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Pseudomonas aeruginosa is a gram-negative rod bacterium (Freeman 2005) characterized by environmental and genomic versatility and an inborn resistance to antibiotics (Stover, 2000). It can be found as a biofilm (a thin slime layer of bacteria) in wet surfaces such as soil, water, plant and animal tissue, and most man made environments (Stover, 2000). It can survive under conditions that few other organisms can tolerate as it requires little oxygen (despite being aerobic) and by producing this biofilm layer, which resists phagocytosis (Qarah, 2009). Because of characteristics such as these it can multiply in an extraordinary assortment of sterilized environments, ranging from distilled water to medical equipment (Qarah, 2009). Although it rarely causes infections in healthy individuals, it is a major cause of hospital acquired infections (Qarah, 2009). P. aeruginosa can lead to hospital-acquired pneumonia in patients on respirators, urinary tract infections, blood stream infections, and pharyngitis (Qarah, 2009). It is also a significant source of bacterium in burn victims and in catheterized patients (Stover, 2000). P. aeruginosa frequently colonizes the lungs of patients with cystic fibrosis as the viscous mucus in their airways contains many nutrients for the bacteria to thrive on (Moskwa, 2007). It is the predominant cause of morbidity and mortality in cystic fibrosis patients (Stover, 2000), contributing to the chronic progression of the disease (Qarah, 2009). P. aeruginosa is an opportunistic human pathogen which affects immunocompromised individuals, such as those infected with HIV (Qarah, 2009).
Like the HIV virus, P. aeruginosa contains a single circular chromosome, although with 6.3 million base pairs (Stover, 2000). Unlike viruses, bacteria such as P. aeruginosa transfer genes from one cell to another via plasmids (double-stranded DNA molecules separate from chromosomal DNA) (Freeman, 2005). This process, known as conjugation, is only possible if a cell has an F (fertility) plasmid (Freeman, 2005). Tubules on the surface of an F+ cell will make contact with an F- cell and then retract, pulling the two cells into contact (Freeman, 2005). When the cell walls touch, a conjugation tube will form between them and a single strand of the plasmid will pass through the tube into the F- cell (Freeman, 2005). If the plasmid contains genomic DNA, it may be integrated into the recipients main chromosome and so a bacterial cell with a new sequence of genes results (Freeman, 2005). This process is how antibiotic-resistant alleles are able to spread rapidly in a population of bacteria.
Such alleles are present in P. aeruginosa, which may have been acquired from evolving in diverse environments and by competing with other microorganisms (Stover, 2000). Stover et al., the team that first sequenced the bacterium's genome, elaborate: "the metabolic diversity, transport capabilities and regulatory adaptability that enable P. aeruginosa to thrive and compete with other microorganisms probably all contribute to its high intrinsic resistance to antibiotics" (pg. 963, Stover, 2000). In addition to it's ability to metabolize a wide variety of organic molecules, P. aeruginosa's genetic complexity also contributes to it's resilience (Stover, 2000). It contains the highest proportion of regulatory genes observed for a bacterial genome, including a large number of genes involved in catabolism and protein transport (Stover, 2000). It's genome also has high gene and functional diversity; when compared to other large bacterial genomes (such as E. coli, B. subtilis and M. tuberculosis) it was found that P. aeruginosa has significantly more distinct gene families (Stover, 2000). Despite this genetic diversity, P. aeruginosa is similar to many other bacteria in that it can alter it's gene expression to respond to environmental changes (Freeman, 2005). Stover et al. explain: "[P. aeruginosa's] 468 genes contain motifs characteristic of transcriptional regulators or environmental sensors. This analysis predicts that 8.4% of P. aeruginosa genes are involved in regulation, a far higher proportion than is found in other sequenced genomes" (pg. 961) and "these regulatory genes presumably modulate the diverse genetic and biochemical capabilities of this bacterium in changing environmental conditions" (pg. 963, Stover, 2000). These regulatory genes make P. aeruginosa an adaptable organism, capable of multiplying in a wide variety of environments and providing resistance to antibiotics and disinfectants.
P. aeruginosa also contains a disproportionately large number of genes that code for outer membrane proteins (around 150), when compared to other bacterial genomes (Stover, 2000). This could be attributed to proteins that are needed to anchor the structures that control the bacterium's adhesion and motility (Stover, 2000). Outer membrane proteins are also likely involved in the export of extracellular virulence factors (Stover, 2000). Virulence factors are proteins expressed and secreted by pathogens that allow for adhesion to cells, evasion of the host's immune response, and acquisition of nutrients from the host (Freeman, 2005). In short, they are what gives the pathogen the ability to cause a disease. P. aeruginosa secretes several virulence factors, including toxins, lipases, proteases, and exoenzymes (Stover, 2000). It's multiple virulence factors and regulatory pathways also contribute to it's ability to adapt and thrive in a multitude of environments (Murray, 2007). To further it's environmental versatility, P. aeruginosa has nearly 300 cytoplasmic membrane transport systems, about two-thirds of which are involved in the import of nutrients and other molecules, such as these virulence factors (Stover, 2000).
