Free-living amoebae and their medically-important endosymbionts in hospital wastewater treatment systems

• Four hospital wastewater treatment systems (HWWTSs) were monitored for six months. • Free living amoebae were isolated and characterized from samples. • Fusarium spp., NoV, AstV, and AdV, and non-methicillin resistant S. aureus were detected in 25.42 % samples. • The presence of FLA in hospital wastewater may increase the risk of transmission of potentially pathogenic microorganisms. Free-living amoebae (FLA) are amphizoic microorganisms, frequently isolated from hospital environments. Regarding the role of FLA in transmission of potentially pathogenic microorganisms as endosymbionts, the presence of FLA in hospital environments increases the public health concerns. The current study investigated fungal, viral, and methicillin-resistant Staphylococcus aureus as endosymbionts in isolated FLA from hospital wastewater treatment system. Totally, 104 samples were collected during 6 months from the wastewater facilities of four hospitals (two general, one children, and one women hospitals). Samples were filtered, cultivated, and microscopically monitored for FLA. After purification of FLA, DNA and RNA were extracted and FLA were characterized using amplification and sequencing of discriminative genes. The presence of Norovirus (NoV), Adenovirus (AdV), Rotavirus (RV), Astrovirus (AstV), Mucor spp., Candida albicans, Aspergillus flavus , and Fusarium spp. was evaluated using real-time PCR. The presence of S. aureus was investigated by amplification of FemA and nuc genes. The methicillin-resistant gene, mecA , was investigated in positive samples. Out of 104 wastewater samples, 94 samples (97.7%) were microscopically positive for FLA. From 94 FLA positive samples, 59 (63.76%) plates were purified and included in molecular assays. Accordingly, 52, 5, and 10 samples were positive for Acanthamoeba sp., Vermamoeba spp., and Vahlkampfiidae, respectively. Viral endosymbionts were detected from seven wastewater samples (four inlets and three outlets) including seven, three, and one for NoV, AstV, and AdV, respectively. Fusarium spp., were detected in eight samples (four for each inlet and outlet). Seven samples carried either FemA or nuc genes. There was no evidence for MRSA in samples. Regarding the capability of FLA to carry and protect potentially pathogenic endosymbionts in harsh conditions such as normal wastewater treatment, the presence of FLA in hospital wastewater may increase the risk of transmission of potentially pathogenic microorganisms to downstream industries that use treated wastewater for different purposes.