Staph. aureus continues to be a significant pathogen which has developed the ability to acquire resistance to nearly all known antibiotics, in particularly methicillin resistant Staph. aureus (MRSA), has vastly reduced the number of antibiotics effective for treating staphylococcal infections. Recently, clinical isolates, with reduced susceptibility to vancomycin, the only antibiotic effective against multidrug-resistant MRSA, have been reported from a number of centres around the world. This situation could return us to the pre-antibiotic era, when even minor bacterial infections could be fatal. It may also have a significant impact on hospital procedures, especially in cases involving systemic infections, immunosuppression, and surgery. Clearly an understanding of the mechanisms of bacterial resistance to vancomycin, and the search for new anti-staphylococcal drugs or alternative forms of therapy, are necessary steps before epidemics of untreatable staphylococcal infections emerge. This work aimed to investigate the pathogenesis and treatment of Staph. aureus infections. Studies were carried out at phenotypic, and molecular level. Two clinical isolates of EMRSA-15 (clone A and clone C) were serially propagated in media containing increasing concentrations of vancomycin in order to select resistant mutants. EMRSA-15 clone A, yielded the vancomycin resistant clone B. EMRSA-15 clone C, yielded the vancomycin resistant clone 0, which confirms that MRSA was able to develop resistance to vancomycin in vitro. During this research an attempt was made to elucidate the development of resistance to vancomycin in Staph. aureus by studying the heterogeneous phenotype of resistance. The population analysis profile confirmed that EMRSA- 15 clones A and C were susceptible to vancomycin and there was no evidence of vancomycin-resistant subpopulations in these strains, all bacteria ceasing to grow at vancomycin concentrations above 2IJg/ml. The vancomycin resistant clones B and 0 readily demonstrated heterogeneous vancomycin-resistance phenotypes compared with the parent clones A and C.The EMRSA strains susceptible and resistant to vancomycin were extensively investigated for their susceptibility to other antibiotics. It was found that the vancomycin resistant derivatives had significant increases in the minimum inhibitory concentration of vancomycin and teicoplanin, and in addition the MICs for several other antibiotics were altered. In this study two main approaches were taken to investigate mechanisms by which Staph. aureus acquire resistance to vancomycin at molecular level. First RNA arbitrarily primed polymerase chain reation (RAP-PCR), was used to investigate differentially expressed genes in the vancomycin susceptible EMRSA and in the vancomycin resistant EMRSA. One combination of two primers yielded fingerprints with differentially expressed cDNA. This cDNA was expressed in the presence of vancomycin only and it was predicted that this expression was inducible. The second approach involved the sequencing of the the 23S rRNA. A fragment of about 500bp of the 23S rRNA from the vancomycin susceptible EMRSA and from the vancomycin resistant EMRSA was cloned, sequenced, and compared. Point mutations were observed in the EMRSA vancomycin resistant strain: such mutations were absent in the vancomycin susceptible clinical isolate. In this thesis, recombinant antibody derivatives (scFv) obtained from a phage antibody display library which was produced against an immunodominant protein (ABC transporter) from EMRSA was studied. The effect of such molecules (scFv) when used alone or in combination with antibiotics (erythromycin and chloramphenicol) and vinblastine was assessed. ABC transporters seem to constitute generic antigens, conserved between multiple genera, and the neutralisation of these essential transporter proteins may be able to inhibit a wide variety of microbial import and export functions. The data collected in this study showed that the antibody-phages with greatest activity were: phage 16 in combination with erythromycin, phage 12 when combined with chloramphenicol, and phage X when combined with vinblastine. These results indicated that such phages might be used as an alternative form of therapy for multidrug-resistant MRSA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594756 |
| Date | January 2001 |
| Creators | Gottardello, Priscila |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0118 seconds