# The RND Efflux Pump Gene Expression in the Biofilm Formation of _Acinetobacter baumannii_
## 4 Materials and Methods
### Extraction of Total Bacterial RNA from Planktonic and Biofilm Forming Cells  
A representative isolate from each ERIC cluster was selected for gene expression analysis by the Quantitative polymerase chain reaction (qPCR). Isolates were grown overnight at 37 \({}^{\circ}\)C in Lauria Bertani (LB) broth and diluted to McFarland 0.5. A volume of 7 mL of adjusted suspension from each isolate was added in each well of 6-well plates (Greiner Bio-One, Kremsmunster, Austria) and was incubated at 37 \({}^{\circ}\)C for 24 h. After incubation, culture suspension was removed from wells for extracting RNA from planktonic bacteria. Cells adhering to the plate wells were washed twice by cold sterile Phosphate buffer saline (PBS) and, then, dislodged by sonication before suspension in falcon tube containing PBS. Total RNA was isolated from planktonic and adherent cells using RNeasy\({}^{\circledR}\) Minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Contaminating DNA was removed by RNase-free DNase I (New England Biolabs, USA). The concentrations and purity of RNA were quantified with a NanoDropTM Spectrophotometer (ThermoScientificTM, Ipswich, MA, USA) at 260 and 280 nm (260/280 ratio of >1.8) and agarose gel electrophoresis of RNA samples verified its integrity. Finally, the RevertAid First strand cDNA synthesis kit (ThermoScientificTM, Waltham, MA, USA) was used to reverse transcribe 1 ug of total RNA sample into cDNA.  
Quantification of the Expression of the RND Efflux Pump Genes (adeB, adeG, adeJ) in Biofilm and Planktonic Forms Using RT-qPCR  
Real-time quantitative PCR was used to measure the relative expression of the RND-family efflux pump genes (_adeB, adeG, adeJ_) using Maxima SYBR Green qPCR Master Mix with ROX (ThermoScientific, Waltham, Massachusetts, USA) and primers listed in Table 4. The expression of pump genes was normalized to the housekeeping gene (16S rRNA). Expression quantification was done using the StepOnePlusTM Real-Time PCR System and software (Applied Biosystems, Waltham, Massachusetts, USA) according to the manufacturer's instructions. After a 10 min activation of the modified Taq polymerase at 95 \({}^{\circ}\)C, 40 cycles of 15 s at 95 \({}^{\circ}\)C, 30 s at 60 \({}^{\circ}\)C, and 30 s at 72 \({}^{\circ}\)C were performed. Data acquisition was done at 60 \({}^{\circ}\)C and 72 \({}^{\circ}\)C. The relative gene expression for pump genes transcripts was calculated against internal control 16S rRNA gene. The relative expression of efflux pump genes in the tested isolates was calculated against those of _A. baumaniii_ ATCC 19606 (expression = 1), which served as the control to determine overexpressed pump genes. The expression of the efflux pump genes in the biofilm form was determined relative to their expression in the planktonic form. The \(2^{-\Delta\Delta\text{CT}}\) method was used to calculate the relative expression level of pump genes. A significant effect on gene expression was concluded when the corresponding ratios were >2.0. All reactions were performed in triplicate [24].  
\begin{table}
\begin{tabular}{c l c c c} \hline \hline
**Primer** & **Sequence 5\({}^{\prime}\)—3\({}^{\prime}\)** & \begin{tabular}{c} **Annealing** \\ **Temperature (\({}^{\circ}\)C)** \\ \end{tabular} & **Ampilicon Size bp** & **Reference** \\ \hline _adeB_ & \begin{tabular}{c} F: GCAGAGCGTACTCGGAAGT \\ R:CCACTGAAACCCCATCCAA \\ \end{tabular} & 57 & 101 & [47] \\ \hline _adeG_ & \begin{tabular}{c} F:GCGTGTGTGTGTGACAGATGT \\ R:TTGTGCACGACCTGATAAA \\ \end{tabular} & 52 & 104 & [5] \\ \hline _adeJ_ & \
\begin{tabular}{c} F:TTCGGTGTGTCATAGCCAAT \\ R:GGAGCACCCTAACTGACC \\ \end{tabular} & 57 & 137 & [47] \\ \hline _16s rRNA_ &
\begin{tabular}{c} F:AGCTAAGCGGATAAGACCG \\ R:TGTCAAGGCCAGGTAAAGGTTC \\ \end{tabular} & 57 & 137 & [47] \\ \hline \hline \end{tabular}
\end{table}
Table 4: Oligonucleotides sequences and annealing temperatures.