Concurrent contamination of Free-Living Amoebae and Legionella pneumophila in water resources of hospitals, A microscopic and molecular analysis

Free-living amoebae (FLAs) are widely prevalent protozoa found in various natural and man-made environments. In Iran, there have been few studies examining the contamination of hospital environments with FLAs and Legionella spp., and none have explored the coexistence of these two organisms. Thus, this study aims to investigate the contamination of FLAs and Legionella spp. in the water resources of hospitals in Birjand city. Water samples were collected from water sources in different hospital departments of Birjand city, were transferred to the laboratory. One litre of each sample passed through the nitrocellulose membrane filter. Samples were taken from the water taps of different departments and storage tanks positive samples were analyzed by morphological keys and Polymerase chain reaction (PCR) using 18S rDNA gene and ITS region to identify amoeba isolates. The possible contamination of the samples with Legionella spp. was examined through cultures and molecular tests. Overall, 80 out of 270 samples from hospital water sources were positive for contamination with FLAs by the culture method. Acanthamoeba was isolated in all 80 samples that were positive for Acanthamoeba . Among the 80 samples in which the Acanthamoeba parasite grown in the culture, 64 Acanthamoeba isolates were also positive with molecular tests. All sequenced samples of Acanthamoeba were reported as T4 genotype. Furthermore, 5/270 examined samples were positive for L. pneumophila contamination. All cases of L. pneumophila infection were reported in samples infected with Acantamoeba . A significant difference was observed between the concurrent contamination of Acantamoeba and L. pneumophila in Razi (P _value =001) and Imam Reza hospitals (P _value = 004) as well as surgical wards (P _value = 0002) and water tanks (P _value = 0.01) at Birjand hospitals. This study aimed to examine the co-occurrence of Legionella spp. and free-living amoebae (FLAs) as the most important water-related pathogens, in hospital water systems to better understand their interactions and the potential consequents for public health, which can lead to both outbreaks and sporadic cases.