Date of Award

12-1997

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

School

Biological, Environmental, and Earth Sciences

Abstract

Pseudomonas aeruginosa is an opportunistic Gram negative facultative anaerobe. It is found primarily in the soil, but is ubiquitous in nature. The clinical significance of the organism is attributed to its innate resistance to antibiotics and its ability to survive in the presence of compounds that would be lethal to other organisms. Hospitals constitute one such environment where Pseudomonas aeruginosa has been found to exist in weak antiseptic solutions. hand creams. and hydrotherapy tanks. The hospital is also a place where patients who are severely burned. have cystic fibrosis. or whose resistance is otherwise compromised are at an increased risk of becoming colonized with this organism.

Ps. aeruginosa produces a number of virulence factors. One protein toxin, cytotoxin, has evoked considerable interest because of its lethal effects on eukaryotic cells.The 31kD protoxin form of this protein is subjected to post-translational modification of the c-terminal end by proteolytic cleavage of 20 amino acid residues to form the 28kD active form. Cytotoxin forms 2-4 nm holes in the cytoplasmic membrane of target cells. Subsequently these cells die because of their inability to maintain osmotic equilibrium. Currently a controversy exists regarding the presence or absence of the cytotoxin gene in Ps. aeruginosa. Several studies have been done to investigate the prevalence of the cytotoxin gene in Ps. aerug.inosa. Several studies have been done to investigate the prevalence of the cytotoxin gene in Ps aerug.inosa. However, different investigators have used different methods and different strains of Ps. aerug.inosa in their studies. Unfortunately none of these studies provided conclusive results as to the distribution of the cytotoxin gene. In the present study numerous PAOl strains of Ps. aeruginosa, an isolate of Pseudomonas fluorescens, and several clinical and veterinary isolates of Ps. aeruginosa were probed with several detected in strains other than PA158 using either molecular probing or the polymerase chain reaction . A gene similar to the cytotoxin gene may be present in non-PA158 strains. but if it is. it is sufficiently different in nucleotide sequence that it is undetectable with oligonucleotide probes or PCR primers derived from the cytotoxin gene of PA158 . oligonucleotides. The sequences of these probes were derived from the _cytotoxin gene of Ps. aerug.inosa strain PA158. They were designed to hybridize withs· and 3' regions of the cytotoxin gene. The polymerase chain reaction (using primers for the 5' ends of each DNA strand) was also used to identify the cytotoxin gene and to assess the possible sequence heterogeneity of putative cytotoxin genes found in other strains with that of PA158. The cytotoxin gene was not detected in strains other than PA158 using either molecular probing or the polymerase chain reaction . A gene similar to the cytotoxin gene may be present in non-PA158 strains. but if it is. it is sufficiently different in nucleotide sequence that it is undetectable with oligonucleotide probes or PCR primers derived from the cytotoxin gene of PA158 .

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