*2.4. Screening for Antibiotic-Resistant* E. coli

The remaining 1 mL from the overnight culture was used for antibiotic resistance analysis using the disk-diffusion method [26]. Briefly, 100 μL of overnight *E. coli* culture was spread on Mueller–Hinton agar (Lasec, Cape Town, South Africa) and antibiotic mastrings (Davies diagnostics, Johannesburg, South Africa) were carefully placed onto inoculated plates, incubated at 37 ◦C for 18–20 h. Following incubation, the diameters (in millimetres) of clear zones of growth inhibition around the antibiotic disks were measured using a ruler and compared with the Clinical Laboratory Standard Institute (CLSI) 2013 reference values. The different phenotypic profiles (resistant, intermediate or susceptible) of the isolates were then determined following the interpretation of the zones of inhibition. A total of 11 antibiotics were selected for this study (Table 1). The antibiotics were chosen for their frequent use in the treatment of bacterial infections in South Africa Both positive (*E. coli* strain ATTC 25922) and negative controls (*E. coli* strain ATTC 35218) were included in the experiments.

**Table 1.** Antibiotics used to determine antibiotic resistance of *E. coli* isolates.


#### *2.5. Data Analysis*

Data were analysed using the Statistical Package for the Social Sciences (SPSS) (Version 16.0, Prentice Hall Press Company, NJ, USA) [27]. The *E. coli* counts were log10 transformed before computation of the means and standard deviations. A multiple antibiotic resistance (MAR) index was performed following the procedure described by Krumperman [28]. A MAR index for an isolate was calculated using the formula: MAR = a/b where 'a' is the number of antibiotics from each group to which a particular isolate was resistant and 'b' is the total number of antibiotics against which the isolate was tested. A resistance index greater than 0.2 shows that *E. coli* isolates are likely to be from a high-risk source.