Virulence factor production is mediated by quorum sensing, a complicated interconnected system of cell-cell communication that also controls for production of biofilm (Murray, 2007). In vitro, bacteria in biofilms grow more slowly, but are more resistant to antibiotics and less susceptible to clearance by the host immune system (Murray, 2007). As Murray et al. explain, "P. aeruginosa possesses multiple quorum-sensing systems that produce and sense N-acylhomoserine-lactones, small molecules that diffuse across bacterial membranes and accumulate in the environment. As bacterial numbers increase, these signaling molecules reach high levels and turn on sets of genes, including those required for biofilm growth" (pg. 84, Murray, 2007). As a biofilm, the bacteria can attach to membranes and begin colonization and production of it's virulence factors (Qarah, 2009). One virulence factor produced by bacteria in biofilm are rhamnolipids, amphipathic molecules that facilitate dispersal from the biofilm, allowing P. aeruginosa to spread from the area of local infection into the bloodstream (Kownatzki, 1987). Rhamnolipids also disrupt respiratory airway epithelial cells (Murray, 2007), and in individuals with cystic fibrosis this can prove to be fatal, as their respiratory tracts are already weakened. This is why chronic infections with P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis patients. (Murray, 2007).
As previously mentioned, another important characteristic of a cystic fibrosis lung is the presence of a thick mucus. Landry et al. showed that biofilms grown in vitro on a mucin coated surface had increased mass and altered architecture (Landry, 2006). These mucin-associated biofilms were more resistant to antibiotics, possibly due to changes in the biofilm's structure that inhibit any antibiotic penetration (Murray, 2007). Clearly P. aeruginosa's virulence is affected by factors present in the environment (Murray, 2007). Goodman et al. have described regulatory pathways that respond to environmental cues to turn on sets of genes associated with biofilm formation and the "type III secretion system" (TTSS) (Goodman, 2004). The TTSS is a "needle-like structure" that injects toxic molecules directly into epithelial cells, leading to acute pneumonia (Murray, 2007). Interestingly, this system is repressed during bacteria colonization and biofilm formation (Murray, 2007), yet further research is needed to determine the extent of correlation and how it affects the progression of pneumonia. In addition to biofilm formation, P. aeruginosa can adopt other strategies to promote survival in vivo and prevent detection by the host's immune system. It can shut down or repress the production of proteins that might elicit an immune response (Murray, 2007). For example, P. aeruginosa possess a single flagellum that is required for swimming motility and initial attachment during biofilm formation (Murray, 2007). Flagellin (the protein that forms flagellum) is highly immunogenic, as it will stimulate receptors in the host's immune system (Murray, 2007). When flagellin is detected in epithelial cells the immune response is to induce the transcription of genes encoding for anti-inflammatory agents (Murray, 2007). P. aeruginosa will then quickly downregulate transcription and expression of flagellin, effectively escaping detection by the immune system (Murray, 2007).
Although P. aeruginosa employs multiple mechanisms to evade immune and inflammatory responses, technological advances (such as rapid high-throughput DNA sequencing) have given scientists an increased understanding of its pathogenesis in vivo, leading to the identification of new targets for therapies (Murray, 2007). One such target is the quorum-sensing system, as many environmental organisms produce quorum-sensing inhibitors, which are being studied as possible antimicrobial mediators (Murray, 2007). Another option is directly target biofilm formation, a fundamental component of P. aeruginosa's virulence. Antibiotics such as azithromycin appear to delay initiation of biofilm formation (Gillis, 2004) and decrease quorum-sensing activity (Nalca, 2006). Studies have also found that azithromycin inhibits protein synthesis in P. aeruginosa and decreases the expression of genes required for biofilm formation (Wagner, 2005). However, azithromycin may only exert an anti-inflammatory effect, and not specifically target the bacteria, as one study found that patients improved regardless of whether they were infected with P. aeruginosa (Clement, 2006).
Unfortunately, genes that are associated with acute infections, such as those that code for the TTSS and thus cause pneumonia, are highly upregulated in response to azithromycin (Murray, 2007). But as Murray et al. point out, the conditions used in these in vitro experiments may not accurately reflect the in vivo environment (Murray, 2007). Nevertheless, there are some antibiotics, specifically aminoglycosides, that actually promote biofilm formation by expressing a protein named aminoglycoside response regulator (ARR) (Murray, 2007). ARR is involved in the metabolism of dicyclic GMP (di-cGMP), a small messenger molecule (Murray, 2007). In P. aeruginosa, several genes that encode for proteins involved in di-cGMP metabolism are also required for biofilm formation, virulence factor expression, and antibiotic resistance (Murray, 2007). It's hypothesized that one aspect of P. aeruginosa's antibiotic resistance is a result of altered biofilm formation (Murray, 2007) as the antibiotics themselves cause physiological changes (similar to mucin-associated biofilms), possibly contributing to an increased drug resistance (Gillis, 2005).
