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Elucidation of the Molecular Mechanisms That Contribute to Variation in Susceptibility Testing Show the Need for Alternative Detection Methods of KPC-producing Gram-negative Bacteria
No full textPathogens that produce the Klebsiella pneumoniae carbapenemase (KPC) have become a major threat to the global health care system. Patients infected with KPC-producing pathogens have a higher incidence of morbidity and mortality, as these infections have been associated with high levels of β- lactam resistance, in addition to other antibiotic drug classes. This, in addition to the lack of novel antimicrobials with activity against Gram-negative bacteria, forces physicians to treat infections caused by KPC-producing pathogens with potentially toxic antibiotics, such as the aminoglycosides and polymyxins. The rapid spread of blaKPC into multiple genera of Gram-negative bacteria, the lack of effective treatment regimens, and the difficulty of identifying these pathogens using current detection methods in the clinical laboratories are the biggest challenges the KPC-producing organisms pose.
Although in vitro testing indicates that the KPC β-lactamase can efficiently hydrolyze the carbapenems, multiple studies have reported variability in susceptibility testing of KPC-producing clinical isolates. The first hypothesis tested in this dissertation was that the variation observed during susceptibility testing was due to variations in gene expression and/or gene copy number. Variation in KPC enzyme production could contribute to the variability observed during susceptibility testing. However, the individual influence of the KPC β-lactamase cannot be determined in clinical isolates since all of the organisms evaluated to date produce multiple β-lactam resistance mechanisms. Little is known about the role gene expression plays in KPC-mediated resistance or its affect on KPC production during susceptibility testing. Therefore, I sought to elucidate the molecular mechanisms that contribute to the variation in susceptibility of Gram-negative bacteria harboring blaKPC.
To address this problem, we created a panel of four different genera of Gram-negative bacteria that expressed the blaKPC gene without the influence of other resistance mechanisms. Using this panel, we evaluated the relationship between blaKPC gene expression to β-lactam MICs, gene copy number, and protein production in comparison to KPC-producing clinical isolates. These studies identified variation in blaKPC gene copy number, expression and protein production in the clinical isolates compared to their
genus-specific control. These variations in expression and protein production did not always correlate with the β-lactam MICs observed during susceptibility testing. Evaluation of RNA expression identified two novel transcriptional start sites and promoter elements for blaKPC. Subsequent analysis of the
upstream promoter regions using constructed promoter deletion clones demonstrated that the proximal promoter elements were responsible for the β-lactam resistant phenotype.
As the expression and protein data do not elucidate the problems associated with susceptibility testing and the rapid spread of this gene among numerous genera, a second hypothesis was tested: Exposure to sub- inhibitory levels of antibiotics stimulates blaKPC expression and/or contributes to the mobilization of Tn4401 carrying blaKPC. I evaluated the effect of “collateral damage,” or the unintended effects of exposure to antibiotics on transmission and expression of KPC–mediated antimicrobial resistance. Five KPC-producing clinical isolates were exposed to antibiotics from five different drug classes to determine whether they would stimulate blaKPC expression or mobilization of the transposon Tn4401 carrying blaKPC. While no changes in Tn4401 mobility were observed, there were subtle increases in blaKPC gene
expression with gentamicin and tigecycline, both of which are used in the treatment of KPC-producing pathogens. Of note, non-β-lactam drugs influenced blaKPC gene expression more often than the β-lactam drugs, but strain to strain variation occurred.
The third hypothesis was that a high resolution melt assay would be able to detect and differentiate between blaKPC alleles. To circumvent the
difficulties that conventional phenotypic testing can pose an alternative testing algorithm was designed to decrease the turn around time required to identify the presence of the KPC carbapenemase in Gram-negative clinical isolates. To do so, a single tube, PCR-based assay was designed to detect and differentiate between blaKPC gene alleles. Validation of this study involved testing 166 Gram-negative clinical isolates, 66 of which were known to be KPC-positive. This assay demonstrated 100% diagnostic sensitivity and specificity for both blaKPC gene detection and differentiation into blaKPC-2-like and blaKPC-3like allele groups.
Taken together, these data demonstrate the level of complexity by which blaKPC is regulated and the preliminary findings may represent a means of differential expression under various selective pressures. Although the fold differences in blaKPC gene expression are subtle, these differences cannot be ignored. This variation may represent differences in metabolic processes or other compensatory mechanisms that may have been selected for in the patients they infect, where they can be exposed to a variety of different physiological conditions and stressors. It is imperative that we continue to evaluate the complexity of this resistance mechanism in order to better understand how it spreads to susceptible patient populations. As it is apparent that there may be several, strain dependent mechanisms involved in the susceptibility testing variation, the development of molecular assays in the clinical microbiology laboratory may be a superior testing method for the identification of KPCs.ProQuest Traditional Publishing Optionxv, 78 page
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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