# The RND Efflux Pump Gene Expression in the Biofilm Formation of _Acinetobacter baumannii_
## 3 Discussion  
_A. baumannii_ has become a major therapeutic problem worldwide due to its high prevalence in hospital environments and acquisition of antimicrobial resistance [6]. _A. baumannii_  
Figure 1: Fold changes in expression of the _adeB_, _adG_, and _adel_ genes in _A. baumannii_ isolates. The bars represent relative expression levels of each gene with the corresponding control. **(a)** Expression of isolates in planktonic cells relative to standard strain (ATCC 19606) (control); **(b)** Expression of isolates in biofilm forming cells and standard strain relative to their planktonic cells (control). The error bars represent the standard error of mean (SEM). All values are means \(\pm\) SEM. 165 rRNA was used as housekeeping gene control. Strong biofilm forming isolate (Standard strain ATCC 19606, 4, 23, 25, 28), moderate biofilm forming isolate (27, 33, 35, 45), weak biofilm forming (7, 12), and non-adherent isolate (36). Dotted line: Change in expression \(>\) 2 folds. has developed different mechanisms to resist antibiotics and among them is extrusion of toxic compounds by efflux pumps and biofilm formation [14]. The resistance modulation division (RND) efflux pump family represents the major type of efflux pump in Gram negative bacteria [15]. Therefore, it has been the focus of our current study. Evaluating the biofilm formation potential of the MDR _A. baumannii_ isolates in our study revealed that, overall, 88.2% (30/34) of isolates were biofilm producers with variable phenotypes that ranged from strong to weak. The presence of weak and non-adherent biofilm isolates representing 44.1% of the MDR isolates indicates that biofilm formation has no association with antibiotic resistance as reported by [PERSON] and colleagues [13]. Recently, the role of efflux pumps in biofilm formation has been the focus of different studies seeking alternatives to antimicrobials in controlling biofilm formation [6; 16]. To study the effect of the efflux pump substrate and efflux pump inhibitors on biofilm phenotypes, we utilized sub-MICs of levofloxacin and CCCP, respectively. Levofloxacin is a common substrate for the three RND efflux pumps: AdeABC, AdeFGH, and AdelJK [17; 18]. In the present study, the resistance of _A. baumannii_ isolates to levofloxacin was detected in 73.5% (25/34) of the isolates, a percentage that is comparable to findings of a previous study by [PERSON] and colleagues [19] but less than resistance percentages detected in other studies that showed resistance rates of 85-92% [20; 21]. The susceptibility of 56% (14/25) of strains to levofloxacin has increased in the presence of the efflux pump inhibitor CCCP; however, only 40% of resistant isolates have restored susceptibility under the effect of CCCP. Similar reduction in quinolone resistance after the addition of CCCP was reported in previous studies [22; 23], indicating the involvement of efflux system in these isolates. The failure of CCCP in restoring susceptibility to quinolone in some isolates can indicate the presence of more than one mechanism of resistance to levofloxacin in these isolates, such as mutations in the _gyrA_ or _parC_ genes encoding gyrase subunit A and topoisomerase IV subunit C, respectively [24]. The addition of sub-MICs of levofloxacin (efflux pump substrate to bacterial culture significantly increased the biofilm formation pattern (\(p<0.05\)) in 20.5% (7/34) of isolates. A study by [PERSON] and colleagues [25] illustrated that sub-MIC of levofloxacin caused overexpression of the _adeFGH_ gene, resulting in accelerated synthesis and transport of quorum sensing molecules during biofilm formation. On the other hand, efflux pump inhibition by CCCP significantly reduced (\(p<0.05\)) biofilm formation in 17.6% (6/34) of isolates. Strains affected by levofloxacin were different from those affected by CCCP indicating that the efflux pumps impacted in each of the two cases were different [26]. Moreover, the exposure of _A.