Aetiology, pathogenesis, diagnosis and treatment Cystic fibrosis: role of P. aeruginosa infection in cystic fibrosis patients

Introduction Cystic Fibrosis (CF) is a genetic disorder of salt and water regulation in the cell. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Cohen and Prince, 2012). The molecular basis of CF depends on its transmembrane conductance regulator (CFTR) protein, which functions as a channel and regulates the movement of ions and water across the epithelial barrier. The barrier opens in response to increased levels of intracellular cyclic adenosine monophosphate (cAMP). The gene in normal condition is responsible for salt (chloride) and water balance in the body. However, if mutated, is not able to excrete out enough salt of the cell; thus resulting, the build of salt within the cell forming sticky mucus. If accumulated in the lungs, this sticky mucus may clinically affect the epithelial innate function of the lungs and may result in ineffective airway inflammation that fails to eradicate pulmonary pathogens (Cohen and Prince, 2012). The working mechanism of CFTR protein under normal condition is shown in figure 1. Studies have shown that increase in sodium (Na) re-absorption on the epithelial cells may indicate an event in cystic fibrosis lung disease (Cohen and Prince, 2012). These mutations on the (CFTR) protein have impacts on its production and transfer to the epical membrane of the epithelial cells, thereby influencing the conductance of chloride and bicarbonate ions across the channels resulting in the CFTR ionic imbalance of on the epithelial cells of different organs. This leads to the formation of excessive mucus in the cells, which provides an ideal condition for bacteria growth and multiplication. Interestingly, even uncommon lung bacteria such as pseudomonas have been found to inhabit in the lungs exploiting the ideal condition (Clunes and Boucher, 2007). Abnormality in the CFTR pathway results in the increase of glycolipids on the surface of CF airway epithelial cells. These glycolipids serve as receptors to increase the binding activities of bacteria, thereby facilitating the early infection process, which is a hallmark of the disease. Moreover, defect in CFTR gene is also associated with decreased nitric oxide concentration in the cell. Nitric oxide possesses antibacterial properties; thus, decrease in its concentration may predispose an individual to severe infections with faulty CFTR gene (Clunes and Boucher, 2007). In addition, the role of inflammation in CF pathogenesis seems equally important. Inflammatory response produced against the infectious pathogens in CF patients may have detrimental effects to the host cells. Inflammatory infiltrates including macrophages, neutrophils, cytokines and interleukins may induce tissue damaging responses, further deteriorating the lung function (Cymberknoh, et.al. 2013). Inhabitation of bacteria in cystic fibrosis lung As mentioned earlier, even uncommon bacterium including Pseudomonas aeruginosa can reside in the cystic fibrosis lung (Hauser, et.al. 2011). Pseudomonas aeruginosa is a gram negative, rod shaped, aerobic bacterium that prefers moist environment for growth. Commonly they are found in places such as sewage and in certain areas of the human body. Furthermore, they reside in lakes, moist vegetables, moist soil and streams. Studies have also suggested that that most human infections due to P. aeruginosa result due to contact with these natural reservoirs (Friman, et.al. 2013). Pseudomonas aeruginosa has been recognized as an important pathogen in CF. Soon after residence in the lungs, it becomes extremely difficult to eradicate them by antibiotics. The persistent presence of these bacteria in the lungs ultimately results failure of the immune system to provide a defense against them; this leads to respiratory failure resulting in death of the patient (Folkesson, et.al. 2012). Firstly, P.aeruginosa acquires the airway of the patient with cystic fibrosis, which occurs during the very early phase of the disease. Infection may ensue once this bacterium enters the CF host and colonizes the oropharynx, then to the lower respiratory tract by process of micro aspiration. Infection becomes intermittent at the initial stage as a result of acquisition of different strains. During the early stage of infection, the isolate looks like environmental strains, non-mucoid and susceptible to antibiotics. Eventually P. aeruginosa establishes itself and chronic infection ensues (Folkesson, et.al. 2012). In chronic stage of CF, P. aeruginosa produces a mucoid alginate by growing in bio-film of the end- bronchiolar space. It