Identification of isolated strains

The identification of the isolates was done based on 16S rRNA gene sequencing. The Polymerase Chain Reaction (PCR) was performed based on the PCR colony method as described by Zainudin et al.¹⁷. A pair of universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACT-3’) targeting the 16S rRNA gene was used as forward and reverse primers. The amplification of the 16S rRNA gene was conducted in 50 μl of PCR Master Mix kit (QIAGEN) mixture containing 25 μl dNTP’s, 1.5mM MgCl₂ buffer, DNA polymerase, 0.5 μl of each forward and reverse primers. The RNase-free water was added until the final volume reaches 50 μl. Thermocycling was set up according to the conditions provided by the kit protocol as follows: 3 min of initial denaturation at 94 °C, 0.5 min of denaturation at 94 °C, 0.5 min of primer annealing at 60 °C, 1 min of primer extension at 72 °C and final elongation at 72 °C for 10 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized using Gel red staining on a UV transilluminator. The PCR products were purified using Nucleospin PCR clean-up (Machery-Nagel, GmbH & Co., Germany) according to the manufacturer’s instructions before sequencing. The PCR products were then sequenced and the 16S rRNA sequences data were compared with those of other know species in the genebank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).