Acinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression. / UOIT

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes. / UOIT

Acinetobacter baumannii is an important nosocomial pathogen characterized by the ability to acquire and develop complex resistance to antimicrobial agents. This capability is caused by eflux systems removing molecules of antibiotics from bacterial intracellular space. AdeABC is an RND-type chromosomal eflux system specific for A. baumannii which has a broad substrate spectrum. In this work, we focused on functional analysis of AdeABC to define its role in the resistance development to aminoglycosides in genetically different strains. We studied a set of 15 epidemiologically and genotypically well characterized strains of A. baumannii which were fully susceptible to aminoglycosides and other antibiotics primarily effective against this species. We determined genotyp of AdeABC for these strains and performed a selection for resistant variants in the presence of netilmicin. Using real-time qRT-PCR we compared the expression of the transporter gene adeB in originally sensitive strains and selected variants. The obtained results confirmed that the increased expression of AdeABC significantly reduces susceptibility to aminoglycosides and other antibiotics. The results also suggest that the efflux system provides a significant selective advantage for nosocomial strains of A. baumannii.

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