Pseudomonas Aeruginosa Essay, Research Paper

Pseudomonas aeruginosa

Research Paper

Julie Johnson

Pseudomonas aeruginosa is a versatile gram negative bacterium that grows in soil, marshes, and coastal marine habitats, as well as on plant and animal tissues. People with cystic fibrosis, burn victims, individuals with cancer, and persons infected with HIV are particularly at risk of disease resulting from Pseudomonas aeruginosa.

Unlike many environmental bacteria, Pseudomonas aeruginosa has a remarkable capacity to cause disease in susceptible hosts. It has the ability to adapt to and thrive in many ecological niches, from water and soil to plant and animal tissues. The bacterium is capable of utilizing a wide range of organic compounds as food sources, thus giving it an exceptional ability to colonize ecological niches where nutrients are limited. Pseudomonas aeruginosa can produce a number of toxic proteins, which not only cause extensive tissue damage, but also interfere with the human immune systems defense mechanisms. These proteins range from potent toxins that enter and kill host cells at or near the site of colonization to degradative enzymes that permanently disrupt the cell membranes and connective tissues in various organs.

In people with cystic fibrosis the most serious complication is respiratory tract infection by the ubiquitous bacterium Pseudomonas aeruginosa. CF is one of the most common fatal genetic disorders in the United States, affecting about 30,000 individuals. A comparable number of people in Europe also have CF. It is most prevalent in the Caucasian population, occurring in one of every 3,300 live births. The gene involved in cystic fibrosis was identified in 1989. Located on human chromosome 7, it codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). This protein, normally produced in a number of tissues throughout the body, regulates the movement of salt and water in and out of these cells. The abnormality in the CFTR gene alters the CFTR protein in people with cystic fibrosis. As a result, one hallmark of CF is the presence of a thick mucus secretion which clogs the bronchial tubes in the lungs and plugs the exit passages from pancreas and intestines, leading to loss of function of these organs.

Progressive lung disease is the predominant cause of illness and death in people with CF. Mucus blocks the airway passages and results in a predisposition toward chronic bacterial infections. Although the genetic defect underlying CF has been characterized, exactly how and why individuals become infected with Pseudomonas is unknown. The lungs of most children with CF become colonized by Pseudomonas aeruginosa before their 10th birthday. Chronic infection with these bacteria reduces an individuals quality of life, causing acute symptoms of cough, sputum production, and inflammation, which causes repeated exacerbations or episodes of intense breathing problems. Eventually leading to scarring and destruction of lung tissue and, ultimately, death. While it is clear that antibiotic therapy directed against these organisms lengthens the life span of individuals with CF, increasing antibiotic resistance develops. Although antibiotics can decrease the frequency and duration of these attacks, the bacterium establishes a permanent residence and can never be completely eliminated from the lungs.

Management of cystic fibrosis lung disease requires a multipronged approach. Outpatient management of pulmonary exacerbation usually includes a combination of 2 IV antipseudomonal antibiotics (an aminoglycoside plus a beta-lactam), appropriate antimicrobial treatment, effective airway clearance, optimization of nutritional status, and anti-inflammatory therapies. Additionally, prevention of respiratory viral disease and avoidance of exposure to irritants, such as smoke, is recommended. Usual duration of therapy is 14 to 21 days, and clinical response is assessed by physical exam, pulmonary function tests, nutritional status, and exercise tolerance. Microbial eradication is not a therapeudic end point. Choice of antibiotics should be based on culture and sensitivity of the sputum. Emergence of antibiotic-resistant species, such as Pseudomonas aeruginosa, has required close monitoring of antibiotic susceptibility patterns and strict infection-control policies.

Administration of chronic intermittent inhaled antipseudomonal therapy (tobramycin solution for inhalation), over a 6 month period was shown to improve FEV by 11.9%, decrease the bacterial density, and reduce hospitalization in CF patients chronically infected with Pseudomonas aeruginosa. Following 92 weeks of therapy with inhaled tobramycin, the mean % change in FEV was 4.7% above baseline. There was no increase in the utilization of antipseudomonal therapy despite an increase in MIC at the end of 12 treatment cycles. Decreases in Pseudomonas aeruginosa tobramycin susceptibility were not predictive of a lack of clinical response, i.e. lung function, to inhaled tobramycin.

A potential role for aggressive antipseudomonal therapy that is currently under study involves the use of inhaled tobramycin in young patients at the time of initial colonization. Researchers are hopeful that early, aggressive intervention may be effective in eradication of Pseudomonas aeruginosa, and therefore, will have a dramatic impact on the natural history of cystic fibrosis.

