Free-living amoebae in water sources by PCR and sequencing in Spain

Autores UPV
Año
CONGRESO Free-living amoebae in water sources by PCR and sequencing in Spain

Abstract

Free-living amoebae (FLA) are ubiquitous protozoa widely isolated from water. Among them, Acanthamoeba species are the most common FLA in those environments and can cause rare but severe infections of the eye (Acanthamoeba keratitis, AK), skin, and central nervous system (Granulomatous Amebic Encephalitis, GAE). FLA belonging to the genus Naegleria and Balamuthia are also important human pathogens, although its presence in water is infrequent. Identification of different FLA in sources able to reach humans is of great importance. A total of 50 water samples were analyzed for the presence of FLA. Nineteen drinking and 31 wastewater samples were filtered. Membranes were placed in Non-Nutrient-Agar (NNA) and the culture was maintained until amoebae growth was observed. FLA were purified individually using a micromanipulator and incubated in NNA. Cultures were harvested, and after DNA isolation, were subjected to Multiplex PCR (Le Calvez et al., 2012). Results were confirmed by 18S rRNA PCR plus sequencing (Thomas et al., 2006). FLA growth was observed in 22/31 and 5/19 wastewater and drinking water samples respectively. In total, 39 FLA were purified using the micromanipulator, 31 from waste water and 8 from drinking water samples. Multiplex PCR yielded specific fragments of Acanthamoeba, Naegleria, Vannellidae, Vermamoeba and Echinamoeba, being Acanthamoeba the most common FLA isolated in both types of water. In some cases, although the micromanipulator was used to isolate a single amoeba, unspecific fragments were observed after Multiplex PCR analysis of the cultures, showed that an axenic culture was not achieved. By 18S rRNA PCR and sequencing, Vannellidae spp., Naegleria spp. and the Acanthamoeba species A. castellanii, A. tubiashi, A. polyphaga, A. rhysodes and A. mauritaniensis were identified. To our knowledge, it is the first time that Vannellidae spp. is identified in wastewater. The fact that those human pathogens were detected from water sources even after disinfection treatment could be a risk concerning Public health. This study was partially supported by a grant of the ¿Recovery and joint Resources from I+D+i of VLC/CAMPUS program¿ funded by the MECD (International Excellence Campus Program). The authors thank the Generalitat Valenciana for FEDER financial support 2007-2013 (T0104000)