Title page for ETD etd-11042005-080132


Document Type Doctoral Thesis
Author Weldhagen, Gerhard Frederick
Email gweldhagen@medic.up.ac.za
URN etd-11042005-080132
Document Title Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa
Degree PhD (Medical Microbiology)
Department Medical Microbiology
Supervisor
Advisor Name Title
Prof M G Dove Co-Supervisor
Prof M B Taylor Supervisor
Keywords
  • peptide nucleic acid
  • blaGES
  • Pseudomonas aeruginosa
  • genetic stability
  • antibiotic selective pressure
  • LightCycler
Date 2004-12-10
Availability unrestricted
Abstract
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.

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  00front.pdf 123.04 Kb 00:00:34 00:00:17 00:00:15 00:00:07 < 00:00:01
  01chapter1.pdf 87.16 Kb 00:00:24 00:00:12 00:00:10 00:00:05 < 00:00:01
  02chapter2.pdf 229.58 Kb 00:01:03 00:00:32 00:00:28 00:00:14 00:00:01
  03chapter3.pdf 184.91 Kb 00:00:51 00:00:26 00:00:23 00:00:11 < 00:00:01
  04chapter4.pdf 243.78 Kb 00:01:07 00:00:34 00:00:30 00:00:15 00:00:01
  05chapter5.pdf 204.16 Kb 00:00:56 00:00:29 00:00:25 00:00:12 00:00:01
  06chapter6.pdf 192.18 Kb 00:00:53 00:00:27 00:00:24 00:00:12 00:00:01
  07chapter7.pdf 90.71 Kb 00:00:25 00:00:12 00:00:11 00:00:05 < 00:00:01
  08back.pdf 102.62 Kb 00:00:28 00:00:14 00:00:12 00:00:06 < 00:00:01

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