One of the major factors that makes Pseudomonas aeruginosa difficult to eradicate is the overproduction of a sugar-like substance, alginate. One of the regulators of alginate production, the AlgR protein, has recently been shown to be involved with the function of pili (tiny hair-like appendages on the outside of the bacteria). Pili are involved in the initial stages of Pseudomonas aeruginosa infection of CF lungs. The AlgR protein, thus, may regulate not only genes controlling alginate production, but other Pseudomonas aeruginosa genes involved in the infection process. A current study is investigating this by isolating genes that are regulated by AlgR and characterizing those genes to determine whether they are used by Pseudomonas aeruginosa to evade the bodys immune response and cause disease. The effects of the isolated genes will be measured on the ability of the bacteria to bind to the cells lining the airway and to avoid ingestion and destruction of defending white blood cells. Results from these studies will give insight into the disease causing mechanisms in Pseudomonas aeruginosa and may lead to alternate methods of infection prevention in the CF patient.

Complications associated with Pseudomonas aeruginosa lung infections in CF patients are the result of a multitude of pathogenic mechanisms in the respiratory tract created by the underlying chloride channel defect. Gene mapping studies of Pseudomonas aeruginosa will help researchers and clinicians better understand local gene expression and the evolution of Pseudomonas aeruginosa as it has adapted to the CF lung. Researchers are using new genetic tools to study bacterial virulence mechanisms during infection with Pseudomonas aeruginosa. Two techniques employed to determine the extent of genomic variation among different Pseudomonas aeruginosa clinical isolates are macroevolutional and microevolutional analysis.

In macroevolutional analysis DNA arrays were used to identify genes in clinical strains of Pseudomonas aeruginosa that were absent in strain PA01 (the Pseudomonas Genome Project strain sequence). Using labeled genomic DNA probes, strains were assessed for reaction with PA01 probes, which represented sequences unique for the clinical strains. The isolates were sequenced and assembled into contigs, or contiguous coding regions, to determine the genetic structure of the strains. Using 2 clinical strains, the first isolated from a catheterized patient with a urinary tract infection and the second isolated from an infant with CF, researchers obtained a collection of genes specific to these 2 strains. Analysis of DNA sequences in these clones revealed that the majority of the genes did not share any sequence similarity with any other genes in the Genome Project database. In each case, the percent G+C content was lower than 64% for most of the strain-specific sequences, suggesting that those traits were acquired by horizontal gene transfer.

In microevolutional analysis, low-passage DNA sequencing of genomes was used to identify sequences unique to clinical isolates and to compare the genomic variations among them as well as to the Pseudomonas aeruginosa PA01 strain. In addition to generating sequence data that define unique genes in the genomes of these 2 clinical isolates, this approach has been extremely useful in defining regions that are present in the genome of PA01 and absent from the genomes of these 2 clinical strains. These DNA segments define unstable genetic elements that may encode proteins that are potentially deleterious for survival in the host. Lastly, DNA sequences of several loci that accumulate single nucleotide mutations also have been identified.

The long-range goal of the genomic comparison project is to correlate the genomic makeup of Pseudomonas aeruginosa strains with specific infections and to monitor the evolution of virulent traits expressed by this pathogen.

There is continuous research being done of the mapping of the genome of Pseudomonas aeruginosa, which may lead to potential new treatments for patients with cystic fibrosis. A team of researchers at the University of Washington Genome Center and PathoGenesis Corporation collaborated to complete this genome sequence genetic map. In fact researchers are already using knowledge about the genetic instructions of Pseudomonas to identify targets for novel drug strategies. They will take the gene sequencing data and attempt to define the molecular mechanisms of infection for Pseudomonas aeruginosa. They want to see which genes are needed for survival in its human host and which are needed for drug resistance.

Pseudomonas aeruginosa is the largest of the 25 bacteria that scientists have sequenced so far. The largest previously sequenced bacterium was Escherichia coli, which has 4.6 million base pairs and approximately 4,200 genes. Pseudomonas aeruginosa, by contrast, has more than 6 million base pairs and approximately 5,500 genes. Preliminary work suggests that the high number of genes in Pseudomonas aeruginosa allow it to adapt and survive in many different environments, whereas most bacteria live within a small niche. Indoor plumbing, in particular, is especially hospitable to Pseudomonas aeruginosa. Typical disinfectants are not effective at eradicationg it so Pseudomonas aeruginosa can be found on nearly every shower curtain and drain pipe around the world.

