It is thought that the compounds responsible for the anti-Pseudomonas activity of the tested Acanthamoeba strains may be fructose, phosphoric acid, galactose, N-Acetylphenylalanine and glucopyranose determined as major compounds. This is the first study showing the anti-Pseudomonas activity of microorganisms of the genus Acanthamoeba living in the waters of our country. Acanthamoeba, which is widely found in nature, appears to be a good source for new antimicrobial agents.

[Inhibition Effect of Cell-Free Supernatants of Environmental Acanthamoeba Strains on the Viability of Metastatic Cell-Lines]. / Çevresel Acanthamoeba Suslarinin Hücresiz Sivilarinin Metastatik Hücre Hatlarinin Canliligi Üzerindeki Inhibisyon Etkisi.

It is known that some of the therapeutic agents against cancer cells are isolated from natural sources such as plants and animals. However, due to increasing drug resistance, studies on the discovery of new sources are needed. In this study, it was aimed to investigate the inhibition effects of four native Acanthamoeba strains on different cancer cell lines (MDA-MB-231, PC3, MAT-LyLu). 3T3 cells were used as normal cell line. All strains were recultured by using non-nutrient agar spread by heat-inactivated Escherichia coli ATCC 25922. A.castellanii ATCC 50373 was used as the standard strain. Molecular identification of the native Acanthamoeba isolates was done by polymerase chain reaction (PCR) and DNA sequence analysis using specific primer pairs (P-FLA-F, P-FLA-R, JDP-F, JDP-R). Axenic cultures of all strains were obtained in 25 cm2 tissue culture flasks and in peptone yeast extract glucose (PYG) medium. In order to investigate the effect of cell-free supernatants obtained from axenic cultures on cancer cell lines and 3T3 cell viability, MTT method was applied using different concentrations of cell-free supernatants (1%, 2%, 5%, 10%, 15%). It was determined that the viability of 3T3 cells was not affected by any Acanthamoeba cell-free supernatants (p≤ 0.05). All of the samples tested were found to have a significant inhibitory effect (p<0.05) on the viability of PC3 and MAT-LyLu cells (human and rat prostate cancer cell line). However, none of the samples had an inhibitory effect on the viability of MDA-MB-231 (breast cancer cell-line). Two native Acanthamoeba cell-free supernatants showed higher inhibitory potency (28% and 21.9%) at 2% concentration against PC3 cells compared to the reference strain (16%). Similarly, the same Acanthamoeba samples were also shown to have a better inhibition potential on the viability of MAT-LyLu cells than the reference strain. It was found that the inhibitory potential of Acanthamoeba cell-free supernatants may not be related to proteins and proteases. The results obtained from this study showed that Acanthamoeba species living in the aquatic environment isolated from our country have a potential inhibitory effect against the tested cancer cell lines. In addition to plants and animals, Acanthamoeba cell-free supernatants can also be a source for natural therapeutic substances that act against cancer cells. However, it is necessary to carry out new studies using more strains in order to detect strains with higher inhibitory effects.