P. aeruginosa are also resistant to multiple classes of antibiotics because they produce the ß-lactamase protein (Murray, 2007). ß-lactam are a broad class of antibiotics, including penicillin and it's derivitives, that inhibit the synthesis of the peptidoglycan layer of bacterial cell walls (Freeman, 2005). They bind to the enzymes responsible for creating the cell wall and so prevent the formation of cross-linkages (Freeman, 2005). The absence of these linkages weakens the cell wall, causing it to rupture, which results in cell death (Freeman, 2005). ß-lactamase is an enzyme that hydrolyzes the ß-lactam ring, cleaving it and rendering the antibiotic useless as it can no longer bind to the cross-linking enzymes (Freeman, 2005). Lateral transfer (the movement of DNA from one species to another) of plasmids that carry the ß-lactamase gene is common (Freeman, 2005), so this mode of resistance is most likely present in many P. aeruginosa strains. Yet another mechanism of antibiotic resistance are drug efflux pumps. Drug efflux pumps are composed of three transmembrane transporter proteins that displace drugs using proton electrochemical forces (Fernandez-Recio, 2004). As Stover et al. have reported, "The P. aeruginosa genome appears to contain a large number of undescribed drug efflux systems" (pg. 962, Stover, 2000).
In order to test for antibiotic resistance mechanisms such as these scientists perform transduction experiments in conjunction with a test for ß-lactamase activity. Transduction is similar to conjugation, except the plasmid is transferred from one bacterium to another by a virus (Freeman, 2005). It is used by molecular biologists to introduce foreign genes, and in this case, to ensure transfer of the resistant genes to recipient strains (Seginková, 1986). First, cultures of a recipient antibiotic resistant strain of P. aeruginosa are grown, centrifuged, and incubated (Seginková, 1986). The mixture is allowed to sit for 90 minutes to allow time for phenotypic expression, and then it is plated onto an agar plate containing the antibiotic of choice (Seginková, 1986). Control cells, plated only on agar, are also prepared. Three drops of a ß-lactamase reagent, containing potassium, phenol, sodium hydroxide and distilled water, are added to the plates, which are immediately mixed (Escamilla, 1976). The ß-lactamase reagent will react with ß-lactamase, if it is present, and change color (Escamilla, 1976). If the solution changes from purple to yellow, the test is positive and ß-lactamase is being expressed in that bacteria (Escamilla, 1976). Thus, that strain of P. aeruginosa is resistant to that particular antibiotic.
Although generally effective, this test contains two potential sources of error. If the transduction somehow fails and not enough bacteria are transformed by the virus then insufficient amounts of ß-lactamase may be produced and the reagent would not react and change color. This would be a problem if multiple strains are present in the bacterial population which are resistant to different antibiotics. In this scenario P. aeruginosa that is resistant to the antibiotic being tested for would not be correctly identified, thus lowering the sensitivity of the test. The second potential source of error stems from the different mechanisms that P. aeruginosa employs to resist antibiotic treatment. As previously mentioned, in addition to producing ß-lactamase, the bacterium may also adapt altered biofilm formation, and it contains drug-efflux pumps on it's plasma membrane. As the creator of this test (Escamilla) realized, "if forms of resistance other than ß-lactamase production exist... such an occurence will necessarily limit the significance of ß-lactamase tests as presumptive evidence
of ampicillin resistance or susceptibility" (pg. 197, Escamilla, 1976). In other words, this test will only correctly identify antibiotic-resistant bacteria if it produces ß-lactamase. If a bacteria utilizes other methods of resistance, this test will incorrectly identify them as negative for antibiotic-resistance. This further lowers the sensitivity of the test.
The effectiveness of these tests, however, seems insignificant when one considers why such tests are needed in the first place. Resistance to antibiotics has become a detrimental problem for pharmaceutical companies, physicians, and patients (Freeman, 2005). Although the spread of alleles that confer resistance is favored by natural selection (Freeman, 2005), hospitals are to blame as well. By over-prescribing antibiotics, or prescribing them in the mildest of cases, physicians have significantly contributed to the evolution and rapid spread of resistant strains. The intrinsic resistance that is found in P. aeruginosa is evidence of this. The problems that stem from this resistance are further exacerbated given the virulence of the bacteria. P. aeruginosa can thrive in diverse environments, it can move readily to more favourable conditions as it has "broad capabilities to transport, metabolize and grow on organic substances" (pg. 963, Stover, 2000), and it favors compromised host defense mechanisms. Although P. aeruginosa infections are particularly difficult to treat because of it's resistance to antibiotics, the more we learn about the bacteria the better we will become at approaching and treating the diseases it causes. Stover et al. said it best: "knowledge of the complete genome sequence and encoded processes provides a wealth of information for the discovery and exploitation of new antibiotic targets, and hope for the development of more effective strategies to treat the life-threatening opportunistic infections caused by P. aeruginosa in humans" (pg. 963, Stover, 2000). This is why it is crucial to gain a better understanding of the pathogenesis of P. aeruginosa, so that we may develop novel approaches to treat it's infections, and one day beat its resistance to drugs.