Mechanisms of Regulation and Resistance for Plasmid-Encoded ampC Beta-Lactamase Genes

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Reisbig, Mark Devoy
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BETA-lactam antibiotics are the largest class of antimicrobial drugs and the most frequently prescribed world wide. As the use of these agents increased so did the evolution of bacterial BETA-lactam resistance. BETA-lactamases are collectively the most commonly identified BETA-lactam resistance mechanism. The class C or, group 1 BETA- lactamases, can be chromosomal or plasmid-encoded. High-level expression of these genes results in resistance to almost all classes of BETA-lactam antibiotics. The mechanisms of high-level expression for the chromosomal AmpC genes from several organisms have been elucidated, whereas prior to data published from the work presented in this dissertation, the mechanisms of expression from plasmid-encoded ampC genes were unknown. The goal of the research presented in this dissertation was to identify the mechanisms of resistance and factors that influence the overall levels of plasmid-encoded ampC expression. It was hypothesized that plasmid-encoded ampC expression is influenced by the genetic organization of the resistance gene, increased gene copy number, and factors present in their genetic context. Data presented in this dissertation resulted from investigations of the plasmid-encoded ampC BETA-lactamase genes at the RNA level, which is a novel approach in the field of BETA-lactamases. Investigations described in this dissertation revealed 3 different mechanisms of expression for 3 different plasmid- encoded ampC genes. blaACT-i was determined to be inducible and expressed at increased levels constitutively due to a mutation in the-10 promoter element. B/aMiR-I was expressed from a novel hybrid promoter, prB that was independent of any AmpR influence. blacuv-i had two different transcriptional start sites , the primary start site driven from a promoter found within an upstream ISZscpl-like sequence insertion element and the secondary start site driven by a putative promoter hypothesized to be under the control of global regulators involved in cell metabolism. RNA expression levels were correlated with gene copy number and BETA-lactam MICs for the organisms from which the plasmid-encoded ampC genes were expressed. It was determined from these studies that the overall levels of plasmid-encoded ampC expression correlate well with BETA-lactam MICs and promoter strength rather than gene copy number. Because the goal of this research is to understand the relationship between plasmid-encoded ampC expression and therapeutic outcome, a pilot surveillance study was performed to evaluate the percentage of plasmid-encoded ampC producers amongst cefoxitin non-susceptible E. coli isolated from urine cultures at CUMC. The results of this study revealed that 55% of the cefoxitin resistant isolates produced a CMY-like plasmid-encoded ampC gene indicating a high percentage of these organisms amongst cefoxitin resistance E. coli in the local community. Taken together, the data presented in this dissertation indicate that plasmid- encoded ampC gene expression is much more complex than originally believed by many in the field of BETA-lactamases and that plasmid-encoded AmpC BETA-lactamases are a threat to available therapeutic options for patients infected with these organisms.
Creighton University
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