Investigating the success of Acinetobacter baumannii in the clinical setting

Acinetobacter baumannii is an increasingly problematic hospital-associated opportunistic human pathogen that causes a range of infections such as respiratory, urinary tract and blood infections. The ability of nosocomial A. baumannii isolates to resist a diverse range of antimicrobial compounds and persist in clinical settings makes it a growing public health problem. Multidrug efflux pumps are significant contributors to antimicrobial resistance determinant in this microorganism. Five multidrug efflux pump superfamilies have been well described in bacteria, and characterized representatives of each of these five families are found in A. baumannii. In addition to drug efflux pumps and other resistance determinants, the capability of this pathogen to flourish in mixed species biofilm communities contributes to its success in the clinical setting. Recently, AceI from A. baumannii has been shown to be a novel chlorhexidine efflux pump. In this study, 23 homologs of aceI from different bacteria were cloned and expressed, and many of the homologs were found to confer resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. Fluorimetric transport assays indicated that an AceI homolog from Vibrio parahaemolyticus mediated resistance to proflavine and acriflavine via an active efflux mechanism. Thus, this group of AceI homologs represent a new multidrug efflux protein family, the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new multidrug efflux family to be found in the past 15 years. The function of AceR, a putative LysR family transcriptional regulator located adjacent to aceI in the A. baumannii genome, was investigated. AceR was demonstrated to be an activator of aceI gene expression, and induction is responsive to the AceI substrate chlorhexidine. AceR was demonstrated to bind in a chlorhexidine-inducible manner with a region of DNA upstream of the putative aceI promoter. AceR represents the first regulator of a PACE family pump to be functionally characterized. A novel high-throughput screening approach was developed to identify genes encoding multidrug efflux pumps and regulators in A. baumannii. This innovative screening method combines fluorescence-activated cell sorting (FACS) in parallel with transposon directed insertion sequencing (TraDIS). The feasibility of this method was demonstrated using a population of more than 100,000 random mutants shocked with ethidium bromide, a common substrate of multidrug efflux pumps. Ethidium bromide is differentially fluorescent inside and outside the bacterial cytoplasm due to its ability to bind nucleic acids. Cells containing the highest and the lowest concentrations of ethidium were collected, as the fluorescence intensity of the mutant cells can be distinguished by FACS. TraDIS was then applied to determine the genomic locations of transposon insertion within the collected mutants. AdeABC, AdeIJK, and AmvA efflux proteins were identified as the major ethidium efflux systems in A. baumannii, and a new transcriptional regulator that controls amvA expression was identified. Another aspect of this work focused on clinical strains of A. baumannii and Klebsiella pneumoniae isolated from a respiratory tract infection from a single patient. Co-culture studies of these two pathogens showed complex, multifaceted interactions that were dependent on their growth state and media. Metabolic profiling showed that the two pathogens had very different carbon utilization capabilities, and cross-feeding studies indicated that A. baumannii was able to utilize a secreted metabolite from K. pneumoniae as a sole carbon source. Transcriptomic analysis of the two strains co-grown in a biofilm identified significant changes in the expression levels of genes encoding surface structure biogenesis, amino acid catabolism and transport, as well as biofilm formation. Overall, this thesis provides new insight into the function and regulation of a novel class of multidrug efflux pumps, enabled the development of a new approach for identifying drug efflux pumps and their regulators, and provides a first look at the molecular and physiological interactions of two co-isolated pathogens.