## 2 Materials and Methods
### Genomic DNA Extraction  
By using the modified protocol proposed by [PERSON] et al. [52], bacterial genomic DNA extraction was performed. The bacteria were cultured for 18 to 24 h at 35-37 \({}^{\circ}\)C. Afterwards, the culture was centrifugated, and the pellets acquired were resuspended in 0.5 mL of lysis solution 8.0 M urea and 0.5 mL of 10% (_w/v_) SDS (Merck, Germany), 10.0 mM Tris-HCL (Merck, Germany), and 0.3 M NaCl (Merck, Germany). The suspension was incubated at 37 \({}^{\circ}\)C for 20 min. The DNA was extracted by adding 2 volumes of phenol and mixed well using a vortex mixer for 10 min. The mixture was centrifugated at 9750\(\times\)\(g\) (Thermo Sorvall Legend Micro, Germany) for 10 min. The liquid at the aqueous phase was aliquoted into a new tube. The same volume of chloroform and isoamyl alcohol was added. They were mixed well and later centrifugated at 9750\(\times\)\(g\) for 5 min. By adding 2.5 volumes of 100% ethanol (Merck, Germany) and 1/10 volumes of 3 M sodium acetate (pH 5.2) buffer (Merck, Germany), the aqueous phase containing genomic DNA was precipitated. The combination was incubated at \(-\)20 \({}^{\circ}\)C for 1 h and then centrifugated at 9750\(\times\)\(g\) for 5 min. Afterwards, the pellets containing DNA were rinsed with 70% (\(v/v\)) ethanol (Merck, Germany). To dissolve the dried DNA pellet, a combination of 1.0 mM EDTA (pH 8.0) (Merck, Germany), and TE buffer 10.0 mM Tris-HCl (pH 7.5), was used. Later, the isolated DNA was stored at \(-\)20 \({}^{\circ}\)C.  
The reaction mixture used in this study is indicated in Table 1. Polymerase Chain Reaction (PCR) was performed based on the manufacturer's instructions (Lucigen, WI, USA) for the amplification of 16S rRNA sequence of all isolates (UPMC 1166, UPMC 1168, UPMC 1254, UPMC 1389, UPMC 1376, UPMC 1393, UPMC 703 and UPMC 704). The PCR was conducted using a thermal cycler (peqSTAR, Germany) with the thermal cycling conditions following the order of (a) pre-heat (94 \({}^{\circ}\)C for 2 min), (b) denaturation (35 cycles at 94 \({}^{\circ}\)C for 30 s), (c) annealing (52 \({}^{\circ}\)C for 30 s), (d) extension (72 \({}^{\circ}\)C for 1 min), and (e) final elongation (72 \({}^{\circ}\)C for 10 min). Later, the amplified 16S rRNA was kept at 4 \({}^{\circ}\)C before electrophoretic analysis. Gel electrophoresis was used to analyze the amplified DNA samples. The preferred protocol of [PERSON] and [PERSON] [47] was used with some modifications. One hundred mL of 1% (\(w/v\)) agarose gel was made by dissolving the agarose powder in 1 \(\times\) TBE buffer and 5 \(\upmu\)L of GelREdTM (Biotium, Fremont, CA, USA) nucleic acid gel stain was added. The solution was heated in a microwave for 2-3 min and mixed well by swirling. The agarose gel was poured into the gel tray before the comb well placement. The solution was left to solidify for 30 min. The comb was removed after the gel had solidified. The gel tray was placed in the buffer tank and immersed with 1 \(\times\) TBE for a depth of 2 to 5 mm. For the sample loading process, 2 \(\upmu\)L of Bromophenol blue loading dye (Lucigen, USA) was mixed with 5-7 \(\upmu\)L of amplified genomic DNA and 3-5 \(\upmu\)L of PCR products. Then, 10 \(\upmu\)L of 1 kb DNA ladder (Lucigen, USA) was used as the molecular weight marker. Then, 75 to 80 Volts (V) of electricity was applied for the gel electrophoresis analysis. For viewing and recording purposes, the gel was then positioned on the molecular imager Gel Doc (Bio Rad, USA) system. HiYieldTM Gel/PCR DNA Mini Kit (Real Biotech, Taiwan) was used to purify the PCR product [53]. The agarose gel with the PCR products was removed until 300 mg and was then transferred into a 1.5 mL microcentrifuge tube. Later, the DF buffer was supplemented to it. The First BASE Laboratories Sdn Bhd service was employed to analyze the purified PCR product for the sequencing analysis. By using ClustalW and BioEdit 7.2.4 software, the sequences were derived from the forward and reverse primers of PCR.