2. Prof. Zeev Ronen

The increased reuse of the treated water resources comes with unexpected environmental and public health consequences. While onsite treatment provides reliable solutions, treating emerging pollutants like antibiotic resistance genes (ARGs) and resistant bacteria (ARBs) is not well-considered. In the last few years, we aimed to characterize AMR in greywater sources and evaluate if recycling vertical flow constructed wetland (RVFCW) can be a sustainable solution for removing AMR. Treated greywater from RVFCW was surveyed in a household in the central Negev Desert, Israel. The presence of antibiotic-resistant bacteria in raw and treated greywater was investigated with culture and molecular methods, as well as their presence in the corresponding treated-greywater-irrigated soils. While the RVFCW effectively reduced amoxicillin, kanamycin, and ciprofloxacin ARBs, tetracycline-resistant bacteria increased in the treated greywater, likely due to their concentration on the filter matrix of the treatment systems. No significant differences were observed when comparing ARBs in soil irrigated with TGW or freshwater. Our research quantified ARGs (sul1, qnrS, blaTEM, blaCTXM32, tetG, tetQ, tetW tet39, fabI, and fabV), class one integron (intI1), and bacterial marker (16S) using quantitative polymerase chain reaction (qPCR). The greywater microbial community was characterized using a 16S rRNA amplicon sequencing. The resistome of the GW was characterized by metagenomic analysis. The RVFCW can reduce ARG in greywater by 1–3 log, depending on the gene and the quality of the raw greywater. The metagenome confirmed these observations and also indicated the presence of different families of ARGs. Resistance genes with efflux mechanism (export of antibiotics out of the microorganism, comprised the majority of the ARG abundance for all (raw and treated) samples. It was followed by the group that is resistant to antibiotic inactivation, which consists of enzymes that physically modify antibiotics, related to biodegradation mechanisms Quantitative PCR of ARGs in soils irrigated with either freshwater or treated GW suggests that the contribution of treated GW to the presence of ARGs is not significant compared to other possible routes of introduction, such as natural occurrence, application of composts, or the presence of animals. Our results highlight the prevalence of ARGs and ARBs in raw, and treated GW. However, it is still unclear whether in-site use of these effluents for irrigation introduced AMR into the soil. Mitigation of the risk of ARBs in reusing TGW can be achieved via effluents disinfection. Nevertheless, there is a need to quantitatively examine the potential health risk from arising from ARGs in the treated effluents.