Detection of CTX-M-type extended-spectrum beta-lactamase (ESBLs) by testing with MicroScan overnight and ESBL confirmation panels.

CTX-M extended-spectrum beta-lactamases (ESBLs) have emerged as the most common type of ESBL globally, their incidence easily surpassing those of SHV and TEM ESBLs in most locales. This study compared the performance of two MicroScan dried panels with CLSI reference broth microdilution and disk diffusion methods on a collection of genetically characterized ESBL-producing isolates. These included 64 Enterobacteriaceae isolates that produced CTX-M8, -14, -15, or -16 according to PCR and sequencing of the bla gene, 17 isolates that produced a SHV or TEM ESBL, and 19 that produced both CTX-M and SHV ESBLs. Each isolate was tested by a frozen reference microdilution panel, the MicroScan ESbetaL plus confirmation panel, and a routine dried panel containing streamlined ESBL confirmation dilutions (MicroScan Neg MIC panel type 32) that included cefotaxime and ceftazidime tested alone or with a fixed concentration of 4 microg/ml of clavulanate. Each isolate was also tested by the standard CLSI double-disk confirmation tests. The disk diffusion method detected all ESBL-producing isolates, the frozen reference panel detected 90% of isolates (10 out of 100 could not be analyzed because of off-scale MICs that exceeded the clavulanate combination concentrations in the panel), the ESbetaL plus panel detected 98% (1 missed and 1 off scale), and the streamlined ESBL panel detected 95% (5 off scale). Very high MICs for a few strains that produced SHV or both CTX-M and SHV ESBLs precluded noting the required three twofold-dilution differences with clavulanate needed to confirm an ESBL primarily in the reference panel and the Neg type 32 panel.

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.