Search

For this study, we utilized class-I and class-II preQ 1 -sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ 1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands' in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

A rapid microarray assay, Nanosphere Verigene® Gram-negative blood culture test (BC-GN), detects four Gram-negative species, four Gram-negative genera, and six resistance genes directly from positive blood culture samples, shortening the time from Gram stain to pathogen and resistance-gene identification. The purpose of this study was to determine the impact of the BC-GN paired with an antimicrobial stewardship intervention on antimicrobial and clinical outcomes. Patients with Gram-negative bacteremia were compared before ( n = 456) and after ( n = 421) BC-GN implementation. The primary objective was to compare time from Gram stain to antimicrobial switch pre- and post-implementation. Time from Gram stain to effective treatment, in-hospital mortality, and hospital length of stay were also compared. The number and type of antimicrobial switches were similar between groups. Median (IQR) time from Gram stain to antimicrobial switch was significantly decreased in the post group, 28.6 (8.6-56.9) h vs 44.1 (18.9-64.6) h, p = 0.004. In patients on ineffective antimicrobial therapy at the time of result, median time to effective therapy was lower in the post group, 8.8 (5.5-18.4) h vs 24.5 (4.9-44.3) h, p = 0.034. Median (IQR) hospital length of stay was also decreased in the post group, 7 (5-15) days vs 9 (4.5-21) days, p = 0.001. The rate of in-hospital mortality was similar between groups, 11.6% (pre) vs 11.4% (post), p = 0.87. Rapid microarray testing on blood cultures combined with active antimicrobial stewardship intervention was associated with decreased time to antimicrobial switch, time to effective therapy, and hospital length of stay. [ABSTRACT FROM AUTHOR]