Browsing by Subject "Drug Resistance, Bacterial"
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Item Characterization of Mycobacterium tuberculosis Cor, a Protein Essential for Carbon Monoxide Resistance and Pathogenesis(2015-01-28) Zacharia, Vineetha Mariam; Hendrixson, David R.; Alto, Neal; McDonald, Jeffrey; Shiloh, MichaelTuberculosis, caused by Mycobacterium tuberculosis, is global health burden as it remains one of the most devastating human infectious diseases causing two million deaths annually and latently infecting a third of the world's population. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, cor) leads to marked CO sensitivity. Heterologous expression of Cor and Cor homologue from Thermotoga maritima (TM0160) in Escherichia coli rescued it from CO toxicity, suggesting a conserved function across diverse microbial species. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Evolutionary modeling suggested that Cor forms an active site, thus we predicted that Cor has enzymatic activity. To determine potential Cor enzymatic activity, we profiled the mycobacterial metabolome using liquid-chromatography mass spectrometry and, in vitro, monitored metabolite fluctuations in the presence and absence of recombinant Cor. Our activity-based metabolomic profiling data showed the accumulation of phosphatidic acid and multiple phospholipids in the presence of Cor, indicating its potential role in catalyzing a reaction involved in phospholipid biosynthesis. Our in vivo metabolomic analysis of a Cor mutant strain showed that in the presence of CO, levels of dihydroxyacetone phosphate is increased, whereas levels of glycerol-3-phosphate is reduced, which is required for phospholipid biosynthesis. Furthermore, we identified key metabolic enzymes in Mtb that physically interact with Cor using bioinformatics and immunoprecipitation techniques. Specifically, Cor interacted with glycerol-3-phosphate dehydrogenase 2, an enzyme that catalyzes the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism, which may have a critical role in phospholipid biosynthesis.Item Impact of Urine pH on Antibiotic Response in Women with Uropathogenic Eschericia [sic] coli Recurrent Urinary Tract Infections(2019-01-22) Chavez, Jacqueline; Christie, Alana; Zimmern, Philippe E.INTRODUCTION AND OBJECTIVES: As early as Brumfitt in 1948, the relationship between the effectiveness of certain uro-antibiotics and urinary pH levels has been considered.1 Some antibiotics are more effective at a urine pH range 5-6, whereas others work better at a more alkaline (urine pH 7-8) range. We compared the urine pH of women infected with E. coli to their antibiotic treatment response. METHODS: An IRB-approved, prospectively maintained database of well-characterized women with antibiotic-refractory recurrent urinary tract infections (RUTI) managed with electrofulguration (F) at a tertiary care center was reviewed. Included were women with at least 6 months follow-up post-F, an electronic medical record (EMR) documenting urine pH value at the time of each urine culture, and at least one E. coli positive urine culture. Total number of urine cultures post-F, urine pH variability, antibiotics prescribed, and the interval (months) between antibiotic administration and another UTI episode were reviewed. RESULTS: From 2006-2016 23 women met study criteria, with mean follow-up of 2 (1-9) years and mean age of 66 (28-92) years. Total number of urine cultures was 181, including 93 positive (I), 88 negative (NI), and 54 with E. coli. The average number of urine cultures per patient was 7 ± 3.8 (2-16). Median urine pH observed was 6, with no difference between I, NI, or E. coli urine cultures. There was no change in urine pH with aging. Six individuals were prescribed antibiotics for which pH has not been shown to change efficacy, 10 in whom urine pH aligned with the reported best efficacy range for their prescribed antibiotic, and 7 whom urine pH was not in the ideal antibiotic pH range. Mean interval time between first and second positive urine culture was longer for those with the appropriate urine pH for the prescribed antibiotic (26 months, 2-63) compared to those with a mismatch between urine pH and optimal pH range for their antibiotic (18 months, 1-33). CONCLUSION: This observational study explores the possible link between the urine pH of a woman with RUTIs and her response to antibiotic treatment administered without taking her urine pH into account. Future studies are needed to determine if an individual's urine pH needs to match the optimal pH range of a prescribed antibiotic to result in maximum therapeutic efficacy.Item [News](1981-08-04) Williams, AnnItem Swine, salad, surfers and you: is the post-antibiotic era upon us?(2018-11-30) Greenberg, DavidItem Using Gene Overexpression as a Potential Method to Elucidate Antibiotic Resistance Mechanisms in Mycobacteria(2015-03-31) Bacalao, Maria Alexandra; Gumbo, Tawanda; Van Oers, Nicolai S. C.; Kant, Shashi; Wakeland, Edward K.Mycobacterium Tuberculosis remains a major public health threat, with 9 million new cases and 1.5 million deaths in 2013. Also concerning is the rise of antbiotic resistance, leading to the development of Multiple Drug Resistant (MDR) and Extensively Drug Resistance (XDR) Mycobacterium tuberculosis strains. The development of these strains is multifactorial involving both extrinsic factors and intrinsic factors. Extrinsic factors include treatment noncompliance and a delay in diagnosis with existing drug sensitivity methods. Intrinsic factors include heterogeneity in the bacterial population and localization in cavitations and other areas where antibiotic penetration is difficult. Our study analyzed the potential role of a potential mechanism of antibiotic resistance, overexpression of existing wild type M. tuberculosis genes involved in mycobacterial antibiotic defense. We cloned seven M. tuberculosis wild type genes using PCR and the subcloned those constructs into E. Coli. Further subcloning steps placed these genes into shuttle vectors that allowed for transformation into M tuberculosis and M. smegmatis. The M. Smegmatis mutant overexpressing the GyrA gene was selected for dose-response studies to establish whether GyrA overexpression led to increased fluoroquinolone resistance. Two M. smegmatis GyrA mutant and one wild type strain were incubated with increasing concentration of Moxifloxacin. Initial dose-response studies did not show yield an adequate dose-response curves under the study conditions. However, the MIC was higher for the GyrA mutant strains than for wild type M. smegmatis, showing that these GyrA mutants were likely more resistant than wild type and that the role of gene overexpression in M. tuberculosis antibiotic resistance merits further study.