# The RND Efflux Pump Gene Expression in the Biofilm Formation of _Acinetobacter baumannii_
## 2 Results
#### 2.2.1 Efflux Pump Substrate (Levofloxacin)  
Levofloxacin-resistant isolates represented (25/34; 73.5%) of the isolates as confirmed by the high MICs of levofloxacin ranging from 4 ug/mL to 216 ug/mL. In order to determine the role of the efflux pump in levofloxacin resistance, CCCP was used at a concentration of 12.5 ug/mL, which does not reduce the growth of isolates as reflected by their OD\({}_{600}\), compared to control untreated culture. The addition of CCCP to the bacterial culture of levofloxacin-resistant isolates caused a reduction in MIC by four folds or more in 56% (14/25) of resistant isolates as shown in Table 2. On the other hand, 40% (10/25) of levofloxacin-resistant isolates became sensitive upon treatment with CCCP (Table 2). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**ERIC Cluster Group** & **Isolate Number** & **Biofilm Formation Pattern** \\ \hline  & & 1 & Moderate \\  & & 26 & Moderate \\
**D** & & 44 & Moderate \\  & & 45 & Moderate \\  & & 47 & Moderate \\  & & 32 & Moderate \\ E & & 33 & Moderate \\  & & 46 & Moderate \\ F & & 31 & Moderate \\  & & 35 & Moderate \\  & & 3 & Weak \\  & & 6 & Weak \\  & & 24 & Non-Adherent \\  & & 28 & Strong \\
**G** & & 29 & Weak \\  & & 36 & Non-Adherent \\  & & 40 & Non-Adherent \\  & & 41 & Non-Adherent \\  & & 2 & Weak \\  & & 4 & Strong \\
**H** & & 14 & Weak \\  & & 15 & Weak \\  & & 23 & Strong \\  & & 12 & Weak \\
**I** & & 21 & Weak \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._ \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Isolate Number** & **MIC (\(\mu\)g/mL)** & **MIC + CCCP (\(\mu\)g/mL)** & **Phenotypic Factor (F)** & **Resistance Phenotype** \\  & & & **\# CCCP** & **\# (-/+ CCCP)** \\ \hline _A. baumanantii ATCC_ & 1 & 1 & S/S \\
**19606** & 4 & 32 & 2 & R/R \\
**7** & 8 & 4 & 2 & R/R \\
**12** & 8 & 4 & 2 & R/R \\
**14** & 64 & 32 & 2 & R/R \\
**15** & 8 & 4 & 2 & R/R \\
**22** & 4 & 1 & 4* & R/S \\
**23** & 8 & 1 & 8* & R/S \\
**24** & 64 & 1 & 64* & R/S \\
**25** & 8 & 1 & 8* & R/S \\
**26** & 64 & 1 & 64* & R/S \\
**27** & 4 & 1 & 4* & R/S \\
**28** & 256 & 128 & 2 & R/R \\
**29** & 256 & 128 & 2 & R/R \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Change in the phenotypic resistance pattern of isolates to levofloxacin under the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazzone (CCCP) (\(n=25\)). After determining the MIC of levofloxacin against isolates, the effect of increased efflux pump activity by sub-MICs of levofloxacin on biofilm formation was studied. The \begin{table}
\begin{tabular}{c c c c c} \hline
**ISolate Number** & **MIC (\(\mu\)g/mL)** & **MIC + CCCP (\(\mu\)g/mL)** & **Phenotypic Factor (F) \#** & **Resistance Phenotype** \\  & & & **CCCP** & **\({}^{\dagger}\) (\(-\)/+ CCCP)** \\ \hline
**30** & 256 & 1 & 256 \({}^{*}\) & R/S \\
**32** & 64 & 8 & 8 \({}^{*}\) & R/R \\
**33** & 16 & 4 & 4 \({}^{*}\) & R/R \\
**35** & 32 & 8 & 4 \({}^{*}\) & R/R \\
**36** & 32 & 1 & 32 \({}^{*}\) & R/S \\
**37** & 32 & 16 & 2 & R/R \\
**39** & 8 & 1 & 8 \({}^{*}\) & R/S \\
**40** & 64 & 32 & 2 & R/R \\
**41** & 128 & 32 & 4 \({}^{*}\) & R/R \\
**43** & 4 & 2 & 2 & R/I \\
**44** & 32 & 16 & 2 & R/R \\
**45** & 16 & 2 & 8 \({}^{*}\) & R/I \\ \hline \end{tabular} \({}^{\dagger}\) S: Susceptible (MIC \(\leq\) 1 ug/mL), I: Intermediate (MIC = 2 ug/mL), R: Resistant (MIC \(\geq\) 4 ug/mL). # Phenotypic efflux factor (F) = MIC without CCCP (EPI)/MIC with CCCP (EPI); * Isolates showing ¿ 4 phenotypic efflux factor with CCCP 12.5 ug/mL.  
\end{table} 
Table 2: _Cont._