Biofilm Inhibition by Synergistic Combination

The biofilm inhibitory effect of the synergistic combination was understood using the gene expression analysis of the biofilm regulatory genes of UPEC. The planktonic cells (log phase) of UPEC were treated with Nitrofurantoin (8 μg/mL), TAP (32 μg/mL), a combination of TAP (32 μg/mL) and Nitrofurantoin (8 μg/mL) and incubated for 24 h at 37°C. The pH of 5.8 was maintained in all the treatments. Total RNA was extracted by following the manufacturer's guidelines of RNeasy® Protect Bacteria Mini Kit (Qiagen). Standard agarose gel electrophoresis procedure was developed to verify the integrity and NanoDrop (Thermo Scientific, USA) was done to evaluate the purity of isolated RNA. From the isolated RNA, cDNA was synthesized using iScript™ cDNA Synthesis Kit (Manufacturer's protocol was followed).
The expression level of genes responsible for adhesins of UPEC was analyzed using qRT-PCR. The respective primers and the melting temperature of each of the gene used were listed in Table S1. Reference gene and negative control are 16srRNA and without cDNA were maintained, respectively. Calculations of relative gene expression were done with 2^−ΔΔCT method (Hema et al., 2017 (link)).