lacks lipopolysaccharide and becomes non-motile, resulting in the development of antibiotic resistance during. Despite the known mechanism and availability of antibiotics, P.aeruginosa is able to chronically infect the airway of patients with CF and is able to cause an unrelenting decline in pulmonary function and deterioration; but how these happen has been focus on intense research (Folkesson, et.al. 2012). Molecular pathogenesis of P.aeruginosa infection in CF The molecular basis of infection due to P. aeruginosa in CF is still not fully understood. However, it may be peculiar to a specific strain at the time of adaptation after acquisition. Studies have indicated that the persistence of the bacterium in the lungs might be due to its high level of propensity and the ability to adapt with environmental changes and stress (Hauser, et.al. 2011). aeruginosa possesses traits that enable them to colonize and persist in acute and chronic infection. These traits include; High resistance to antibiotics (Zhang, et.al. 2011) Effective cell-cell communication singalling for growth and multiplication (Friman, et.al. 2013). Ability to form biofilms, plethora and virulence products and metabolic versatility (Wagner and Iglewski, 2008). The biofilms are synthesized by the bacterium encased within a hydrated polymeric matrix and are clinically important; this is because, P. aeruginosa in this mode of growth utilizes it to cope with phagocytes by the neutrophils and macrophages, as well as to the killing by antibiotics. This allows them for persistent survival in chronically infected CF despite the continuous natural immune response and antibiotics effect (Zhang, et.al. 2011). The disease pathogenesis and severity is further triggered by inflammatory responses in the later stage of the disease. Mainly the pro-inflammatory enhancers become physiologically active in the absence of function CFTR gene. Although the exact inflammatory pathophysiology of CF still remains debatable, persistent infection, poor pathogen clearance, acidic environment and are considered some of the key triggers for initiating chronic inflammatory response in the lungs of the patient with CF (Pier, 2008). The mechanism is explained in figure 2. Synthesis of ceramide from sphingomyelin by acid sphingomyelinase (ASM) and degradation by acid ceramidase (AC) is optimal at an acidic pH, such as that of normal intracellular vesicles (pH 4.5). Lack of functional CFTR increases vesicle pH to 5.9, partially inhibiting ASM and highly inhibiting AC, resulting in ceramide accumulation in the vesicle and age-dependent pulmonary inflammation. Other properties of the cystic fibrosis–affected lung, including lack of the CFTR receptor needed for clearing Pseudomonas aeruginosa, enhanced mucin secretion, a dehydrated airway surface liquid, viscous mucus and DNA deposits released from ceramide-engorged apoptotic cells, also contribute to P. aeruginosa airway colonization. Eventually, this microbe establishes a chronic infection wherein bacterial cells reside in low-oxygen mucus plugs and become highly resistant to clearance or killing by host defenses (Pier, 2008) P.aeruginosa potentially synthesize factors such as proteolytic enzymes, which damage the host cells by altering the host iron-containing protein forming hydroxyl radicals that contribute to host tissue injury and inflammation; thereby decreasing the level of anti-inflammatory cytokines including IL-10, and at the same time, activating neutrophils in the airway lumen. Continuous recruitment of these neutrophils induces tissue damaging inflammatory response mainly affecting pulmonary tissue. Furthermore, neutrophils trigger the production of pro-inflammatory cytokine such as IL-8, which further contributes in the tissue damage process. In addition to pro-inflammatory cytokines, neutrophils also release the oxygen free radicals that induce apoptosis (programmed cell death) of the epithelial tissue in the lungs. Thus, to conclude, the role of neutrophils in airway inflammation in patients with CF seems crucial. Instead of providing innate immunity to the infected lung, it rather trigger s detrimental inflammatory response and promote the growth of P. aeruginosa though the production of different growth enhancing cytokines, leading to the destruction of the lung tissue (Sagel, et.al. 2009). The effectiveness of the immune response in CF is exploited, allowing the establishment of a relentless cycle, whereby persistent bacteria cause increased inflammation that itself leads to increased bacterial densities, which in turn results stronger inflammatory response (Sagel, et.al. 2009). The net result is progressive