*2.3. Molecular Identification and Phylogenetic Analysis*

The total genomic DNA of bacteria was extracted by phenol-chloroform method, quality checked by agarose gel electrophoresis and quantified using NanoDrop1000 (Thermo-Scientific, USA). The PCR amplification of 16S rRNA gene was carried out with universal primers: 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1492R (5 - ACG GCT ACC TTG TTA CGA CTT-3 ) using the following conditions: initial denaturation temperature was set at 95 ◦C for 5 min, followed by 35 cycles at same temperature for 1 min, primer annealing at 54 ◦C for 1 min, and primer extension at 72 ◦C for 2 min. The reaction mixture was kept at 72 ◦C for 10 min subsequently and then cooled to 4 ◦C. The PCR products were checked in 1.5% agarose gel and visualized in a UV transilluminator (Tarsons, India) and the gel imaging was done using a Gel documentation system (Bio-Rad, USA). The amplified PCR products were sequenced using same set of primers (27F' and 1492R') on Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA). The genetic relationship between the strains was determined by neighbor-joining tree algorithm method. The phylogenetic tree was constructed with a bootstrapped database containing 1000 replicates in MEGA 7.0 software (Mega, Raynham, MA, USA). The nearly complete 16S rRNA consensus sequences were deposited in the GenBank database.
