MUTAGENESIS AND SPECTROSCOPIC STUDIES OF MYCOBACTERIUM TUBERCULOSIS STEROL 14ALPHA DEMETHYLASE.

P450s are heme containing enzymes which affect oxidation of substrates via catalytic intermediates having transient lifetimes. These oxidative catalytic intermediates are formed by a sequential interplay of electrons and protons at the active site of the enzyme bearing molecular dioxygen. The proton transfer to the active site from bulk solvent is coordinated by an “acid-alcohol” pair of active site residues which are conserved in all P450s. Sterol 14α-demethylases (CYP51) are P450 enzymes which catalyze oxidative deformylation of lanosterol in the cholesterol/ergosterol biosynthetic pathway. Both cholesterol and ergosterol are important regulators of membrane fluidity. CYP51 differs from other P450s in that the acid in the acid-alcohol pair in the active site is replaced by a His residue. This enzyme is present in tuberculosis (TB) causing pathogen Mycobacterium tuberculosis (Mtb). This finding was significant for primarily two reasons. The first one being the baffling presence of CYP51 in Mtb, as Mtb is not known to have any endogenous sterol biosynthetic pathways. The second being that CYP51 is a validated drug target in treating fungal infections. Thus given the global resurgence of multidrug resistant strains of Mtb and the deadly coexsistence of Mtb in immunocompromised HIV patients, CYP51 may be an ideal drug target for new generation of antimycobacterial drugs. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme. The Mtb CYP51 enzyme was chosen to study the proton transfer pathways in the active site based on the outcome of explicit solvent molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations performed in our laboratory. Based on these calculations of CYP51 catalysis, Glu173 was implicated to be the proton source. Proton transfer to the active site occurred by a coordinated shuttling via four water molecules, His259 and Thr260. To experimentally verify the roles of Glu173, His259 and Thr260 they were mutated to alanine and biophysically characterized. Ferredoxin, an accessory protein required to shuttle electrons from NADPH to the CYP51 active site for catalysis, was also cloned using ligation independent cloning. We were successfully able to reconstitute the electron transport chain for CYP51. The mutants were found to differentially bind type I and type II enzymes. Based on biophysical characterization, Thr260 can be implicated to have a role in modulating the spin state of the enzyme

International Nosocomial Infection Control Consortium report, data summary of 50 countries for 2010-2015: Device-associated module

•We report INICC device-associated module data of 50 countries from 2010-2015.•We collected prospective data from 861,284 patients in 703 ICUs for 3,506,562 days.•DA-HAI rates and bacterial resistance were higher in the INICC ICUs than in CDC-NHSN's.•Device utilization ratio in the INICC ICUs was similar to CDC-NHSN's. Background: We report the results of International Nosocomial Infection Control Consortium (INICC) surveillance study from January 2010-December 2015 in 703 intensive care units (ICUs) in Latin America, Europe, Eastern Mediterranean, Southeast Asia, and Western Pacific. Methods: During the 6-year study period, using Centers for Disease Control and Prevention National Healthcare Safety Network (CDC-NHSN) definitions for device-associated health care-associated infection (DA-HAI), we collected prospective data from 861,284 patients hospitalized in INICC hospital ICUs for an aggregate of 3,506,562 days. Results: Although device use in INICC ICUs was similar to that reported from CDC-NHSN ICUs, DA-HAI rates were higher in the INICC ICUs: in the INICC medical-surgical ICUs, the pooled rate of central line-associated bloodstream infection, 4.1 per 1,000 central line-days, was nearly 5-fold higher than the 0.8 per 1,000 central line-days reported from comparable US ICUs, the overall rate of ventilator-associated pneumonia was also higher, 13.1 versus 0.9 per 1,000 ventilator-days, as was the rate of catheter-associated urinary tract infection, 5.07 versus 1.7 per 1,000 catheter-days. From blood cultures samples, frequencies of resistance of Pseudomonas isolates to amikacin (29.87% vs 10%) and to imipenem (44.3% vs 26.1%), and of Klebsiella pneumoniae isolates to ceftazidime (73.2% vs 28.8%) and to imipenem (43.27% vs 12.8%) were also higher in the INICC ICUs compared with CDC-NHSN ICUs. Conclusions: Although DA-HAIs in INICC ICU patients continue to be higher than the rates reported in CDC-NSHN ICUs representing the developed world, we have observed a significant trend toward the reduction of DA-HAI rates in INICC ICUs as shown in each international report. It is INICC's main goal to continue facilitating education, training, and basic and cost-effective tools and resources, such as standardized forms and an online platform, to tackle this problem effectively and systematically