# The RND Efflux Pump Gene Expression in the Biofilm Formation of _Acinetobacter baumannii_
## 3 Discussion
baumannii_ to sub-inhibitory concentrations of antimicrobials did not only increase substrate efflux by the efflux pump but also changed the expression of genes controlling biofilm formation, including genes regulating pili, other efflux pumps, and virulence factors [27]. The RND efflux pump genes were detected in all tested isolates. Likewise, in another study, AdeABC, AdeFGH, and AdelJK pumps were detected in 92.18%, 98.43%, and 89.06% of strains, respectively [28]. The relative gene expression of the major part of tripartite efflux systems (_adeB_, _adeG_, and _adeJ_) was measured by qPCR for eleven isolates representing different ERIC clusters relative to that of the reference _A. baumannii_ strain (ATCC 19606). CCCP increased the levofloxacin susceptibility of the five isolates that did not show overexpression for any of the efflux pumps. No correlation was detected between the effect of CCCP on resistance to levofloxacin as indicated by a reduction in the MIC and the RND efflux pump expression (Spearman's \(r_{s}=-0.08\), \(-0.16\), and \(-0.02\), for _adeB_, _adeG_, and _adeJ_, respectively; \(p>0.05\)). This result indicates the involvement of other efflux pumps in levofloxacin resistance by these isolates, such as _abeM_ and _abaQ_, that belong to the MATE and MFS efflux systems, respectively [29]. The previous efflux pumps can also be inhibited by CCCP [30; 31]. Previous studies on the role of _A. baumannii_ efflux pumps in biofilm formation were based on knocking out the RND efflux pump genes in a limited number of strains and then measuring the impact of this on biofilm formation [16; 32; 33] or on measuring the relative expression of efflux genes in the planktonic form and correlating the expression levelsto the degree of biofilm formation [6; 13]. In our assessment, these previous approaches are not adequate or even representative for the bacteria, given its distinct phenotypic and genotypic characteristics. Moreover, these methods do not reflect the actual expression pattern in the biofilm form. Thus, the expression of _adeB_, _adeG_, and _adeF_ efflux pump genes in the biofilm form relative to the planktonic form was studied to illustrate the role of these pumps in the mature biofilm of _A. baumannii_. Results have shown that the relative expression pattern of the RND pumps genes varied among different isolates with different biofilm forming abilities. The lack of correlation between efflux pump gene expression and biofilm formation potential suggests that the RND efflux pump role in biofilm formation is not pivotal. Some studies reported the lack of association between the activity of the efflux pump family and biofilm formation [6; 13; 28]. Other studies reported a negative correlation between the activity of one or more of the RND efflux pump genes _adeB_, _adeG_, and _adeF_ and biofilm formation [6; 12; 34; 35; 36; 32]. The negative correlation observation was explained by the presence of associated changes in the bacterial membrane with efflux pump overexpression, such as the reduction in _Csu-pili_ and the downregulation of genes involved in iron acquisition and motility [6; 16; 33]. Furthermore, other studies reported that the increase in expression of one or more of the _adeB_, _adeG_, and _adeF_ genes was associated with increased biofilm formation [6; 16; 25; 28; 33; 36]. The overexpression of the _adeFGH_ efflux pump gene was suggested to play a role in the synthesis and efflux of quorum sensing molecules during biofilm formation [25; 37]. Other explanations highlighted operons as the controllers of these pumps through controlling other genes contributing to biofilm formation. In this scenario, the efflux pumps are not directly in control of the biofilm phenotype. For example, the low expression of _adeABC_ is associated with the reduced expression of other genes involved in biofilm formation, motility, and virulence because these genes are subjected to control by the regulator adeRS [33].