tissue damage and pathological consequences and sequelae of CF that include mucopurulent plugging of bronchioles, chronic bronchitis (inflammation of the bronchioles) and bronchial gland hyperplasia (increase in the cell size of bronchial gland cells). The airway later becomes dilated and results bronchiectatic due to loss of support cartilage (Pickett, 2013). Treatment options for cystic fibrosis The decision to treat patients who are infected with P.aeruginosa can be very challenging. Prolonged treatment with antibiotics can have toxic side effects, as well as increases the chance of microbial resistance in the patients. Also, prolonged treatment may cause long term harm. Data suggest that 80% of the patients with CF die indirectly or directly from pulmonary disease (Hurley, et.al. 2012). Clinically, there has been very little progress in the development of new antibiotics with novel mode of action. Recently, researchers exploited the adjuvant therapeutic agents that may be used alongside the conventional antibiotics. Source control measures, de-escalation of antibacterial should be followed up in patients with clinical response, especially with known antibacterial susceptibilities (Hurley, et.al. 2012). Considering the virulence of this pathogen, the progress of various strategies such as efflux pumps and lectins, the use of iron chelators, immunization, immunotherapy, and inhibition of quorum sensing are currently being tested clinically for the better management of the disease. It has also been suggested that the impacts of intervening virulence of P. aeruginosa should also be assessed (Hurley, et.al. 2012). Some novel therapies are also based upon the fact that CF is caused by the mutation of the gene encoding CFTR protein; targeting the defects produced in CFTR with invacaftor in one such approach. This novel treatment seems promising in patients with specific genotype of CF. Although the long term-outcome remains unknown, the development of CFTR targeted drug is an important milestone in CF (Petit, et.al. 2012). More recently, gene therapy has been extensively studied for the complete cure of the disease. However, the technique is not easy and has several limitations and hurdles. For instance, the research on this approach is very time consuming and often very expensive. However, focus of research has been on identifying the optimal vector for gene therapy (Picket, et.al. 2013). Thus, the management of CF has been a major challenge ever since the discovery of the disease. However, advancement in medical technologies and treatments has improved the prognosis of the disease. More sophisticated treatment including gene therapy (replacing faulty CFTR gene with a normal gene) has been the focus of intense research. P. aeruginosa resistance in CF Pseudomonas aeruginosa is one of the common infections in western society, because of its high level of resistance to antibiotics. The synergistic relationship between the outer membrane permeability and the efflux pumps is the most important factor influencing intrinsic antibiotic resistance in Gram-negative bacteria like P. aeruginosa (Liu, et. al. 1996). The high level of P. aeruginosa resistance is due to the acquired genes coding for amino glycoside-modifying enzymes or periplasmic beta-lactamases or mutations in fluoroquinolone targets (Liu, et.al. 1996). Low outer membrane permeability, which is caused by either less production of the OprD porin, or by expressing multidrug resistance efflux pumps contributes to high level of intrinsic antibiotics resistance (Liu, et.al. 1996). Numerous efflux pumps such as MexAB-OprM and MerXY-oprMb MexAB-OprM, MeXY-OprM, MexCD-OprJ, MexEF-OprN, MexJK, MexGHI-OpmD, MexVM, MexPQ-OpmE, MecMN and TriABC are encoded in the genome of Pseudomonas aeruginosa. These are of clinical importance because of their chemical ability to remove chemical-unrelated antibiotics. Among all, resistance-nodulation division (RND) super family contains the main efflux pumps of P. aeruginosa. This efflux pump has been discussed in greater details below. The energy source is derived from its proton motive force. Resistance Nodulation Division Efflux Pump Structure There are five families of multidrug resistance efflux pumps which include the ATP-binding cassette (ABC) super family, the major facilitator super family (MFS), the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family (Paddock, 2006). Efflux of antibiotics from the periplasm is a critical mechanism of antibiotic resistance utilized by Gram-negative cells. P. aeruginosa is capable of actively effluxing antibiotics from the periplasmic space using efflux pumps primarily