Detection of viable Helicobacter pylori inside free‐living amoebae in wastewater and drinking water samples from Eastern Spain

Summary Helicobacter pylori is one of the most concerning emerging waterborne pathogens. It has been suggested that it could survive in water inside free‐living amoebae (FLA), but nobody has studied this relationship in the environment yet. Thus, we aimed to detect viable H. pylori cells from inside FLA in water samples. Sixty‐nine wastewater and 31 drinking water samples were collected. FLA were purified and identified by PCR and sequencing. For exclusively detecting H. pylori inside FLA, samples were exposed to sodium hypochlorite and assayed by specific PMA‐qPCR, DVC‐FISH and culture. FLA were detected in 38.7% of drinking water and 79.7% of wastewater samples, even after disinfection. In wastewater, Acanthamoeba spp. and members of the family Vahlkampfiidae were identified. In drinking water, Acanthamoeba spp. and Echinamoeba and/or Vermamoeba were present. In 39 (58.2%) FLA‐positive samples, H. pylori was detected by PMA‐qPCR. After DVC‐FISH, 21 (31.3%) samples harboured viable H. pylori internalized cells. H. pylori was cultured from 10 wastewater samples. To our knowledge, this is the first report that demonstrates that H. pylori can survive inside FLA in drinking water and wastewater, strongly supporting the hypothesis that FLA could play an important role in the transmission of H. pylori to humans.