# Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies
## 2 Pathogenesis of Cystic Fibrosis (CF)
### Complications Associated with Mutation of CFTR and Hyperactivation of ENaC
#### 2.3.1 Biofilms in CF  
The chronic inflammation and inadequate eradication of pulmonary infections are significant issues in CF patients [72]. These patients suffer from frequent and persistent lung infections caused by ESKAPE pathogens (_Enterococcus faecium_, _S. aureus_, _ Klebsiella pneumoniae_, _Acinetobacter baumannii_, _P. aeruginosa_, _and Enterobacteriaceae species_); most commonly is _P. aeruginosa_, as previously mentioned [73, 74]. The ability of some of these organisms to form biofilms results in antibiotic resistance and the development of an innate immune response [75]. Biofilms are organized, adherent bacterial colonies, encapsulated in extracellular polymeric substances (EPS) made of carbohydrates, proteins, and nucleic acids [76]. EPS preserves the integrity of the biofilm and protects it from extreme environmental conditions [77]. As shown in Figure 2, biofilms are formed through five sequential stages [78].  
Antimicrobial resistance (AMR) is one of the most common challenges that can hinder an effective treatment of CF. AMP can be naturally occurring in some microorganisms due to their unique structural or functional characterization. For instance, the natural feature of the Gram-negative bacteria, such as _P. aeruginosa_, can resist the glycoptide antibiotics, for example vancomycin, which is effective against the Gram-positive bacterial strains [79]. Moreover, biofilm-forming bacteria are more AMR than non-adherent planktonic bacteria [80]. Biofilm may not only act as a barrier for antibiotics, but also can facilitate the bacterium to acquire AMR [81]. Biofilms could also enhance the horizontal gene transfer between bacteria, which will cause a transfer of several drug resistance plasmids and thus, sharing of a broad range of resistance genes [80].  
The drug efflux system is considered as one of the main strategies that bacteria use to develop AMR. These pumps are mainly encoded by genes presented either in the bacterial chromosome or in plasmid [82]. Drug efflux pumps in bacteria can be categorized into five different classes--the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, the resistance-modulation-division family (RND), the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the multidrug and toxic compound extrusion (MATE) family [83]. When the bacterial drug efflux pumps are upregulated, the antibiotics will be expelled from their cellular targets, and therefore, reduce their effectiveness [84]. Thus, targeting these pumps using efflux pump modulators can be considered as a potential strategy to overcome AMR.  
Figure 2: The five stages of biofilm formation. (**a**) Irreversible attachment of bacteria to a substrate, (**b**) bacterial proliferation, (**c**) shift from the bacterial planktonic to the biofilm life style by the loss of bacterial motility and production of extracellular polymeric substances (EPS), (**d**) biofilm maturation, and (**e**) dispersion of bacteria either to colonize a new substrate or shift to their original planktonic life style.  
In _P. aeruginosa_, the overexpression of four efflux pumps of the RND family, MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY/OprM, plays a major role in enhancing bacterial resistance toward antibiotics [85]. One of the modulators that might be used as an alternative therapy to treat mild infections are essential oils from aromatic plants [84]. For example, cinnamaldehyde(CAN), cinnamon bark essential oil, showed a significant inhibitory activity in vitro against _P. aeruginosa_. This is due to the interaction of aldehyde group of CAN with multiple bacterial cellular components and functions, including membrane lipids, ATPases, and cell division [85]. Furthermore, CAN may also interfere with the quorum sensing regulatory systems of _P. aeruginosa_, which would reduce the chances of biofilm formation [85].