from the RND family. The RND complex is tripartite in nature, composed of an outer membrane channel protein (OMP), inner membrane RND transporter, and a membrane fusion protein (MFP) (Kumar and Schweizer, 2005). The RND transporter provides the energy for the molecular transport and is often referred to as the RND pump protein (Misra and Bavro, 2009). The OMP interacts with the RND protein in the periplasm producing a chann el capable of transporting antibiotics into the extracellular space. The MFP is believed to stabilize the interactions between the RND transporter and the OMP (Misra and Bavro, 2009). All three of these components are essential for efflux function. The RND transporters are comprised of a homotrimer folded into a ?-helical transmembrane domain, with a large, soluble periplasmic domain. The top of the bacteria have semi-permeable membranes which may limit the passage of some types of antibiotics into the cell (Avrain, et.al. 2013). Multidrug resistance efflux pumps allow the bacterium to pump antimicrobials out of the cell. Porins maintain osmotic pressure by allowing the entrance/exit of hydrophilic small molecules but do not permit the passage of larger molecules. Enzymes can modify the antibiotic so that it is no longer recognized by the target, or modify them so that they are no longer functional. This is particularly prevalent with antibiotics that alter the ribosome or interfere with ribosome binding to inhibit protein synthesis. Inhibition of mRNA synthesis occurs by binding to DNA dependent RNA polymerase inhibiting initiation (Avrain, et.al. 2013). The mechanism of antibiotic resistance is shown in figure 3. Fig3: Different mechanisms of antimicrobial resistance in Gram-negative bacteria (adapted from Fluit, et.al. 2001). In the figure, A represents mRNA and B represents tRNA. When exposed to antibiotics, MexAB-OprM and MexXY-OprM remain inducible but the rest of the systems when expressed in resistant strains may immensely contribute to biocide or antibiotics resistance. Antimicrobial components are released by the RND systems and these include first line anti-pseudomonal drugs such as beta-lactams and beta-lactamase inhibitors (Poole, 2011). Furthermore, alteration or loss of the outer membrane porin protein OprD is yet another common mechanism of resistant to carbapenems(Wang, et. al. 2010). Since P. aeruginosa infections are usually treated by the use of fluoroquinolone (Ciprofloxacin), its high level resistance is as a result of mutations in the DNA gyrase and topoisomerase IV enzymes, which target these antibiotics. Outbreak of P. aeruginosa An outbreak can be defined as two or more sterile site isolate of the same species, with the same antibiogram, from different babies within the space of two weeks. A Pseudomonas aeruginosa infection outbreak on neonatal intensive care units reflects a degree of breakdown in infection prevention measures. Poor hand hygiene, low nurse: patient ratio, inadequate spacing between cots, overcrowding, environmental colonization (especially of water systems), inadequate cleaning of common use equipment, injudicious use of antibiotics, particularly broad spectrum and prolonged courses and delaying the introduction of material breast milk, all contribute to the emergence of outbreaks. It has been reported that infection from P. aeruginosa usually occurs 48hrs after birth, which affects two to three per thousand babies in the UK (Walker, et.al. 2013). The occurrence of the infection is even higher in those born prematurely or with surgical conditions (Durojaiye, et.al. 2011). Microbiological analysis In previous studies, (Walker, et.al. 2013) carried out the following microbiological investigations. Dismantling of Taps Taps and flow strengtheners from relevant hospital were dismantled, categorized into components, swabs and water samples were also taken. Samples for microbiological assessment were placed in 10 ml of maximum recovery diluents together with 10 sterile glass beads (3 mm diameter, VWR International, Lutterworth, UK). The surface of each component was scraped with a sterile plastic loop (Sterilin, Newport, UK) and the suspension was then vortex-mixed (10 s) to remove biofilm and to suspend the micro-organisms. Each sample was then spirally plated (Don Whitley, Shipley, UK) on to plate count agar (Oxoid, Basingstoke, UK) and Pseudomonas selective agar (PCN, Oxoid, UK), for determination of aerobic colony count (ACC) and P. aeruginosa count respectively. This was then incubated at 30Â °C on (Plate Count Agar) or at 37Â °C (PCN) for 48 h. It was found out that complex flow straightener had significantly higher P.aeruginosa counts (P