Detection of Free-Living Amoebae and Amoeba-Resisting Bacteria in Beach Waters in Southeastern Georgia

Background: Kings Ferry Park in Savannah, Georgia, has been under a permanent health advisory since 2004 due to high levels of Enterococci; however, other information about microorganisms circulating at this site is lacking. We examined the occurrence and distribution of free-living amoebae (FLA), and FLA associated bacteria in water and sediment samples in the park and the surrounding area along the Ogeechee River.

Methods: Water and sediment samples were collected monthly from 14 sites along the beach and river around Kings Ferry Park. Sample filtrates were used for isolation of FLA and bacteria. Amoebae were isolated using the walk-out method. Bacterial isolates were established using co-culturing with Acanthamoeba castellanii followed by colony isolation on charcoal yeast extract agar plates with and without BCYE supplement. DNA was extracted using Qiagen QIAmp protocol. Identification of amoeba isolates was completed by sequencing 18S rRNA gene; bacteria were identified by sequencing 16S rRNA gene and Legionella 23S-5S rRNA intergenic region.

Results: 155 isolates of amoebae were obtained from water and sediment samples collected from the beach and surrounding sites. Vermamoeba vermiformis was a predominant species; Naegleria clarki and Acanthamoeba spp. were also found. 17 species of bacteria which are obligate or opportunistic pathogens of human and animals were recovered. Bacteria of Bacillus cereus complex were the most frequently isolated followed by Stenotrophomonas maltophilia and Acinetobacter baumanii. Legionella anisa and Mycobacterium sp. were also detected.

Conclusions: All three species of amoebae isolated as a part of this study can harbor diverse amoeba-resistant bacteria; many bacteria isolated are known to carry a variety of antibiotic resistance genes. The origin, sources, and spatial and temporal distribution of these pathogens in the area need further study. In depth characterization of these microorganisms and their interactions is critical to assess the risk of human and companion animal exposure to these common groups of organisms as a result of various aquatic recreational activities.