Antibiotic-resistant Escherichia coli and Klebsiella spp. in greywater reuse systems and pond water used for agricultural irrigation in the West Bank, Palestinian Territories

Treating and reusing greywater for agricultural irrigation is becoming increasingly prevalent in water-scarce regions such as the Middle East. However, the potential for antibiotic-resistant bacteria to be introduced into food systems or the environment via greywater reuse is a potential area of concern. It is known that off-grid treated greywater often has elevated levels of bacteria, however, little is known regarding the prevalence of antibiotic-resistant bacteria in this water source. To address this knowledge gap, samples (n = 61) of off-grid, household greywater (influent), treated greywater effluent, and irrigation pond water were collected between October 2017 and June 2018 from four farms in the West Bank, Palestinian Territories. Samples were tested for pH, turbidity, dissolved oxygen, electrical conductivity, and oxidation reduction potential. Standard membrane filtration was used to enumerate presumptive Escherichia coli, and isolates (n = 88) were purified, confirmed using 16S rRNA sequencing, and subjected to antimicrobial susceptibility testing using microbroth dilution. The majority of influent (76.5%) and effluent (70.6%) samples had detectable presumptive E. coli. Interestingly, the majority of the isolates were confirmed as Klebsiella sp. (n = 37), followed by E. coli (n = 32), and the remainder were classified as other Enterobacteriaceae (n = 19). A higher percentage of effluent isolates were fully susceptible to all tested antibiotics when compared to influent isolates (28.6% vs 18.6%). Resistance was most commonly observed against ampicillin (69.3% of all isolates), trimethoprim-sulfamethoxazole (11.4%), tetracycline (9.1%), and cefazolin (7.9%), and 7.9% of isolates were observed to be multidrug-resistant. While most water quality parameters were within Israeli and Palestinian wastewater reuse requirements, E. coli levels in effluent violated available standards. These findings suggest that, despite observed decreases in bacteria and an overall decrease in isolates expressing antibiotic resistance from influent to effluent, off-grid greywater treatment systems are still a potential source of both susceptible and antibiotic-resistant bacteria in the agricultural environment. •28.6% of effluent isolates were fully susceptible to all tested antibiotics.•Resistance was most commonly observed against ampicillin.•Prevalence of antibiotic-resistant isolates higher in effluent than pond water.•Treated greywater generally meets Palestinian re