Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum ␤-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing ␤-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no ␤-lactamases gave consistent MIC results with inocula of 10 5 and 10 6 CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total ␤-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.Extended-spectrum ␤-lactamases (ESBLs) are considered one of the most important resistance mechanisms for penicillins and cephalosporins when these enzymes are produced in Escherichia coli and Klebsiella spp. (7). The genes encoding these enzymes are most often carried by multidrug-resistant plasmids and are capable of being readily transferred among different species of the family Enterobacteriaceae (2). Infections caused by ESBL-producing pathogens may not be responsive to treatment by most penicillins and cephalosporins (32). Hence, their appearance in a hospital setting creates a situation in which ESBL-producing organisms should be identified quickly so that appropriate antibiotic usage and containment measures can be implemented (21).Clinical microbiologists have devised a number of testing strategies based on phenotypic testing to identify putative ESBL-producing organisms. One testing strategy is the recently adopted set of NCCLS guidelines for E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca isolates with elevated cephalosporin MICs (16). In the protocol for MIC testing, strains with cefotaxime, ceftazidime, ceftriaxone, or aztreonam MICs of Ն2 g/ml or cefpodoxime MICs of Ն8 g/m...

The Q Q-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His-and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A î for the protein^detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K d ) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 W WM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.