A dissertation submitted to the Faculty of Medicine, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the
degree of Master of Medicine. / The emergence of strains of S. pneumoniae resistant to penicillin and to other
antibiotics, and the spread of that resistance over the world, have become
major concerns and increase the need for epidemiological surveillance. The
following typing methods have been used to detect strain variability in
pneumococci: Serotyping, antibiotic susceptibility profiles, multilocus
enzyme electrophoresis (MLEE), penicillin-binding protein (PBP) profiles,
pulse-field gel electrophoresis (pFGE), and ribotyping. Serotyping,
antibiograms, and MLEE only detect phenotypic variability. PBP gene profiles,
PFGE, and ribotyping detect genotypic differences but these techniques are
labour intensive and time consuming.
Random amplified polymorphic DNA (RAPD) is a new technique that bas
proved useful for typing bacteria, fungi, and parasites, but has not been.
studied using pneumococci. Unlike conventional polymerase chain reaction
(peR), RAPD utilizes single, short primers, usually 10 oligonucleotides in
length. As the primer is short and low astringency annealing temperatures are
used, there will be many complimentary sites scattered randomly throughout
a bacterium's genome, When such sites occur a few hundred base pairs away
from each other and on opposite DNA strands, the enclosed region can be
amplified by peR This results in numerous discrete target fragments which
can be separated by agarose-gel electrophoresis and ethidium bromide
staining. RAPD requires no sequence information and it scans the whole
genome rather than relying on hypervariability within one specific gene.
The aims of this study were: to determine strain variability using RAPD, to
determine the reproducibility ofRAPD, and to demonstrate intercontinental
spread of a multiresistant pneumococcal strain.
The following strains were evaluated: a) 10 strains from a day-care centre
(DCC), the index case being a 3 year old girl 'with otitis media. An aunt from
Spain had recently been staying with the family. The other strains were
isolated from class mates and siblings of the index case.; b) 18 clinical
isolates from Seoul, Korea; and c) 11 epidemiologically unrelated isolates
from South Africa, including the reference strain, R6.
Two DNA extraction methods were used. The first involving lysis with
sodmm-dodecyl-sulphaze and proteinase K. Proteins were removed with
phenol-chloroform, and the DNA precipitated with ethanol. The second
method involved incubating the cells at 95 0C for 15 microlitres, followed by
centrifugation. 2 microlitres of the supernatant was then used for each PCR
reaction, Three primers were evaluated.
After 01uimisation of the RAPDreaction for pneumococci, the final peR
mixtures per 50 ul was: primer (4 plY1), template (0.5 ng), nuc1eotides (300
pMeach), magnesium (4 mM~, and Taq polymerase (2 U). 35 cycles were
used with an annealing temperature of 35'C.
Both DNA extraction methods: gave reproducible results but were not
comparable to each other. All 10 strains from the DCC gave the same
banding pattern as the Spanish done for all 3 primers. 7 of the Korean strains
gave the same banding pattern as the Spanish clone using the first two
primers, however one strain showed an additional band using the third
primer. Of the remaining 22 strains, 21 different banding patterns were
obtained.
This study has shown that RAPD is a simple and rapid technique that can
distinguish strain variation among pneumococci. The reproducibility is
excellent within the same laboratory. Finally using RAPD. this study
identified a Spanish multiresistant 23F clone in South Africa and Korea. / Andrew Chakane 2018
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/24704 |
| Date | January 1994 |
| Creators | Friedland, Hillel David |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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