Pseudomonas aeruginosa

Classification
The genus Pseudomonas holds about sixty different types of species in the kingdom classified as bacteria. The species Pseudomonas aeruginosa is classified as a Gram-negative bacterium|. Most Pseudomonas aeruginosa are categorized as obligate aerobes, however sometimes in certain environmental conditions, the bacteria acts as a facultative anaerobe. Furthermore, because of the way it obtains its energy, it is considered to be a chemoheterotroph. (Willey)

Description and significance
The Pseudomonas genus includes bacteria that are straight or slightly curved rods. P. aeruginosa is a rod-shaped bacterium. Its size ranges from 0.5 to 1.0mm by 1.5 to 5.0mm in terms of its length and width. Almost all types of strains are motile by means of a polar flagellum (Todar). P. aeruginosa was first isolated from green pus in 1882 by C. Gessard. He described the bacterium as a pathogen when he linked it to wound infections, where he would see blue/green colors underneath bandages of wounded soldiers [8]. P. aeruginosa is a well-studied species due to its high level pathogenicity and its significant role in human disease. The organism can affect humans, animals as well as plants, and can thrive under many environmental conditions, such as in soil, water and even hospital environments. P. aeruginosa is primarily a nosocomial pathogen. According to the CDC (Center for Disease Control), the overall incidence of P. aeruginosa infections in U.S. hospitals averages about 0.4 percent (4 per 1000 discharges), and the bacterium is the fourth most commonly-isolated nosocomial pathogen accounting for 10.1 percent of all hospital-acquired infections (Todar).

Gene mapping studies of Pseudomonas aeruginosa have helped researchers and clinicians better understand local gene expression and the evolution of Pseudomonas aeruginosa as it has adapted to the CF lung. Because of its ability to resist many antibiotics and due to its high level of adaptability to most environments, p. aeruginosa genome sequencing proved to be crucial.

Genome structure
Pseudomonas aeruginosa has the largest genome of the 25 bacteria that scientists have sequenced so far [3]. The genome of P. aeruginosa has an unusually large number of genes for nutrient transport, metabolic regulation and catabolism; this may be why the bacterium has the ability to grow in a wide range of environments and resist antibiotics (Willey). This microbe’s genome is a single circular chromosome that is made up of 6,264,403 base pairs (Bp), which is 6.3 million bases (Mb) and contains 5,570 predicted genes on one chromosome. Also, P. aeruginosa has metabolic plasmids that are about 75-230 kbp in size and are involved in degrading substances such as sugars (Stover, et al). These plasmids may have genes which code for factors that make the bacteria drug resistant (Todar).

The large genome sequence of P. aeruginosa shows that their low level of outer membrane permeability causes in them an all-rounded, “intrinsic” drug resistance to many antibiotics and also a efflux system of organic compounds. A multi-drug efflux system has been identified in this for this Gram-negative bacterium and is probably the strongest in any of the other Gram-negative bacteria that have been sequenced (Stover, et al).Due to its large genome size, P. aeruginosa has tremendous genetic density, allowing it to form biofilms. It also utilizes quorum sensing (group symbiosis) to achieve its resistance against microbial agents in most cases.

Cell structure and metabolism
P. aeruginosa has a cell wall that is Gram-negative as it is composed of three layers; the plasma membrane, a thin peptidoglycan layer, and an outer membrane. Gram-negative bacterium generally have seven different pathways of protein secretions, and three of them are seen in P. aeruginosa. These protein secretion pathways include ABC transport proteins that are embedded in lipid membranes, and are involved in the translocation of many different substrates across the membrane (Stover, et al.).

P. aeruginosa secretes virulence factors which include potent toxins and degradative enzymes. The toxins cause extensive tissue damage and also interfere with the human immune systems defense mechanisms. They also enter and kill host cells at or near the site of colonization. Moreover, the bacterium's degradative enzymes permanently disrupt the cell membranes and connective tissues in various organs; these enzymes include lipases and proteases [3].

