Gelcompar 2 version 6

Fingerprinting PCR was carried out in 20µL reaction tubes in a Bio-Rad T100™ Thermal Cycler (Bio-Rad, Singapore, Singapore). The reaction mixture consisted of 10µL KAPA BIOSYSTEMS 2X KAPA Taq Ready Mix (Kapa Biosystems Cape Town, South Africa), 0.3µL GTGGTGGTGGTGGTG oligonucleotide primer (Versalovic et al., 1994) , 2µL DNA template, 7.3µL PCR-grade water and 0.4µL Dimethyl sulfoxide (DMSO). The PCR program was as follows: 95 o C for 5 min, 95 o C for 1 min, annealing at 52 o C for 1 min, extension at 72 o C for 3 min. The program was repeated for 34 cycles and a final extension at 72 o C for 10 minutes. PCR products were separated by Gel electrophoresis using a 1.5 % Agarose gel (55V for 4h) and the image viewed using a Bio-Rad Gel Doc™ EZ (Bio-Rad, California, USA). The GTG5 fingerprints were analysed using Gel-Compar II version 6.5 (Applied Maths NV, Sint-Martens-Latem, Belgium). The similarity of digitised bands patterns was calculated using Pearson's correlation coefficient, and unweighted pair group method with arithmetic means. Complete linkage algorithms were used to construct an average linkage dendrogram to show relationship of isolates. Isolates were considered to be within a clonal cluster if relatedness was 70% and above (Stackebrandt et al., 2002) . However, due to the close similarities that existed between the isolates sub clusters were further considered at 95%.

Fingerprints were generated for the 46 E. coli isolates including the O157:H7 reference strain.
The 46 fingerprinted isolates had previously given a positive presumptive test on STEC media (Ntuli et al., 2016) . The 20 μL reaction mixture consisted of 2 μL (100 ng) DNA template, 0.3 μL (0.2 μM) of the single oligonucleotide (GTG) 5 (5′-GTGGTGGTGGTGGTG-3′) primer (Versalovic, Schneider, De Bruijn, & Lupski, 1994) , 10 μL of 2 times KAPA Taq ReadyMix PCR Kit, 0.4 μL (1%) dimethylsulphoxide and PCR grade water. The PCR thermal cycling was performed as follows: Initial denaturation at 95 ºC for 10 min; 35 cycles at 95 ºC for 30 s; 40 ºC for 60 s and 65 ºC for 3 min and a final elongation step at 65 ºC for 8 min. The PCR amplified DNA was separated on 2% agarose gel (55 V for 4 h). The GTG 5 fingerprints of the E. coli isolates were analysed using GelCompar II version 6.5 (Applied Maths NV, Sint-Martens-Latem, Belgium). The similarity of digitized bands patterns was calculated using Pearson's correlation coefficient, and unweighted pair group method with arithmetic means. Complete linkage algorithms were used to construct an average linkage dendrogram to show relationship of isolates. Isolates were considered to be within a clonal cluster if relatedness was 70% and above (Stackebrandt et al., 2002) .