Characterization of the blaKPC gene across three different bacterial genera
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Initially discovered in 1996, Klebsiella pneumoniae Carbapenemase (KPC) encodes for an enzyme which can hydrolyze the entire β-lactam drug class. Predominantly found in K. pneumoniae and to a lesser extent other species of Gram-negative bacteria, KPC is typically encoded on a Tn-3 like transposon found on plasmids which harbor additional resistance genes. Infections with KPC producing bacteria are associated with increased morbidity and mortality due to difficulties in detection of organisms that produce KPC in addition to having few effective therapeutic agents to treat such infections.|The impact KPC-producing pathogens have on the outcome of antibiotic therapy and the ability to detect the enzyme in susceptibility assays relies in part on the levels of protein product produced by the organism. Two promoters have been identified upstream of the KPC structural gene. However, upstream regions capable of binding potential transcription factors have not been identified. In addition, eight different deletion isotypes have been described involving deletions within the KPC promoter ranging from no deletion to a 256 bp deletion. Previous research has shown that a 99 bp and a 188 bp deletion in the promoter lead to an increase in gene expression but the effects on the level KPC protein production have yet to be evaluated. In this study, we characterize the RNA and protein production of KPC in both K. pneumoniae and E. coli isolates with and without deletions in the KPC promoter. Additionally, we evaluated the KPC promoter region for possible DNA binding proteins using electrophoretic mobility shift assays and whole cell lysates from three different genera, K. pneumoniae, E. coli, and Enterobacter cloacae. Sequence homology of the distal promoter to the σ70 sequence, suggest that the distal promoter was responsible for driving gene expression. Additionally, factors which regulate initiation of transcription must bind upstream of the distal promoter of the KPC gene. Therefore, we hypothesized that the distal promoter was the dominant promoter driving KPC RNA and protein production in clinical isolates of K. pneumoniae, E. coli and E. cloacae.|A series of eight clones with systematic deletions to the promoter region were utilized to determine which promoter was dominant. In addition, clinical isolates of K. pneumoniae and E. coli possessing the full-length or 99 base pair deletion in the promoter were evaluated and compared for RNA and protein production levels. The E. coli isolates were of various sequence types (ST) including ST131, ST964, ST2521, ST648, ST372, and ST404. Expression and protein analysis were measured by real-time reverse transcription PCR and western blot analysis. Carbapenem MICs were measured using Etest®, agar and microbroth dilutions. Gel-shift mobility assays were performed using whole cell lysates from K. pneumoniae, E. coli and E. cloacae and a 50 base pair (bp) fluorescently labeled probe with DNA sequence specific to a region located eight bp upstream of the distal promoter.|The deletion clones identified the distal promoter as the only promoter responsible for RNA and protein production resulting in carbapenem MICs at or above the resistant breakpoint. Interestingly, when E. coli ST131 isolates were evaluated for RNA and protein production, these isolates had an increase in RNA expression that did not correspond to increased levels of KPC protein production compared to non-ST131 isolates. Comparisons of clinical isolates with a deletion of 99 bps in the promoter vs. isolates with the full-length promoter showed the RNA and protein levels varied regardless of the presence of the 99 bp deletion. Very little correlation was observed among the isolates with respect to KPC protein production and carbapenem MICs. Although some isolates had increased protein production that correlated with increased carbapenem MICs, many isolates had elevated carbapenem MICs in the presence of very low levels of KPC protein production. These data suggest that additional resistance mechanisms contribute to the carbapenem MICs and would be of interest of further study. Finally, using whole cell lysates from three different genera, EMSA identified a single band in the presence of K. pneumoniae and E. cloacae protein lysates while the E. coli isolates resulted in two shifted bands.|Taken together, the data presented in this thesis work shows that the distal promoter of the KPC gene is required to drive expression resulting in protein production that in the deletion clones correlates with carbapenem MICs that reflect a resistant phenotype. Additionally, regions within the upstream sequence of the distal promoter interact with DNA binding proteins. However, in clinical isolates the correlation is not clear with no decisive impact with respect to deletions within the full-length promoter or sequence type of the E. coli expressing the gene.
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