Year

2009

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

Multi-drug resistant pathogens are the principal cause of failure in the treatment of bacterial infectious diseases. Accurate surveillance of antibiotic resistance genes in the community is essential to developing strategies for resistance control and prevention. In this study, a collection of 514 Escherichia coli strains from animal and human sources was examined for the presence of class 1, 2 and 3 integrons using a PCR-based screening method. A multiplex PCR was developed to simultaneously screen for intI1, intI2 and intI3 genes. This study characterised all gene cassettes including those that could not be PCR amplified using standard screening methods.

The frequency of class 1 and class 2 integrons detected in E. coli strains in this study was generally lower than that reported in previous studies. Class 1 integrons were detected in 81/514 E. coli strains sourced from animals and humans. Gene cassette arrays identified in class 1 integrons include dfrA5, dfrA7, aadA1, aadA2, dfrA1/aadA1, dfrA17/aadA5 and dfrA12/orfF/aadA2. In addition, atypical integrons containing dfrA5-IS26 and dfrA15-IS26 elements were discovered. The dfrA5-IS26 element, a unique class 1 integron with most of the integron 3′-conserved segment (CS) deleted by the insertion of IS26, was detected in 31/514 E. coli isolates. This novel integron-dfrA5-IS26 element, which was widespread in E. coli isolates of bovine origin and also found in E. coli of human origin, may act as a conduit for the transfer of integron-related resistance genes to human pathogens. Utilisation of PCR targeting the integron-IS26 element will allow the characterisation cassette arrays in atypical class 1 integrons that remain undetected using currently available PCR-based screening strategies.

Seven of the 514 E. coli strains contained class 2 integrons and six of these harboured the gene cassette array analogous to that found in Tn7, dfrA1-sat2-aadA1. In the remaining intI2 positive E. coli strain 80, in which the gene cassette region could not be PCR amplified using standard methods, the intI2 gene was found to be located on a plasmid. The complete nucleotide sequence of this plasmid (pECTm80) was determined, revealing an intact dfrA1-sat2 cassette array and a truncated aadA1 gene cassette, due to the insertion of IS1. Open reading frames and Tn7 transposition genes normally conserved at the 3′ end of Tn7-like class 2 integrons were not detected. This atypical class 2 integron is flanked by a Tn3 family transposon or insertion sequence (IS) remnant and IS1. The plasmid pECTm80, of the incompatibility (Inc) group X, has the potential to facilitate the horizontal transfer of tightly-linked antibiotic resistance genes to diverse antimicrobial species. Features which contribute to the clinical relevance of this plasmid include its ability to be mobilised, the presence of genes to ensure the stable maintenance of the plasmid through successive bacterial cell divisions and the presence of a highly-regulated DNA replication system consisting of alpha, beta and gammaorigins of replication.

This thesis provides a snapshot of the antibiotic resistance genes located in integrons in E. coli strains sourced from Australian animals and humans. The association of atypical integrons with IS elements suggests these DNA elements play an important role in the evolution of integrons.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.