Effect of Treated Wastewater Irrigation on the Proliferation of Antibiotic Resistance in Agricultural Soils

In Israel with its semi-arid climate, water is a limited resource and yet, in the last two decades about 60% of all of Israel’s water resources are directed for agricultural use. This is feasible due to the capacity to recycle water. Treated wastewater (TWW) has become an important source of irrigation water in Israel and other parts of the world due to the pressures of increasing population and the limited amount of high quality water. Today, approximately 50% of agricultural irrigation in Israel is carried out with TWW. Irrigation with TWW has significant immediate benefits as it mitigates water shortages, and enables continuation of agricultural development and expansion, in response to the growing needs of the population. However, there are some disadvantages and potential risks associated with its long-term use that are not fully understood. TWW may contain pathogenic microorganisms, effluent-borne anthropogenic chemicals such as pesticides, and a large group of compounds collectively referred to as Pharmaceuticals and Personal Care Products (PPCPs) as pollutants. A major concern is that compounds such as hormones and antibiotics can detrimentally affect the environment and public health due to biological interactions when released into soil and water environments through TWW-irrigation. In this work, I evaluated the environmental impact of one group of TWW-associated PCPPs – antibiotics, by assessing levels of antibiotic resistance and antibiotic resistant genes (ARG's) of the soil bacteria, in TWW and TWW-irrigated soils compared to fresh water (FW) irrigated soils. Indigenous soil-dwelling bacteria both produce and encounter a variety of antibiotics, resulting in evolution of diverse and novel resistance mechanisms. These bacteria may serve as reservoirs of resistance determinants that can be mobilized into the human-associated microbial community. On a molecular level, antibiotic resistance may evolve from either spontaneous mutation of genes, or by interor intra-species horizontal gene transfer (HGT) of existing antibiotic resistance genes. Both of these mechanisms are believed to occur spontaneously at relatively low frequencies. However, exposure to antibiotics can result in the proliferation of these mechanisms due to natural selection. Proliferation of resistant bacteria due to natural selection in areas with anthropogenic effects (such as manure-fertilized soils, rivers receiving treated wastewater effluent, etc) has been well documented. Nevertheless, there is no current research, which focuses on the direct effect of irrigation with TWW on the resistance of the soil bacteria to antibiotics. In this study, I assessed the resistance level of four different antibiotics (that differ significantly in chemical composition, source and modes of action): tetracycline, erythromycin, sulfonamide and ciprofloxacin in four agricultural soils irrigated with fresh water and TWW. Traditional microbiological isolation methods and cultureindependent molecular analysis using quantitative real time PCR targeting the antibiotic resistant genes: tetO, ermB, ermF, sul1 and sul2 were implemented. The working hypothesis of this thesis was that TWW irrigation increases antibiotic resistance levels in irrigated soils due to selective pressure generated by residual antibiotic compounds and the presence of antibiotic resistance bacteria. We compared the levels of antibiotic resistant bacteria in soils irrigated with TWW to identical soils irrigated with freshwater. Overall, the main findings of this work did not support the hypothesis and revealed interesting points: i. Based on conventional isolation methods and a cultivation–independent approach, irrigation with TWW does not cause an increase in the resistance level of the soil bacteria. This is despite of the high levels of AR bacteria and ARG's that are present in the TWW. ii. Soil moisture induces bacterial resistance to tetracycline and ciprofloxacin, in both TWW and FW irrigated soils. iii. The significant levels of antibiotic resistance detected in the agricultural soils analyzed in this study, are mainly attributed to native bacteria and not to wastewater-derived bacteria. Table of contents List of abbreviations 1