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Title: Mechanisms of evolution in antibiotic resistant clones of Streptococcus pneumoniae
Author: Miyashita, Lisa Frances
ISNI:       0000 0004 2734 3804
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2012
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Streptococcus pneumoniae has a highly adaptable genome due at least in part to its natural transformability and its ability to recombine with other pneumococci and related species. The emergence of antibiotic resistant clones of S. pneumoniae presents an opportunity to investigate how the genome has altered, spread and diversified within a defined time frame. We postulated that the genomes of epidemic, resistant isolates of S. pneumoniae would carry evidence of the genetic mechanisms that have shaped their evolution. We investigated this using eight to fifteen isolates from each of three S. pneumoniae clones; two multiply resistant clones Spain 9V-3 and Taiwan 19F-14 and one clone that has not acquired multiple resistance, England 14-9. Genome diversity in each of the three clones was investigated using pulsed field gel electrophoresis and multilocus sequence typing. Polymorphisms identified as a result of changes in the size of restriction fragments were found to be caused mainly by genomic rearrangements rather than restriction site mutations. Several deletion/insertion events in addition to one large inversion were identified. A number of polymorphisms correlated with previously known variable regions. Database analysis of multilocus sequence data from all three clones showed that recombination leads to sequence divergence more frequently than de novo mutation, but was significantly less common in England 14-9. The lower frequency of recombination events in England14-9 was in line with a transformation deficiency observed in vitro, and may explain the rare occurrence of penicillin resistance in this clone. Analysis of competence and recombination gene sequences available from databases revealed a potential cause of transformation deficiency: a four amino acid deletion in CelA, involved in DNA uptake and transport. Recombination can act as a DNA repair mechanism, but the significantly low occurrence in England14-9 suggests other mechanisms act to repair severe damage. Although S. pneumoniae does not have a typical SOS response it does possess DNA polymerase IV, encoded by dinB, which is predicted to be involved in error-prone DNA replication and repair of double strand breaks. DinB knockout mutants were created to investigate the effect in isogenic backgrounds. DinB mutants presented a lower frequency of spontaneous rifampicin resistance mutations than wild type 3 isolates. DinB mutants were more sensitive to killing by three different DNA damaging agents as well as by hydrogen peroxide. Isolates of the natural dinB mutant clone Spain 9V-3 were also shown to be more sensitive to DNA damaging agents than clones England 14-9 and Taiwan 19F-14. It is concluded that genetic differences between the three clones investigated do influence their patterns of evolution, and may account for differences in their antibiotic resistance profiles. Furthermore DNA polymerase IV does function as an error prone repair polymerase capable of protecting pneumococci from DNA damage despite the lack of a coordinated SOS response in pneumococci, and the absence of the gene in one successful multi-resistant clone.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine ; Infectious diseases ; Streptococcus pneumoniae ; Pneumococci