Multidrug resistance mechanisms in the human pathogen Acinetobacter baumannii

Infections caused by the nosocomial multidrug resistant human pathogen Acinetobacter baumannii are a global public health issue. Without new and potent antibiotics or alternative therapies for infectious diseases caused by A. baumannii and other drug resistant pathogens, the World Health Organization has predicted there will be a return of the pre-antibiotic era in the near future. This thesis investigates the effects of antimicrobial agents on A. baumannii, using two global approaches: RNA-Seq and TraDIS (transposon directed insertion site sequencing). Another novel high throughput technique, which combines Biolog phenotype microarrays and qPCR, was developed for the characterization of drug resistance phenotypes of putative drug efflux pumps. Drug efflux pumps are cytoplasmic membrane transport proteins. They confer antimicrobial resistance through exporting the chemical agents out of the host cell. High expression levels of these proteins are frequently associated with increased bacterial drug resistance. More than 50 genes were annotated as putative drug efflux pumps in A. baumannii ATCC17978 strain. In this study, seventeen of these genes were heterologously cloned and highly expressed in Escherichia coli, and the drug susceptibility changes of the E. coli clones were screened through the combination of Biolog Phenotype Microarrays and qPCR. This multiplexed phenotype analysis approach identified potential drug resistance phenotypes of three novel drug efflux genes. A protein synthesis inhibitor, tigecycline, is one of the last line therapeutic options for A. baumannii infection. RNA-Seq was used to analyse how A. baumannii physiologically responds to subinhibitory concentrations of tigecycline. The transcriptomic data showed an increase in expression of many genes involved in ribosome biosynthesis and assembly, and decrease in expression of genes involved in peptide synthesis, consistent with tigecycline-induced ribosomal stalling after translational initiation. Decreased expression of genes involved tricarboxylic cycle, cell respiration and cell division was observed, which is consistent with tigecycline's bacteriostatic effect. Tigecycline induced increased expression of genes involved in exogenous DNA uptake, mobile genetic element translocation, and DNA mismatch repair, suggesting this antibiotic may promote gene mutation and lateral gene transfer. Biocides are critical for disinfection in hospitals and are commonly used in personal hygiene products. Antibiotic resistance in bacterial pathogens has been intensively studied, but comparatively little is known about resistance to biocides or the mode of action of biocides. The potential modes of action and resistance mechanisms of ten clinically important biocides were assessed with TraDIS using a highly-saturated transposon Tn5 mutant library. Like the tigecycline data, the TraDIS analysis suggested that these biocides have pleiotropic effects on the bacterial host. Lipooligosaccharide and capsular polysaccharide biosynthesis genes, and drug efflux genes appear to be important fitness factors in A. baumannii against biocides. Collapse of the cytoplasmic membrane proton motive force is likely to be a downstream antibacterial effect of multiple biocides including silver nitrate, which could potentially cause malfunction of membrane transport, cell division and various pleiotropic physiological effects. Antimicrobial drug resistance in pathogenic bacteria, such as A. baumannii, is a multifactorial phenomenon. The contemporary high throughput technologies, for example the ones that were used in this thesis, has enabled us to decipher this problem at a global scale. Knowledge of finer details of antimicrobial mode of actions and bacterial drug resistance will further shed light on finding appropriate solutions for multidrug resistance infections.