Date of Award

2001

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Science

First Advisor

Dr. Séamus Fanning

Abstract

β-Lactam antibiotics are commonly used antimicrobial agents in clinical medicine. Because of their frequent use it is perhaps not surprising that resistance to these agents has increased over the past few decades, thus compromising the therapeutic advantages offered by these agents. The most common cause of resistance to β-lactam-based antibiotics has been the evolution of a group of enzymes known as β-lactamases, which act by inactivating the drug through structural modification. Plasmid-encoded TEM β-lactamases are examples of these hydrolytic enzymes and are associated with the amoxicillin resistance reported in over 50% of all E. coli isolates.

More recently β-lactamase enzymes have evolved further, due at least in part, to the selective pressure linked to our dependence on these important classes of antibiotics. This selection has resulted in the emergence of extended- spectrum β-lactamases (ESBLs) and even more recently to inhibitor resistant TEM (IRT) enzymes. Such enzymes may be classified by biochemical methods, which although useful, do not reveal any detail concerning the molecular events that gave rise to their modified biological behaviour.

In this study the molecular diversity of TEM-encoding enzymes produced by thirteen isolates from Irish patients with Urinary Tract Infections was investigated. Modern molecular techniques including the polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis and finally DNA sequencing were used to uncover unique changes at the genetic level. All sequences were analyzed by computational means. Four IRT enzymes were identified from the thirteen clinical isolates submitted to the Molecular Diagnostics Unit, Cork Institute of Technology for study. Each of these isolates, was resistant to a combination of amoxicillin and clavulanic acid. Mutations at the genetic level resulted in amino acid substitutions at residue positions 69 and 244. These locations along with one other are responsible for the molecular architecture of the catalytic center. The remaining nine isolates were found to be resistant to amoxicillin alone and were indistinguishable from the wild-type TEM-1-encoding p-lactamase on the basis of genetic and biochemical characteristics.

In an effort to investigate the origin of these genes and to assess their potential for dissemination to other organisms, DNA-DNA hybridization studies involving plasmid and integron analysis were carried out. Non-isotopic probing data showed that all blaTEM-encoding genes were located on both chromosomal and plasmid DNA structures. In addition, the genetic relationship(s), among the study collection was successfully analysed using PCR-based protocols (including REP- and IS-3A-based methods), together with pulsed-field gel electrophoresis (PFGE).

Access Level

info:eu-repo/semantics/openAccess

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