## 2 Materials and Methods
### Bacterial Characterization
#### 2.3.3 16s rRNA Sequencing  
Pure isolated bacterium samples were grown in RCM broth for 18 h at 37 \({}^{\circ}\)C. The broth was then centrifuged for 90 s at 15000 g to separate the supernatant from the bacterial pellets. The pure culture bacterial pellet provided the deoxyribonucleic acid (DNA) for the polymerase chain reaction (PCR). Deoxyribonucleic Acid (DNA) Extraction  
The DNA from bacterial pellets was extracted using Geneaid(tm) DNA isolation kit (Geneaid(tm), Taiwan) following the manufacturer's recommended procedure. Firstly, 300 \(\upmu\)L cell lysis buffer was added to the bacterial pellet in a 1.5 mL microcentrifuge tube. The mixture was vortexed homogenously and incubated at 60 \({}^{\circ}\)C for 15 min. Following that, 100 \(\upmu\)L protein removal buffer was added to lysate and vortexed immediately for 15 s before centrifuging it at 15,000\(\times\)\(g\) for 8 min. The supernatant was transferred to a fresh 1.5 mL microcentrifuge tube while the pellet was discarded. The extracted DNA was precipitated by adding 300 \(\upmu\)L of isopropanol and thereafter gently mixed. The mixture was centrifuged at 15,000\(\times\)\(g\) for 5 min and the supernatant was discarded. The pellets were washed with 70% (\(w/w\)) ethanol and centrifuged it at 15,000\(\times\)\(g\) for another 5 min. Finally, the DNA was rehydrated by adding 100 \(\upmu\)L of DNA hydration buffer and incubated it at 60 \({}^{\circ}\)C for 45 min. The extracted DNA was used as a template for DNA amplification by polymerase chain reaction (PCR) using PCR Master Mix (1 st BASE, Singapore). This PCR Master Mix is a premixed PCR cocktail that contains Taq DNA polymerase (0.06 U/\(\upmu\)L), 400\(\upmu\)M of each of NTTPs, and 3 mM MgCl\({}_{2}\) in reaction buffer. Additionally, 10 \(\upmu\)M of forward primer 8F (5'-AGAGTTTGATCCGGCTCAG-3'), and 10 \(\upmu\)M of reverse primer 1492R (5'-GGTTACCTTGTTAGCATT-3') were purchased from 1 st Base Sdn. Bhd. The PCR was started by adding 25 ul of PCR Master Mix, 2.5 \(\upmu\)L of each primer, 5 \(\upmu\)L of DNA template, and 15 \(\upmu\)L of nuclease-free water in a PCR tube. PCR cycle was performed with an initial denaturation at 95 \({}^{\circ}\)C for 2 min, followed by 30 cycles of 95 \({}^{\circ}\)C for 30 s, annealing gradient from 55 to 72 \({}^{\circ}\)C for 1 min, extension of 72 \({}^{\circ}\)C for 1 min and a final extension of 72 \({}^{\circ}\)C for 10 min [8]. The PCR product was kept at 4 \({}^{\circ}\)C until further use. PCR Product Purification  
The PCR product was then purified using Gel/PCR DNA Fragments Extraction Kit (Geneaid(tm), Taiwan). Accordingly, 50 \(\upmu\)L of PCR product was transferred into fresh 1.5 mLmicrocentrifuge tube. Then, 250 \(\upmu\)L DF buffer was added to the tube and homogenously mixed. The mixture was then pipetted into DF column and centrifuged at 15,000\(\times\)\(g\) for 4 min. The filtrate was discarded and 600 \(\upmu\)L of wash buffer was added to the DF column and centrifuged again at 15,000\(\times\)\(g\) for 2 min. After discarding the filtrate, DF column was placed in fresh 1.5 mL microcentrifuge tube and 25 \(\upmu\)L elution buffers was added to it. The DF column was allowed to stand for 5 min before being centrifuged at 15,000\(\times\)\(g\) for 4 min, and thereafter the purified PCR product was collected. Agarose gel was prepared by heating 1.2 g agarose powder (1 st Base, Singapore) in 80 mL Tris Borate EDTA buffer (TBE) (1 st Base, Singapore). The agarose solution was then cooled down at room temperature, and 5 \(\upmu\)L of ethidium bromide was added to the solution. The mixture was then poured onto a gel plate with suitable gel comb. After the gel hardened, the comb was removed, leaving a row of wells. The gel was immersed in TBE buffer in the electrophoresis chamber (Bio-Rad, USA). The DNA sample (3 \(\upmu\)L) was mixed with loading dye (1 \(\upmu\)L) and pipetted into the agarose gel wells. One of the wells was loaded with DNA ladder.