Functions of Pseudomonas aeruginosa that were previously unknown have been identified, suggesting new avenues for drugs to treat serious lung infections caused by this bacterium. Researchers now have a better understanding of why Pseudomonas aeruginosa is naturally resistant to most antibiotics. As a result, they have new ideas on how to identify antibiotics that might circumvent some of the bacterium’s intrinsic drug resistance mechanisms. The bacterium was sequenced based on one particular organism, or isolate, that is the standard in laboratories. Variations are now being examined that occur when the organism is taken from patients with cystic fibrosis. Scientists are looking for not only how Pseudomonas aeruginosa differs from patient to patient, but what happens to the organism inside the body. “We want to figure out what is different about those clinical isolates, and how these isolates change over time during chronic ingection” Olson, 142. With this information in hand, new windows of opportunity may arise, suggesting that certain biochemical pathways or proteins within Pseudomona aeruginosa are good targets for drug development. Pseudomonas aeruginosa is difficult to overcome with antibiotics even in patients with new infections. Over time, treatment becomes progressively more difficult; this is not unique to Pseudomonas aeruginosa.

Attempts to produce a vaccine for Pseudomonas aeruginosa have not resulted in a high level of protection against subsequent infections. Previous studies have focused on creating immunity against a single surface element of the bacterium. There is a study going on now that is testing the feasibility of using an oral dose of an intact but weakened or attenuated strain of Pseudomonas aeruginosa for vaccine development. This approach has been used in vaccine production in other diseases and has resulted in an immune response in the lung. Strains of Pseudomonas aeruginosa are being produced which must be supplied with certain amino acids for growth, and thus should be able to survive only under laboratory conditions. Plans are to feed these strains to mice to determine whether the bacteria can cause disease, how long the bacteria survive, and whether an immune response is generated in the lung. The long-term goal is to produce a safe vaccine for use in the individuals with cystic fibrosis.

Pseudomonas aeruginosa infection is also common among persons infected with HIV. Most of the patients who developed invasive Pseudomonas aeruginosa infections were severely immunocompromised, with a mean CD4+ cell count of 31/uL and a history of lung injury. These infections often occur at multiple sited, particularly the lungs and sinuses. Since the advent of the routine use of HAART (highly active antiretroviral therapy), there has been a striking decline in the incidence of invasive Pseudomonas aeruginosa disease. Scientists suggest that HAART may in fact restore humoral immunity.

HIV positive adults often are plagued with severe pyrogenic infections that tend to recur. In fact, recurrent bacterial pneumonia is considered an AIDS defining illness. Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Moraxella catarrhalis are the most frequently isolated pathogens. Pseudomonas aeruginosa infection is yet another complication of late stage HIV disease. Mortality from Pseudomonas aeruginosa infection in HIV positive patients is estimated at 22% to 36%. The literature reveals that neutropenia, indwelling catheters, and hospitalization may be associated with Pseudomonas aeruginosa infection, but traditional risk factors are not always present.

Clinical records were reviewed of all patients with HIV infection seen at the outpatient clinic of the Hospital of Saint Raphael between January 1992 and January 1998 to identify patients who had invasive pseudomonal disease. Pseudomonas aeruginosa pneumonia was defined as the presence of a febrile (temperature greater than 38.3*C) respiratory illness, isolation of Pseudomonas aeruginosa as the sole pathogen from expectorated sputum, and evidence of infiltrate on chest radiograph. Bronchitis was defined as a productive cough. Otitis media was defined as the presence of a bulging or inflamed tympanic membrane with purulent auditory canal drainage that grew Pseudomonas aeruginosa. Sinusitis was defined as a febrile illness, headache, sinus tenderness, and purulent nasal discharge that grew Pseudomonas aeruginosa. Bacteremia was defined as the isolation of Pseudomonas aeruginosa from one or more blood cultures from a patient with clinical signs of infection.

The hospital microbiology laboratory processed all specimens. Blood samples were incubated in a Bac-T alert system. Expectorated sputum samples were inoculated onto blood MacConkey and chocolate agar, and nasal aspirates were inoculated onto chocolate plates. Antimicrobial susceptibilities were obtained using the Microscan walkaway system.

Medical records of patients who met the criteria for Pseudomonas aeruginosa pneumonia, sinusitis, and otitis media were reviewed. Paying close attention to the following variables: age, gender, race, self-reported risk of HIV acquisition, CD4+ cell count, previous opportunistic infections, antiviral therapy, use of Pneumocystic carinii pneumonia prophylaxis, prior pulmonary infection, signs and symptoms of disease on presentation, therapeutic interventions, and clinical outcome, including death. Specific antiviral regimens varied, most subjects were on a regimen that included one or two reverse transcriptase inhibitors. Of the two patients who developed invasive Pseudomonas aeruginosa infections following the routine use of HAART, one was receiving a three-drug regimen, including a protease inhibitor, and the other did not comply with his antiviral therapy.

