Master Theses & Projects (FSCI)

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Characterization of two component systems of Acinetobacter baumannii.
(2014-10-01) Alsaadi, Yasser; Kumar, Ayush
Acinetobacter baumannii is an important opportunistic pathogen of hospital acquired infection, particularly in intensive care units. The emergence and rapid spread of multidrug-resistant A. baumannii strains has become a major health threat worldwide which severely limits the treatment options for this pathogen. This work investigated global mechanisms of antibiotic resistance and virulence of the problematic pathogen A. baumannii, in particular those mediated by two component regulatory systems (TCSs), that typically consist of a membrane bound sensor kinase and a cognate response regulator. Bacterial TCSs play an important role in the regulation of adaptation to different environmental conditions. Five TCSs in A. baumannii have been characterized; however, there are a number of putative two component systems encoded in the genome of A. baumannii that await detailed characterization. Differential expressions of six different TCSs was observed in two clinical isolates of A. baumannii AB030 and AB031, and whole-genome sequencing of both clinical isolates was performed. Data obtained from the comparative whole-genome analysis revealed the presence of an insertion element in the orphan TCSs response regulator A1S_2006 in AB030, mutation in the promoter region and an 1189 DNA insertion element were present in AdeRS system in AB031. The whole-genome sequencing analysis of TCSs operons in AB030 and AB031 also identified sequence polymorphisms that could alter the activities of these TCSs in AB030 and AB031. Finally, we identified A1S_3229_30 an excellent candidate that may act as global regulator of antibiotic and virulence in A. baumannii. The sequence of A1S_3229_30 was highly conserved among the wild-type ATCC17978, AB030 and AB031, and showed 73% identity to the amgRS operon that encodes for the well characterized AmgRS system that confer resistance to aminoglycoside antibiotics and required for the virulence of the problematic pathogen P. aeruginosa.
Cloning and characterization of AdeMNO RND efflux pump of Acinetobacter baumannii
(2010-11-01) Cortez-Cordova, Jenny Lilian; Kumar, Ayush
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.
Impact of environmental conditions on expression levels of resistance-nodulation-division (RND) efflux pumps and outer membrane porins in Acinetobacter baumannii
(2012-09-01) Bazyleu, Andrei; Kumar, Ayush
Acinetobacter baumannii is a globally emerging Gram-negative pathogen accounting for a variety of nosocomial conditions, including pneumonia, meningitis, peritonitis, urinary tract infections and, in particular, wound infections in soldiers. One of the major reasons for A. baumannii success as a human pathogen is attributed to a tremendous potential of this organism for development of the multidrug resistant phenotype (MDR). This organism’s ability to regulate expression of its intrinsic antibiotic resistance determinants, such as Resistance-Nodulation-Cell Division (RND) efflux pumps and outer membrane proteins, is regarded to be amongst the most important strategies in the arsenal of this pathogen. Despite considerable efforts being dedicated in the attempt to understand the underlying mechanisms in the development of the MDR phenotype in A. baumannii, the role of environmental stress factors is often overlooked. Evidence from other Gram-negative pathogens suggests that adaptive responses of the bacterial cell to environmental stress often target the same cellular processes that are exploited by antibiotics, therefore indirectly promoting the emergence of the MDR phenotype. This study shows the impact of a variety of environmental factors, namely iron(III) availability, oxidative stress, salicylate exposure, steady-state osmotic stress, ethanol shock, exposure to different incubation temperatures and exposure to blue light on expression levels of three RND efflux pumps and three outer membrane porins of A. baumannii. In addition, we investigated the difference in expression levels of the selected two-component systems between a type strain of A. baumannii, ATCC 19606, and two clinical isolates with the MDR and biofilm-forming phenotypes. The quantitative-real time polymerase chain reaction analysis revealed that levels of iron(III) in growth medium, oxidative stress and ethanol shock were amongst the most potent factors capable of influencing expression of RND efflux pumps and outer membrane porins.

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Browsing Master Theses & Projects (FSCI) by Subject "Acinetobacter baumannii"