Year

2011

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

The use of antibiotics in the treatment of bacterial infections has greatly improved human and animal health, but has also driven the development and spread of antibiotic resistance in bacterial populations. The increase in multiply antibiotic resistant strains of pathogenic bacteria has been the cause of growing worldwide concern for the past 30 years. Research indicates that the development of multi-drug resistance is primarily due to the acquisition of multiple antibiotic resistance genes, often grouped in complex clusters and in association with mobile genetic elements, such as insertion sequences, transposons and plasmids, via horizontal gene transfer mechanisms.

The aim of this study was to characterise multiple antibiotic resistance patterns in a large collection of Escherichia coli isolates (n = 605), recovered in Australia over the past decade, from different hosts. Firstly, the collection was PCR-screened for the presence of 12 multiple antibiotic resistance markers (strA, strB, sul1, sul2, sul3, tetA(A), tetB(A), tetC(A), tetD(A) and tetG(A), merA and floR), encoding the most common resistance phenotypes found in enteric bacteria. A subset of 43 representative multi-resistant isolates was also screened by PCR for the presence of the aphA1 and blaTEM-1 genes. The detected multi-resistance gene clusters were then further characterised via PCR, Southern hybridisation, PFGE, DNA sequence analysis and mobilisation experiments.

Multiple antibiotic resistance was widespread in the collection, with 25.9% (157 of 605) multi-resistant isolates carrying two to six different resistance determinants. Even though resistance gene frequencies and associations varied considerably within each host group, the predominant gene combination in the collection was aphA1-strA-strB-sul2-blaTEM-1-merA-tetA(A) and/or tetB(A). These resistance genes were found to be clustered together, and associated with IS26 and elements of transposon Tn21, including class 1 integron gene cassettes encoding trimethoprim resistance. Multi-resistance gene clusters were shown to be predominantly plasmid-borne. Even though some of the multi-resistant isolates, carrying the Tn21-associated resistance locus, were found to be related by PFGE, genetic profiles differed among most strains. Plasmid profiles, however, were often similar, suggesting that horizontal gene transfer mechanisms, more than clonality, played a major role in the dissemination of resistance genes in this strain set. Characterisation of the Tn21-associated multi-resistance locus from 10 isolated E. coli plasmids showed that the gene cluster is preferentially found in large, low-copy number, conjugative plasmids, belonging to the IncF and IncI1 incompatibility groups. These loci are structurally diverse and are associated with complex genetic backgrounds.

Multiple resistance was also found to be mainly associated with pathogenic E. coli types. Sequencing of plasmid pO26-CRL from EHEC strain O6877 showed that this O26 virulence plasmid had acquired the complex Tn21-associated multi-resistance region. The plasmid was found to be mobilisable, but not conjugative. A second plasmid, pO6877, also containing the complex Tn21-associated antibiotic resistance locus, was isolated from the same EHEC O6877 strain. Unlike pO26-CRL, this plasmid did not contain the enterohemolysin operon and was conjugative, and thus capable of promoting the mobilisation of pO26-CRL in trans. Complete sequencing of plasmid pO6877, as well as of nine other multi-resistance plasmids isolated from E. coli strains in the collection, is underway.

The complex Tn21-associated multi-resistance locus was detected in plasmids isolated from both human and cattle E. coli strains. In two instances the very same resistance plasmid was isolated from a human and a bovine E. coli strain, suggesting crosstalk and active DNA exchange between the two bacterial populations. These data provide insight on the evolution of the complex multi-resistance gene clusters, and add to the understanding of the link between resistance and virulence in zoonotic E. coli. Better knowledge of microbial resistance mechanisms is essential for the decision-making processes required for better management of the antibiotic resistance problem.

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