Quantification and characterization of antimicrobial resistance in greywater discharged to the environment

In disenfranchised communities, untreated greywater (wastewater without sewage) is often environmentally discharged, resulting in potential human exposure to antimicrobial-resistant bacteria (ARB), including extended-spectrum beta-lactamase (ESBL) producers. We sought to examine the abundance of ARB, specifically ESBLs, and antimicrobial resistance genes (ARGs) in greywater from off-grid, pastoral Bedouin villages in Southern Israel. Greywater samples (n = 21) collected from five villages were analyzed to enumerate fecal coliforms and Escherichia coli. ESBL producers were recovered on CHROMagar ESBL and confirmed by VITEK®2 (bioMerieux, Marcy l'Etoile, France) for identification and antimicrobial susceptibility testing. Total genomic DNA was extracted from greywater samples and quantitative PCR (qPCR) was used to determine relative abundance (gene copies/16S rRNA gene) of class 1 integron-integrase intI1, blaTEM, blaCTX-M-32, sul1, and qnrS. The mean count of presumptive ESBL-producing isolates was 4.5 x 10⁶ CFU/100 mL. Of 81 presumptive isolates, 15 ESBL producers were recovered. Phenotypically, 86.7% of ESBL producers were multi-drug resistant. Results from qPCR revealed a high abundance of intI1 (1.4 x 10^-1 gene copies/16S rRNA), sul1 (5.2 x 10^-2 gene copies/16S rRNA), and qnrS (1.7 x 10^-2 gene copies/16S rRNA) followed by blaTEM (3.5 x 10^-3 gene copies/16S rRNA) and blaCTX-M-32 (2.2 x 10^-5 gene copies/16S rRNA). Results from our study indicate that greywater can be a source of ARB, including ESBL producers, in settings characterized by low sanitary conditions and inadequate wastewater management.