Pseudomonas aeruginosa is an important cause of recurrent, community-acquired sinopulmonary disease among HIV infected patients, even in the absence of traditional risk factors. Several studies delineating the potential risks of pseudomonal infection in the HIV population have found corticosteroid use, recent antibiotic exposure, PCP prophylaxis with TMP-SMX, prior hospitalization, and neutropenia to be predictive of Pseudomonas aeruginosa infection. The profound immunosuppression inherent in advanced HIV disease may in fact be the most important risk factor for Pseudomonas aeruginosa infection. Patients with pseudomonal disease were severely immunocompromised and suffered from frequent opportunistic infections. Cell-mediated immunity is thought to play a vital role in defending against Pseudomonas aeruginosa infection. Therefore, it is not surprising that the loss of functionally active T cells that occurs with progressive HIV infection was an important predisposing factor for pseudomonal disease.

Many patients with pseudomonal disease, including the two in whom infections developed hollowing the initiation of HAART, had suffered previous pulmonary infections. This suggests that chronic lung damage resulting from recurrent bacterial pneumonias and other opportunistic infections may predispose a patient to subsequent pseudomonal disease. Recurrent bacterial lung infections, combined with defective humoral immunity, may contribute to the development of bronchiectasis in HIV infected patients. The destructive nature of Pseudomonas aeruginosa in particular may make patients vulnerable to bronchiectasis and lead to persistent bacterial colonization and relapsing pneumonia.

Pseudomonas aeruginosa produces an elastase that destroys the Pis that are normally present in the bronchial tree and cleaves IgG, necessary for opsonization. In addition, Pseudomonas aeruginosa releases endotoxin and results in a polyclonal increase in immunoglobulins that are deposited as immune complexes, causing further lung damage. Precisely how HAART affects this cascade of damage remains to be elucidated.

Defective humoral immunity is another possible contributing factor in the development of Pseudomonas aeruginosa infections in patients with HIV. Although hypergammaglobulinemia occurs commonly in patients with HIV disease, it selectively involves IgG subclasses 1 and 3, with relative deficiencies of IgG2 and IgG4. Adequate opsonization of encapsulated organisms requires antibody to polysaccharide antigen, which consists primarily of IgG2. In addition, it has been shown that most HIV infected patients with Pseudomonas aeruginosa bacteria were unable to mount a specific IgG response to lipopolysaccharide immunotypes despite the presence of hypergammaglobulinemia. Defective chemotaxis, abnormal neutrophil degranulation, and impaired antibody response to new antigens have all been seen in patients with AIDS.

Pseudomonas aeruginosa pneumonia and sinusitis frequently occurs together in AIDS patients. In general, HIV infected patients are at increased risk for sinus infections, especially when theCD4+ cell count falls below 200/uL. In addition to the immunologic deficits associated with HIV infection, local factors may predispose the patient to sinus disease. Persistent bacterial colonization along with the loss of mucosal integrity from smoking or cocaine use may predispose a patient to recurrent bouts of sinusitis. In addition, prior sinusitis and impaired sinus drainage, possibly from lymphoid hyperplasia, may be risk factors for recurrent disease.

How HAART will affect the incidence of pseudomonal infections is still not clear. A correlation between viral load and risk of opportunistic infections has been established, and there has been a decline in AIDS related morbidity and mortality in patients treated with aggressive anti-viral regimens. Most of the observed changes in immune competence relate to cellular immunity rather then humoral immunity.

Thus, based on experiments, there is an anticipation of a decline in the incidence of sinopulmonary disease caused by Pseudomonas aeruginosa. Prospective studies are necessary to determine whether the patients with newly diagnosed HIV infection who have pulmonary damage will develop pseudomonal disease and to better define the changing epidemiology, optimal treatment strategies, and the role of adjunctive immunotherapies.

Now that scientists have completed the genome sequence genetic map of Pseudomonas aeruginosa, it may lead to potential new treatments for patients with cystic fibrosis, patients with HIV, and others who develop this type of infection. This map of the genome provides scientists with a powerful tool that opens up new doors to develop innovative therapies that will make a difference in many lives.

Bibliography

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November 10, 2000. www.cff.org

4. Cells Alive. Cells Alive. October 30, 2000. November 10, 2000. www.cellsalive.com

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