Evidence of Helicobacter spp. in freshwaters from Roraima Tepui, Guayana Shield, South America

Helicobacter presence and viability in waters is not well characterized. The identification of natural reservoirs and infection sources may provide novel insights into its waterborne transmission. The goal of this study was to investigated the occurrence of Helicobacter spp. in natural freshwaters from Roraima Tepui, a little studied and unique ecosystem of the Guayana Shield. Freshwaters collected from two localities at Roraima Tepui were cultured in HP selective broth and agar for Helicobacter pylori and analysed by fluorescent in situ hybridization (FISH), specific PCR assays, 16S rRNA gene sequencing and phylogenetic analysis. The presence of other bacteria in freshwater enrichments was determined using clone library sequencing of the 16S rRNA gene and phylogenetic inferences. Helicobacter spp. were detected by semi-nested PCR and FISH in freshwater enrichments from both sites. Coccoid viable but nonculturable (VBNC) cells were evidenced using 16S rRNA gene Helicobacter species and H. pylori-specific probes. Partial 16S rRNA gene sequences of two HP enrichments showed high similarity to H. pylori and Helicobacter nemestrinae (99-100 %). Other bacteria such as Serratia, Aquitalea, Chromobacterium, Mycobacterium, Acinetobacter, Curvibacter and Dysgonomonas were also detected using complete 16S rRNA gene sequences, with Serratia, Aquitalea and Chromobacterium the most common genera (40.9, 18.2 and 15.2 %, respectively). This is the first time that Helicobacter spp. have been reported in freshwaters of a tepui ecosystem. Our results contribute to the current knowledge of these bacteria in the aquatic environment and expand their known/potential sites outside the human host.