P. aeruginosa, like many bacteria, behaves differently when grown in different environments, and many different models, both in vitro and in vivo, have been developed in an attempt to model the growth and evolution of P. aeruginosa in the CF lungs.
1.3.1 Synthetic sputum models
CF sputum can be used as a growth medium by dissolving sterile, lysophilised sputum in buffer, creating a medium that supports high densities of P. aeruginosa (Palmer et al., 2005) because of the high concentration of amino acids that bacteria use as a growth source (Barth & Pitt, 1996; Palmer et al., 2005). However, CF sputum is in limited supply and the chemical composition is unlikely to be consistent between different batches. Because of this, several synthetic sputum models have been developed for the study of P. aeruginosa and other CF pathogens.
Artificial sputum media (ASM) was one of the first synthetic sputum models to be developed (Sriramulu et al., 2005). ASM contains a high concentration of free DNA, mucin, amino acids, salts and lipids, resulting in a medium that has a similar
viscosity and chemical composition to CF sputum (Sriramulu et al., 2005). When grown in ASM, P. aeruginosa forms tight microcolonies that are not surface attached (Haley et al., 2012; Kirchner et al., 2012; Sriramulu et al., 2005), thought to be how P. aeruginosa grows in the CF lung (1.2.7). Bacteria grown in ASM undergo
phenotypic diversification (Sriramulu et al., 2005; Wright et al., 2013), exhibiting changes in colony morphology (Sriramulu et al., 2005), pyocyanin production and antibiotic susceptibility (Wright et al., 2013). Furthermore, gene expression is altered
23 in ASM, with an upregulation of genes involved in biofilm formation, QS and
phenazine biosynthesis (Fothergill et al., 2014).
ASM can be filtered to remove contaminating bacteria without damaging the heat- sensitive components, but a modified version (ASMDM) contains less DNA and more mucin, considered by the authors to better represent the CF lung (Fung et al., 2010). The additional mucin makes filtration difficult, so antibiotics are added to ASMDM to inhibit growth of contaminants, but this is potentially concerning as subinhibitory antibiotic concentrations can have a wide range of effects on bacterial cells, including altered gene regulation (Davies et al., 2006).
Other synthetic sputum models include synthetic CF sputum medium (SCFM), which contains amino acids but lacks DNA and mucin (Palmer et al., 2007), and a more recent version, SCFM2, which is supplemented with DNA, mucin, N-acetyl glucosamine and lipids to better represent CF sputum (Turner et al., 2015). The addition of these components probably makes SCFM2 more alike to ASM than its precursor.
Support for the use of synthetic sputum as a model for the CF lung comes from a recent transposon-sequencing study that determined the genes required for P. aeruginosa fitness in both CF and synthetic sputa (they used SCFM2). The essential genes were very similar for both (Turner et al., 2015), suggesting that synthetic sputum is a good in vitro model to study P. aeruginosa.
1.3.2 In vivo models
As P. aeruginosa is an opportunistic pathogen, infection of healthy animals usually leads to rapid clearance (Southern Jr et al., 1970), unless the dose is sufficiently high or the animal is immunocompromised, in which case an acute infection is established and the animal quickly dies (Morissette et al., 1995). However, this is not
representative of chronic P. aeruginosa infections in CF, which are characterised by a slow decline in health, often over many years (Kosorok et al., 2001). Although natural animal models of CF do not exist, attempts have been made to engineer CFTR deficient animals in order to replicate the CF disease phenotype.
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Numerous murine CF models have been used that differ in CFTR functionality and range from a defective to completely absent CFTR, but the pathophysiology of lung disease is not reflective of that in humans and they are not intrinsically susceptible to respiratory bacterial infections (Fisher et al., 2011). The more recently developed ferret and pig models are more promising. CFTR defective pigs develop CF-like lung disease as adults (Ostedgaard et al., 2011; Stoltz et al., 2010), following colonisation with multiple bacterial species (Stoltz et al., 2010). Furthermore, CF pigs were unable to successfully eradicate bacteria when artificially infected, mimicking the defective clearance observed in CF humans (Stoltz et al., 2010). CFTR knock-out ferrets are susceptible to bacterial respiratory infections soon after birth and also demonstrate many other symptoms seen in CF humans (Sun et al., 2010).
CFTR models are expensive and not readily available, so many animal models used in the study of P. aeruginosa infections in CF use phenotypically healthy animals, but the mode of infection is designed to make the resulting infection as close to chronic respiratory infection as possible. It was discovered that by embedding P. aeruginosa in agar beads and introducing the beads into the rat lung by intubation, a chronic lung infection could develop, resulting in a similar pathophysiology to chronic P. aeruginosa infections in individuals with CF (Cash et al., 1979). The rat agar bead model is still in use and has been used to study the growth and fitness of several P. aeruginosa strains (Kukavica-Ibrulj et al., 2008b).
A major issue with these models is the artificial nature of infection. A pulmonary mouse model was developed that used an alginate-overproducing (mucoid) CF isolate, and the self-produced alginate protected the bacteria from the immune system, negating the need for artificial embedding (Hoffmann et al., 2005).
However, such a model is obviously limited in the bacterial strains that can be used. Furthermore, implantation into the lungs by intubation bypasses the host’s normal defences, and analysis of P. aeruginosa in the paranasal sinuses and lungs of CF children suggests that P. aeruginosa colonises and “pre-adapts” to the upper respiratory tract before seeding down into the lungs and establishing chronic infection (Hansen et al., 2012). A recently developed murine model utilised a more natural infection route, as free bacteria were administered intranasally. Bacteria colonised the nasopharynx and although no bacteria were detected in the lungs 2
25 weeks post-infection, after 4 weeks bacteria were present in the lungs, suggesting the nasopharynx acts as an infection reservoir, leading to adaptation and re-seeding of the lungs (Fothergill et al., 2014).
1.3.3 Other P. aeruginosa infection models
An ex vivo porcine lung infection model has recently been developed as an
alternative model that bridges the gap between in vivo and in vitro models of chronic P. aeruginosa infection. It involves bacterial inoculation of sections of fresh pig lung tissue suspended in ASM, and the lung tissue provides the spatial structure that is lacking in ASM (Harrison et al., 2014). The extent of tissue damage can then be evaluated to estimate the virulence of different strains. A major advantage of this model is that it is not subject to the same ethical constraints as in vivo models, as it uses pig carcasses from the food industry.
Other models exist to assess P. aeruginosa virulence, including Caenorhabditis elegans (nematode) (Tan et al., 1999), Drosophila melanogaster (fruit fly)
(Apidianakis & Rahme, 2009), Galleria mellonella (wax moth) larvae (Miyata et al., 2003), plants (Rahme et al., 1997) and Dictyostelium discoideum (amoeba) (Cosson et al., 2002), but these models result in acute infection, which is not representative of P. aeruginosa infections in CF and involves different fitness requirements (Turner et al., 2014).