*2.2. Sample Collection*

Water samples were collected bi-weekly using Nalgene sterile bottles from 6 different sampling points along the Nahoon beach and canal for a period of twelve months, between 8:00 a.m. and 11:00 a.m. The sampling points where the water samples were collected are: canal point 1, canal point 2 (where domestic effluents flowed into canal), canal point 3 (where canal flowed into beach), beach point 1, beach point 2 (where final effluent was released into beach), and beach point 3. The samples were then transported on ice to the Applied and Environmental Microbiology Research Group (AEMREG) laboratory, University of Fort Hare, Alice within 6 h for analyses.

#### *2.3. Isolation and DNA Extraction*

The water samples were filtered using the membrane filtration technique, after which the filter papers were aseptically picked, placed onto *E. coli* chromogenic agar, and incubated at 37 ◦C for 18–24 h. After incubation, the isolates were re-streaked onto nutrient agar (NA) plates and incubated at 37 ◦C for 24 h. A total of 260 presumptive *E. coli* isolate colonies were picked from the NA plates, inoculated into nutrient broth, and incubated at 37 ◦C for 24 h. Thereafter, glycerol stock was prepared from the cultured broths, and DNA was extracted following the method of Torres et al. [22], and stored at −80 ◦C for further analyses.
