Date of Award

2001

Degree Name

Forensic Science

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Terry Fenger

Second Advisor

Hongwei Yu

Third Advisor

Charles Somerville

Fourth Advisor

Leonard Deutsch

Abstract

Pseudomonas aeruginosa is a common environmental microorganism. However, it has the genetic capacity to cause diseases in patients with compromised host defense systems. Cystic fibrosis (CF) is one of the major hereditary diseases among Caucasian populations. CF patients are born with a defective chloride channel that is responsible for maintaining fluid and electrolyte balance across the lumen of the lung. This imbalance leads to the production of an abnormal dehydrated viscous mucous, prohibiting the patient from normally clearing the respiratory airway. Chronic pulmonary infections with P. aeruginosa are the major causes of high morbidity and mortality in CF. Two of the most discernible phenotypes expressed by P. aeruginosa during its chronic presence in CF are mucoidy, the overproduction of the mucoid exopolysaccharide (MEP) alginate and biofilm, the formation of microcolonies, both of which defend the bacterium from host defenses and antibiotic therapy.

Pulsed-field gel electrophoresis (PFGE) was used to analyze the genetic profiles from a collection of 101 clinical CF, 2 burn, 7 laboratory and 4 environmental isolates of P. aeruginosa. The goals of this study were to determine the following: i) presence of genetic diversity; ii) correlation of mucoidy with genotype; iii) presence of significant-sized deletions in a set of genes; and iv) presence of CF-specific extra-chromosomal DNA. Seventy-five unique genomic profiles were identified, thus genetic diversity exists throughout a majority of the isolates. Sets of defined CF isolates were also used to track the origin and mode of Pseudomonas infection by PFGE. Sequential isolates studied showed conservation of banding pattern throughout the colonization. Also, examples of possible cross-sectional infection between CF patients and common-vehicle environmental infection were identified. No correlation was found between the mucoid phenotype and the overall genomic profiles. Among the 75 unique profiles, one CF isolate, CF32, was found to have identical restriction fragment length polymorphism (RFLP) patterns using 3 different restriction digests (Spel, Xbal and Dpnl) when compared to the standard reference strain PAO1, originally isolated from a burn patient. This raises the possibility that a burn isolate of P. aeruginosa possesses the ability to infect a CF patient, a CF isolate could infect a burn patient, or an environmental PAO1-like strain can infect both CF and burn patients. Most importantly, since the entire genome sequence of PAO1 is available, this result indicates that PAO1 can be used as an index strain for genomic comparison to CF clinical isolates.

To establish a link between the RFLP patterns and precise chromosomal alterations, polymerase chain reaction (PCR) was applied to study a panel of 41 CF isolates for the presence of possible significant-sized genomic deletions. Twelve virulence genes were amplified: algD, oprF, oprC, exoS, exoT, mucE, mutS, mutY, 3 gene cluster mucBCD and one undefined gene identified in PAM, a burn isolate. While the burn-specific gene was not present in the CF isolates tested, the results indicated that no significant-sized deletions were seen suggesting that these genes are not modified during chronic Pseudomonas colonizations in CF. Combined PFGE and Southern blot analyses with wholegenome labeling were used to identify CF-specific extra-chromosomal DNA fragments of P. aeruginosa. Under the parameters used in this study, no CF-specific DNA was found, yet the procedure is being further developed. Determining a correlation between RFLP banding pattern and inherent virulence properties as well as identification of CF-specific extra-chromosomal DNA and CF-specific genomic deletions will provide basis for the rapid diagnosis of CF lung infections and quick determination of antibiotic resistance of these isolates, which eventually may lead to a more effective treatment for Pseudomonas pulmonary infections in CF.

Subject(s)

Genetic disorders – Research.

Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections – Research.

Cystic fibrosis.

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