Limited distribution and mechanism of the TetX4 tetracycline resistance enzyme

Tetracyclines,a group of antimicrobial agents targeting protein synthesis,have been extensively used in agricultural,veterinary,and clinic settings[1].The sporadic rise in tetracycline resistance is partially due to the occurrence of efflux pumps and/or ribosome protection[1,2].The growing body of tetracycline-inactivating enzymes(e.g.,TetX(X1 to X4)[3,4]and Tet(47 to 55)[5])represents an alternative mechanism for tetracycline resistance,and threatens the renewed interest of tigecycline as a last-resort defense against lethal infections with carbapenem-resistant Enterobacteriaceae and Acinetobacter species.

Separation of scleroglucan and cell biomass from Sclerotium glucanicum grown in an inexpensive, by-product based medium

Scleroglucan is an extracellular polysaccharide produced by the fungi Sclerotium.Scleroglucan is stable over a broad range of temperatures,pH and salt concentration,thus having great potential in many diverse applications.Common media for scleroglucan production contain expensive components,such as yeast extract,that make the medium cost-ineffective for some industrial applications.Thus we developed a medium which uses Condensed Corn Solubles(CCS),a nutrient-rich byproduct of corn-based ethanol production,to replace expensive components.Methods typically used to recover scleroglucan are also expensive,and can limit commercialization.To evaluate alternative processes for scleroglucan recovery,we prepared scleroglucan in a modified version of Wang’s medium and a CCS-glucose medium.Broth samples were initially subjected to the standard recovery method to develop a complete mass balance,and then we evaluated various treatments to improve scleroglucan recovery.These included heat treatments to lyse cells,using different alcohol precipitants,freezing or refrigerating before recovery,and diluting broth to enhance cell separation.The CCS medium produced 14.2 g/L scleroglucan,compared to 10.1 g/L in the modified Wang’s medium.Based on the standard recovery protocol,we determined that 96%of the scleroglucan was recovered from the initial centrifugation and precipitation.Washing the cell pellet with water and recentrifuging only recovered a minimal amount of scleroglucan,and thus it could be eliminated from protocol to save energy and costs.Lysing cells by boiling or autoclaving did not release more scleroglucan than un-heated samples,and we also found no statistical difference between ethanol,isopopanol,and methanol as scleroglucan precipitants.Refrigerating the broth prior to scleroglucan recovery had no significant effect,while freezing actually decreased scleroglucan recovery.Initially diluting the broth by 0.50 or 0.34 resulted in the greatest scleroglucan recovery,while higher or lower dilutions decreased recovery.The optimum protocol for scleroglucan recovery was a 0.50 dilution of broth prior to centrifugation,no washing of the cell pellet,and use of the least expensive alcohol to precipitate scleroglucan from the supernatant.