Mycobacterium hassiacum: a thermophilic Mycobacterium species to demonstrate thermal disinfection of medical devices.

OBJECTIVE: Reprocessing reusable medical devices is crucial in the healthcare industry. To ensure patient safety, strict standards are dictated to validate thermal disinfection in automated washer-disinfectors. The United States Food and Drug Administration (FDA) has specific recommendations on the vegetative bacterial challenge but comparatively vague guidance on the use of a thermophilic Mycobacterium strain for thermal disinfection studies. This study aims to compare thermal resistance of Mycobacterium hassiacum and Mycobacterium terrae and determine which strain is suitable for medical device thermal disinfection validation testing in automated washer-disinfectors. RESULTS: Thermal resistance was demonstrated in vitro by calculating D-values for each strain at different exposure temperatures, and correlated with actual in situ processing conditions. M. terrae was completely killed (> 7 log reduction) at temperatures above 68 °C, with D-values between 46.6 and 27.8 s at temperatures between 59.5 and 67.2 °C. M. hassiacum was completely killed (> 8 log reduction) at temperatures above 75 °C, with D-values between 82.1 and 21.7 s at temperatures ranging between 69.2 and 73.6 °C. In vitro results were correlated in a washer-disinfector performance validation setup.

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference.

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.

Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids.

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS activity (IC(50)=0.16-0.58mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition (IC(50)=0.060-0.121mM). An intact purine ring with anionic character favors inhibition. In general, methylation of the purine does not significantly affect inhibition.