Investigation of the effects of free-living amoeba cell-free supernatants on pseudomonas

Free-living amoebas (FLA) are microorganisms that are common in nature and man-made water systems, causing diseases that can also be fatal depending on the host's immune system. They also act as hosts for the various bacteria living in the same environment. As a result, they can contribute to the growth of bacteria or they may be lysed. However, the knowledge about the production of antimicrobial agents that can ensure survival of FLA in these environments is quite limited. In this context, it was aimed to investigate whether cell-free supernatants (ACM) belonging to FLA strains have an antibacterial effect against Pseudomonas aeruginosa in this study. Totallly four environmental FLA strains (A10, A13, A14, U.GÖL) isolated from our country's swimming pools and a lake water, in addition to the Acanthamoeba castellanii ATCC 50373 strain were used In this study. Pseudomonas aeruginosa strains were selected from the clinical strains (PA2, PA3, PA4, PA5) belonging to patients in our country, and P. aeruginosa ATCC 27853 was used as the standard strain. All FLA strains were grown in non-nutrient agar (NNA) seeded with heat-inactivated Escherichia coli ATCC 25922 suspension. Peptone yeast extract glucose (PYG) medium was used to obtain axenic cultures The number of viable FLA trophozoites (cell /mL) in cultures was determined using Thoma slide and trypan blue. Obtaining the cell-free supernatants (ACM) of each FLA strain was accomplished by centrifuging of the axenic culture liquid at 3000 rpm for 5 minutes, then passing through a 0.22 µm pore diameter sterile filter. Suspensions (108 cfu/mL) of each P. aeruginosa strain were measured using a spectrophotometer (OD = 595). Antibacterial activity experiments of ACMs belonging to FLA strains against P. aeruginosa strains were performed on nutrient agar using colony count method. It has been determined that ACMs of FLA strains tested showed antibacterial effect at varying rates (3.9 - 91.5 %) against P. aeruginosa strains tested. It was found that strain A13 (64.6 %) has the highest mean inhibitory effect and strain A14 (34.4 %) had the lowest mean inhibitory effect. It has been determined that three of the four environmental FLA isolates (A13, U.GÖL, A10) belonging to our country have higher inhibitory effect than Acanthamoeba castellanii ATCC 50373 on average. It was determined that the most sensitive P. aeruginosa strain to ACMs was PA5 strain (mean: 82.64 %), followed by PA4, PA2, PA ATCC 27853 and PA3 strains, respectively. This is the first study to show that ACMs of FLA strains belonging to our country have antimicrobial agents against P. aeruginosa. However, new studies are needed to increase the number of samples. In addition, it will be possible with future studies to discover the antimicrobial activities of ACMs of these strains against microorganisms (other pathogenic bacteria, viruses, etc.) other than P. aeruginosa.