Since P. aeruginosa is a chemoheterotroph, it can grow on the least amount of media available, and will use just about anything as a carbon source to build up a biofilm in various types of environments (Willey, et al 2008). Its metabolism is respiratory, as it almost always functions as an obligate aerobe, but it is able to grow in the absence of O2 if NO3 is available as a terminal electron acceptor (Todar).

Ecology
P. aeruginosa can thrive in a wide range of environmental conditions and the bacteria have developed adaptability to minimal nutritional requirements (Todar). They are found almost everywhere, including the most unlikely places such as distilled water and many sterile environments such as in hospitals. P. aeruginosa has been isolated from very sick individuals who have decreased immune responses, such has patients suffering from AIDS, cancer (undergoing chemotherapy) and especially those suffering from cystic fibrosis.

Because P. aeruginosa is an opportunistic pathogen, it has a permanent detramental effect on sick individuals as the bacterium almost always kills its host cells.

Pathology
P. aeruginosa can infect animals, plants and also humans. They cause disease in humans who are already ill, infecting those patients with low resistance in their bodies. P. aeruginosa are opportunistic pathogens and cause infection in patients who are diagnosed with cystic fibrosis, have lower respiratory tract infections, surgical wounds, urinary tract infections, skin infections i.e:(dermatitis), and even in patients who have cancer and are undergoing chemotherapy (Willey). It causes nosocomial infection in patients as this bacterium can grow anywhere where enough nutrients and enough moisture are found.

An opportunistic pathogen, P. aeruginosa produces a thick biofilm and due to its dense colonization, it is able to resist many antibiotics, disinfectants, as well as UV light and infected patients can therefore be difficult very to treat. Another factor that contributes to P. aeruginosa resistance is its Gram-negative cell wall that is composed of three layers; the inner plasma membrane, peptidoglycan, and its outer membrane. This high level of resistance in P. aeruginosa can be of consequence and dangerous to a patient’s health. Moreover, Pseudomonas maintains antibiotic resistance plasmids, R-factors and RTFs, and it is able to transfer these genes by horizontal gene transfer (HGT), mainly transduction and conjugation (Todar).

P. aeruginosa bacterium is naturally resistant to many antibiotics due to the permeabiliity barrier afforded by its Gram-negative outer membrane. Also, its tendency to colonize surfaces in a biofilm form makes the cells even more resistant to antibiotics. Biofilms form organized and specialized bacterial communities which mediate bacterial attachment to surfaces and provide protection. Biofilms are made of microcolonies that are buried in a dense matrix of exopolysaccharides. How exactly does the bacterial colony know to initiate biofilm formation is yet to be understood [5].

One of the major factors that makes Pseudomonas aeruginosa infections difficult to treat is their overproduction of a sugar-like substance, called alginate, an exopolysaccharide. The AlgR protein, which is found to be one factor that regulates alginate production, has recently shown to be involved with P. aeruginosa's pili function (pili mediate attachment in bacteria). Pili are involved in the initial stages of Pseudomonas aeruginosa infection of CF lungs. Thus, it is thought that the AlgR protein, might be regulating not only genes controlling alginate production, but other P. aeruginosa virulence genes involved in the infection process [3].

Furthermore, two extracellular proteases have been associated with P. aeruginosa virulence; elastase and alkaline protease, which cleave collagen and interfere for fibrin formation and lyse fibrin in host cells, respectivily. The bacteria produces three other soluble proteins which aid in the invasion/infection process; a cytotoxin which is a "pore-forming" protein, and two hemolysins called phospolipase and lecithinase which work synergisticlaly to break down lipids and lecithin. P. aeruginosa produces two extracellular protein toxins called Exotoxin A and Exoenzyme S and it has been suggested that Exoenzyme S protects the bacterium against phagocytic cells, whereas Exotoxin A effects protein synthesis in the host cell and is thought to contribute to the colonization process (Todar). It is obvious that because P. aeruginosa secretes quite a lot of extracellular proteins, the genes that regulate production of these proteins and various protein-secretion mechanisms may be an important evolutionary adaptation that has led this bacterial species to be so pathogenic and virulent.

