Browsing by Subject "Drug resistance"
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Item Characterization of HIV-1 Reverse Transcriptase substrate specificity by conformationally sensitive fluorescence(2010-12) Kellinger, Matthew William; Johnson, Kenneth Allen; Dalby, Kevin; Robertus, Jon; Russell, Rick; Zhang, Yan JessieWe have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). The result is a polymerase that is kinetically indistinguishable from the wild-type enzyme, but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding. Using this system, we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding. In doing so, we define the role of induced-fit in nucleotide specificity and mismatch discrimination. Additionally, we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI’s) used to combat HIV infection including 3TC (Lamivudine), FTC (Emtricitabine), and AZT (Zidovudine) for the wild-type polymerase and mutants with clinical resistance to these compounds. Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors. The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change. The apparent Kd (Kd,app) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium. The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association. We show that DNA binding is governed by a two-step process where a fast initial association is followed by a second, slow isomerization that is off the pathway for nucleotide binding and incorporation. Finally, we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase. We find that primer termination with AZT results in DNA that fails to translocate, therefore occluding the next nucleotide from binding. This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP- or pyrophosphate-mediated AZT excision; thereby rescuing productive polymerization. This finding represents the first kinetic measurement of DNA translocation by a polymerase.Item Investigation of antimicrobial drug resistance in bacterial isolates from commercial cattle processing systems: pre and post-harvest food safety issues(Texas Tech University, 2004-12) Fluckey, WadeTo determine the patterns of cross-contamination and antibiotic susceptibility, a total of 60 cattle were shipped to a commercial abattoir, 20 in each of three separate trial periods. The same animals were followed through the process and bacterial isolates were collected from these animals immediately before shipping, at the abattoir after exsanguination, after hide removal and in the cooler. Samples were cultured for Salmonella, non-type specific E. coli, and Enterococcus. Salmonella was identified in 33.9% («=20) of fecal samples and on 37.3% (n=22) of hides prior to shipment. At the abattoir, the proportion of hides from which Salmonella was isolated increased (P < 0.001) to 84.2% (n=48). Generic E. coli was recovered from 40.4 % of preevisceration carcass samples, while Salmonella was recovered from 8.3% and Enterococcus from 58.3% of these carcasses. No Salmonella or generic E. coli were recovered from hotbox carcass samples. Enterococcus however, was recovered from 8.3% of the hotbox carcass samples. Isolates were also tested for antimicrobial drug susceptibility. For generic E. coli 80.3% (n=270) of isolates were resistant to at least one antimicrobial. For Salmonella, 97% (n=101) of the isolates were resistant to at least one antimicrobial however only 4.0% were resistant to two or more. The most common resistance for gramnegative bacteria was to sulfamethoxazole. Antibiotic susceptibility patterns were fairly consistent across sampling points. Enterococcus isolates showed a higher degree of resistance to the antimicrobials tested. Of 279 confirmed Enterococcus all were resistant to at least one antimicrobial. Interestingly, 179 (64.15%) of these isolates were resistant to at least six agents tested. The most common resistance was to chloramphenicol (100%) followed by flavomycin (90.32%), hncomycin (87.81%), tylosin (78.49%), erythromycin (76.34%), tetracycline (58.87%), synercid or quinupristin/dalfopristin (47.67%), bacitracin (17.92), streptomycin (8.96%), ciprofloxacin (1.43%), linezolid (0.72%) and salinomycin (0.36%). Enterococcus spp. were also analyzed for pulsed-field gel electrophoresis profiles from all sampling points. Similar or identical PFGE patterns were found from isolates recovered at the feedlot and in the commercial abattoir. This presents strong evidence that bacterial isolates are being propagated from the feedlot to the processing environment and onto the final processed carcasses.Item The regulation of Vibrio cholerae MDR efflux Pump in a TolC mutant of Escherichia coli(Texas Tech University, 2004-05) Ramasubramian, BhagavathiThe emergence of antibiotic-resistance among bacterial pathogens is becoming a serious threat to human health and welfare. One mechanism by which bacteria become resistant to antibiotics is to remove them via a multiple drug resistant (MDR) efflux pump. The first multidrug resistance pump was described in tumor cells that were resistant to chemotherapeutic agents. More recently, MDRs have been characterized in both Gram-negative and Gram-positive bacteria. Vibrio cholerae, a Gram-negative non-invasive enteric pathogen, is the causative agent of the severe diarrheal disease cholera. The first multi-drug efflux pump of V. cholerae, VceAB, was isolated and characterized by Colmer et al. In that study the authors cloned a 6.6 kb SalI-Hind III fragment of V cholerae into pBR322, pVC2 and demonstrated that this plasmid was capable of providing both V. cholerae and an Escherichia coli to/C mutant with resistance to a variety of toxic compounds and antibiotics. The authors also demonstrated that this 6.6 kb DNA fragment contained two genes, vceA and vceB, which encoded proteins whose amino acid sequences shared a high degree of similarity with the EmrA and EmrB proteins of E.coli, respectively. Since those studies were published, our laboratory has identified the outer membrane component of this efflux pump, OepX, whose gene resides in an operon with vceA and vceB and a regulatory gene vceR, whose product is a member of the TetR/AcrR transcriptional regulator family and which negatively regulates the oepX-vceA-vceB operon. During that study, the authors constructed an in-frame vceA::phoA gene fusion in pVC2 (pi328). In this construct the phoA gene is fused in-frame to the vceA gene at position 753 I have employed this reporter fusion to examine the regulation of the oepX-vceA- vceB (i.e., VceAB MDR) operon. The activity of this gene fusion, measured by alkaline phosphatase assay was found to be up regulated in the tolC mutant strain. The purpose of this study was to decipher the cause of this regulation which seems to be a tolC effect.