Virulence regulators of the significant human pathogen Acinetobacter baumannii

Author: Sarah Giles

Giles, Sarah, 2019 Virulence regulators of the significant human pathogen Acinetobacter baumannii, Flinders University, College of Science and Engineering

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Acinetobacter baumannii is a prolific pathogen and a leading cause in nosocomial infections worldwide. This particular bacterium causes a number of infections including pneumonia which is commonly ventilator-associated, urinary tract and meningitis. Described as multidrug-resistant (MDR) and presenting a high threshold for a number of antibiotics as well as antiseptics, it is now commonplace to hear reports of pandrug-resistant strains. These strains are resistant to all clinically used classes of antibiotics. Epidemiological studies show a rapid global emergence and dissemination of this pathogen, demonstrating the remarkable successfulness and adaptation ability of A. baumannii in the hospital environment. This thesis provides insight into the regulation of a variety of genes and their impact on a number of virulence factors. These factors include pellicle formation, antibiotic resistance, adherence to eukaryotic cells and the ability of A. baumannii to cause disease in a murine infection model.

The ability to form a biofilm at the air-liquid interface, known as a pellicle, is a factor that is involved in the persistence of A. baumannii in various environments and is described in detail in chapter two (Giles et al. 2015). To investigate the pellicle forming ability of this bacterium a transposon bank was generated in a pre-existing hypermotile derivative of A. baumannii ATCC 17978 called 17978hm. Evaluation of the transposon mutant strains identified 11 mutants that were unable to form a pellicle. Sequencing of the pLOFmini::Tn10:gfp:kan (Tn10) insertion site identified three genes essential in pellicle formation. Of significant interest, was the identification of cpdA. The cpdA gene degrades cyclic adenosine monophosphate (cAMP), a secondary signalling molecule, maintaining the amount of cAMP within a cell. Measurement of cAMP levels within the A. baumannii 17978hmcpdA::Tn (cpdA::Tn) mutant strain confirmed the abundance of cAMP and its role in the ability to form a pellicle.

Two-component signal transduction systems (TCSTS) are known to regulate virulence factors in numerous bacteria, and as these systems are not found within humans they are of interest as potential drug targets therefore warranting investigation. The highly virulent clinical A. baumannii 04117201 strain was selected from in a collection of 54 clinical isolates as an appropriate candidate to assess virulence and antibiotic resistance as described in chapters three and four. Examination of A. baumannii 04117201 identified a novel TCSTS designated herein as StkRS. The StkRS system is not present in the avirulent strain SDF and is therefore postulated to regulate virulence factors. Deletion of the stkR response regulator (RR) gene in the A. baumannii 04117201 parent strain was achieved generating the mutant strain 04117201ΔstkR (ΔstkR). Assessment of the resulting ΔstkR mutant strain identified a number of alterations in the virulence potential of this strain compared to the 04117201 parent strain as described in chapters three and four. Importantly, an increase in resistance to colistin (CST) of the ΔstkR mutant strain compared to the 04117201 WT was identified. CST being a polymyxin E antibiotic which has re-emerged as a viable treatment due to the severity of infections caused by A. baumannii. Additional differences were identified including an increase in; adherence capabilities to A549 pneumocytes, macrophage survival and resistance to human serum. A murine infection model of the 04117201 WT and ΔstkR mutant strains identified an increased bacterial load in the blood, lungs and spleen of mice infected with the ΔstkR mutant compared to the 04117201 WT strain. The above alterations plus further discussion in chapters three and four, reveal that the stkR RR gene is significant in regulating virulence factors in A. baumannii 04117201. Further investigation into this system is necessary to deepen the understanding of this invasive and problematic pathogen.

Keywords: Two-component signal transduction system, biofilm, pellicle, antibiotics, resistance, mouse infection model

Subject: Microbiology & Infectious Diseases thesis

Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Science and Engineering
Supervisor: Melissa Brown