As far as symptoms are concerned, cystic fibrosis patients that are infected with P. aeruginosa may not show any at first. However, as the infection grows and becomes severe, patients may notice symptoms of infection and inflammation as well as decreased tolerance for exercise and shortness of breath. The inflammation usually leads to blockage of breathing passages and can permanently damage airways in the lungs [6]. Some more symptoms include salty-tasting skin, persistent coughing with phlegm, poor growth or even weight gain and also difficulty in bowel movements [7].

Application to Biotechnology
P. aeruginosa produces certain degradative enzymes such as lipases, proteases and many other toxins. These Lipases and proteases can be used to degrade the lipid membranes of cells (most cellular membranes are composed of lipids and integral proteins).

Does this organism produce any useful compounds or enzymes? What are they and how are they used?

Current Research
A research conducted in Copenhagen, Denmark, at the University of Copenhagen's Department of Microbiology provides insight on how the antibiotic Azithromycin (AZM) has been shown to improve the lung function of CF patients. P. aeruginosa that produce O-acetylated alginate in the lungs of Cystic Fibrosis patients are tolerant of many antibiotics however, this study suggests that AZM inhibits production of alginate production, blockage of quorum sensing and increased sensitivity of hydrogen peroxide and the complement system. AZM also may affect the formation of functional alginate by causing incomplete polymerization of the substance. However, the study also showed that only bacteria in the stationary phase became sensitive to AZM and those in the growth phase did not. What is of great interest is that mutant P. aeruginosa that were treated with AZM developed resistance and those with a functional quorum sensing system did not. In the experimental group made up of mice with cystic fibrosis and chronic lung infection, AZM suppresses QS-virulence factors and significantly clears up alginate biofilms. Thus, AZM attenuates P. aeruginosa virulence by impairing its ability to form complete and effective biofilms [4].

Another research, carried out at the University of California by the Department of Anesthesia and Perioperative, proposes that the loss of bacterial diversity during antibiotic treatment of patients infected with P. seruginosa causes it to become the dominant species in the bacterial community. The research analyzed bacterial diversity in the endotracheal aspirates taken from intubated patients infected with P. aeruginosa. They did this by using 16S rRNA clone libraries and microarrays (PhyloChip)in order to determine any changes, during antibiotic treatment, in bacterial community compositions. It was found that the 16s rRNA genes were absent were absent from patients who were itubated briefly but was present in those who were intubated for a longer period. Also, for each patient, bacterial diversity decreased after administration of antibiotics and P. aeruginosa dominated. The collected data demonstrates that the development of pneumonia in patients infected with P. aeruginosa, is highly correlated to loss of bacterial diversity due to antibiotics. The research basically suggests that various bacterial groups may compete for a similar ecologocal niche and that loss of microbial diversity may contribute directly to pathogen selection and persistence [Flanagan].

Lastly, a research project conducted in Marseille, France, attempts to describe the series of events that take place during development of biofilms. Using the genetic screening method, many biofilm-defective mutants were identified and have been characterized further. This study proposed a global model where key events are described for the different stages of biofilm formation. The flagellum is used to approach a surface and type IV pili allows for attachment to surfaces and colonization (microcolony formation), and finally, these colonies are buried in an extracellular matrix to form a differentiated biofilm. The research points out that these different stages of biofilm formation also call for perception of stimuli. These stimuli and the bacterium's regulatory networks are yet to be fully characterized in order to understand the bacterial strategy that initiates formation of biofilms. Quorum sensing is one such regulatory system that plays a major role in biofilm production. The research concluded that a better understanding of biofilm establishment at the molecular level should help in designing antimicrobials that would protect us from bacterial infections [5].