Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa
Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004.
Saved in:
| Other Authors: | |
|---|---|
| Format: | Thesis |
| Published: |
University of Pretoria
2013
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613728641384448 |
|---|---|
| access_status_str | Open Access |
| author2 | Dove, Mike G. |
| author_browse | Dove, Mike G. |
| author_facet | Dove, Mike G. |
| collection | Thesis |
| dc_rights_str_mv | © 2004, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/29221 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:40:45.814Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/29221 Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa Dove, Mike G. Taylor, Maureen B. gweldhagen@medic.up.ac.za Weldhagen, Gerhard Frederick Peptide nucleic acid Blages Pseudomonas aeruginosa Genetic stability Antibiotic selective pressure Lightcycler UCTD Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004. Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms. Medical Microbiology unrestricted 2013-09-07T15:10:29Z 2005-11-04 2013-09-07T15:10:29Z 2004-12-10 2004 2005-11-04 Thesis Weldhagen, G 2004, Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/29221 > http://hdl.handle.net/2263/29221 http://upetd.up.ac.za/thesis/available/etd-11042005-080132/ © 2004, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf University of Pretoria |
| spellingShingle | Peptide nucleic acid Blages Pseudomonas aeruginosa Genetic stability Antibiotic selective pressure Lightcycler UCTD Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title | Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title_full | Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title_fullStr | Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title_full_unstemmed | Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title_short | Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa |
| title_sort | laboratory detection and gene cassette stability of the novel extended spectrum beta lactamase ges 2 from pseudomonas aeruginosa |
| topic | Peptide nucleic acid Blages Pseudomonas aeruginosa Genetic stability Antibiotic selective pressure Lightcycler UCTD |
| url | http://hdl.handle.net/2263/29221 http://upetd.up.ac.za/thesis/available/etd-11042005-080132/ |