Deep-Amplicon Sequencing (DAS) Analysis to Determine the Presence of Pathogenic Helicobacter species in Watewater Reused for Irrigation

Abstract

[EN] Wastewater has become one of the most important and least expensive water for the agriculture sector. As an alternative to the overexploitation of water resources. Inappropriate treatment before reuse can result in a negative impact in environment, such us presence of pathogens. Causing an increased risk in health and environmental safety, which can lead to an increased risk of human infection. Among all the emerging wastewater pathogens, bacteria of the genus Helicobacter are some of the most disturbing ones, since they are directly related to gastric illness and hepatobiliary and gastric cancer. Therefore, the aim of this study was to determine the presence of potentially pathogenic Helicobacter spp in treated wastewater aimed for irrigation. The next generation sequencing approach based on Illumina in combination with culture and molecular techniques (q PCR, FISH and DVC-FISH) have been used to analyzed 16 wastewater samples. All the enriched samples were negative for Helicobacter growth in any of the culture media used. Characteristic colonies, covered by a mass of non-specific bacterial growth, were analyzed by Helicobacter spp. PCR, H. pylori qPCR and sequencing. One of the direct samples was positive for H. pylori. Using FISH and DVC-FISH technique, Helicobacter spp, including H. pylori, was detected in seven out of eight samples of wastewater from the tertiary effluents, all of them viable. Although, qPCR yield only three positive results. Moreover, when wastewater microbiome was studied, Helicobacter genus was detected in 7 samples. The different molecular techniques using in the present study, provided evidence, for the first time, the presence of species belonging to the genus Helicobacter such as H. pylori, H. hepaticus, H. pullorum and H. suis in wastewater samples, even after disinfection treatment.