Campylobacter jejuni in gastroenteritis: Detection and mechanisms of intracellular survival

Persistent Link:
http://hdl.handle.net/10150/282761
Title:
Campylobacter jejuni in gastroenteritis: Detection and mechanisms of intracellular survival
Author:
Day, William Alan, 1964-
Issue Date:
1998
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The fastidious bacterium Campylobacter jejuni has recently been identified as a leading cause of human bacillary enteritis. Delays in recognition of this important pathogen reflect inadequate isolation techniques, which cannot recover environmentally stressed viable but non-culturable forms. Therefore, a non-culture based detection systems for C. jejuni would be invaluable. Studies were undertaken to develop a PCR based detection assay for C. jejuni. Fingerprints enriched for repetitive C. jejuni chromosomal elements were generated using arbitrarily primed PCR. Dot blot screening of fingerprint products for specificity to C. jejuni identified a 496 bp product which hybridized with all C. jejuni isolates examined. No binding to other Campylobacter species or enteric genera screened was observed. The product was cloned, sequenced, and primers synthesized to three overlapping regions of the probe. A primer pair was identified which directs amplification of a 265 bp product from C. jejuni alone. Sensitivity studies demonstrated that the C. jejuni specific PCR generated product from as few as 100 lysed bacteria. The ability of C. jejuni to penetrate normally non-phagocytic host cells is believed to be a key virulence determinant. Kinetics of C. jejuni of intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies by Pesci et al. demonstrate that super-oxide dismutase contributes to intra-epithelial cell survival, suggesting that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intra-epithelial cell survival as growth curves for katA mutant and wild type strains generated from long term culture within HEp-2 cells are roughly identical. Catalase does however contribute to intra-macrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p = 0.00246) relative to the wild type strain after 72 hr incubation with these cells.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Microbiology.; Health Sciences, Pathology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Joens, Lynn A.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCampylobacter jejuni in gastroenteritis: Detection and mechanisms of intracellular survivalen_US
dc.creatorDay, William Alan, 1964-en_US
dc.contributor.authorDay, William Alan, 1964-en_US
dc.date.issued1998en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe fastidious bacterium Campylobacter jejuni has recently been identified as a leading cause of human bacillary enteritis. Delays in recognition of this important pathogen reflect inadequate isolation techniques, which cannot recover environmentally stressed viable but non-culturable forms. Therefore, a non-culture based detection systems for C. jejuni would be invaluable. Studies were undertaken to develop a PCR based detection assay for C. jejuni. Fingerprints enriched for repetitive C. jejuni chromosomal elements were generated using arbitrarily primed PCR. Dot blot screening of fingerprint products for specificity to C. jejuni identified a 496 bp product which hybridized with all C. jejuni isolates examined. No binding to other Campylobacter species or enteric genera screened was observed. The product was cloned, sequenced, and primers synthesized to three overlapping regions of the probe. A primer pair was identified which directs amplification of a 265 bp product from C. jejuni alone. Sensitivity studies demonstrated that the C. jejuni specific PCR generated product from as few as 100 lysed bacteria. The ability of C. jejuni to penetrate normally non-phagocytic host cells is believed to be a key virulence determinant. Kinetics of C. jejuni of intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies by Pesci et al. demonstrate that super-oxide dismutase contributes to intra-epithelial cell survival, suggesting that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intra-epithelial cell survival as growth curves for katA mutant and wild type strains generated from long term culture within HEp-2 cells are roughly identical. Catalase does however contribute to intra-macrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p = 0.00246) relative to the wild type strain after 72 hr incubation with these cells.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Microbiology.en_US
dc.subjectHealth Sciences, Pathology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorJoens, Lynn A.en_US
dc.identifier.proquest9906539en_US
dc.identifier.bibrecord.b38874751en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.