Author: Bart Antonie Eijkelkamp

Eijkelkamp, Bart Antonie, 2012 FACTORS CONTRIBUTING TO THE SUCCESS OF ACINETOBACTER BAUMANNII AS A HUMAN PATHOGEN, Flinders University, School of Biological Sciences

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Acinetobacter baumannii is a major problem in the hospital setting and also shows significance as a community-acquired pathogen in tropical climates, including parts of Australia. The increase in resistance to widely used antibiotics is evident and signs of pan-resistance are emerging. Epidemiological studies have shown global dissemination of successful clones, explaining the occurrence of troublesome A. baumannii outbreaks worldwide. However, the wide variety of A. baumannii resistance and persistence mechanisms remains poorly understood. This can be partially attributed to the phenotypic and genetic variation observed between clinical A. baumannii strains. Iron acquisition systems are important virulence factors in pathogenic bacteria. To identify these systems in A. baumannii, the transcriptomic response of the fully sequenced strain ATCC 17978 under iron-limiting conditions was investigated. Of particular significance, three siderophore biosynthesis gene clusters, including one novel cluster, were highly up-regulated. Various genes involved in motility featured prominently amongst the genes down-regulated when iron was less readily available. Motility assays confirmed that these transcriptional changes are manifested at the phenotypic level. The clonal relationship, and the motility and adherence characteristics of 54 Australian clinical isolates and various fully sequenced Acinetobacter strains were investigated. The majority of the strains were classified as part of the international clone groups I and II, as seen in other parts of the world. However, unlike distribution of clinical A. baumannii isolates in Europe or the United States, international clone III isolates were not identified in our collection. Motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers not part of the international clone I group. A high level of variability in adherence to both abiotic surfaces and human lung epithelial cells was found. A derivative of strain A. baumannii ATCC 17978, isolated in this study, was found to possess enhanced motility and adherence characteristics. An insertion event of a mobile genetic element into a gene encoding a histone-like protein (A1S_0268) was identified by whole genome sequencing. Introduction of the wild-type A1S_0268 gene in the variant strain using an Acinetobacter/E. coli shuttle vector complemented the altered motility and adherence characteristics, making it indistinguishable to the wild-type A. baumannii ATCC 17978 parent. Transcriptional profiling of the variant strain under motile conditions assisted in identification of molecular mechanisms that play a putative role in A. baumannii adherence and motility. Investigation of motility, adherence and transcription levels of various molecular mechanisms, including type IV pili, under different conditions, such as low iron and high salt, showed that A. baumannii is highly responsive to stress. Active transport of antimicrobial agents mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Novel multidrug efflux systems were identified using a number of Gateway-based destination vectors constructed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout strategy was shown to be successful in disrupting a novel drug transporter. Examination of the extensive collection of A. baumannii isolates at a genetic, transcriptional, protein and cellular level assisted in delineating factors that contribute to the success of A. baumannii as a human pathogen. The importance of various molecular mechanisms in persistence, resistance and disease potential has been described in this thesis.

Keywords: Acinetobacter baumannii,resistance,virulence,transcriptome,biofilm,motility

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2012
School: School of Biological Sciences
Supervisor: Associate Professor Melissa H Brown