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Journal of Clinical Microbiology, January 2004, p. 269-275, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.269-275.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effects of Inoculum and ß-Lactamase Activity in AmpC- and Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli and Klebsiella pneumoniae Clinical Isolates Tested by Using NCCLS ESBL Methodology

Johnson & Johnson Pharmaceutical Research and Development, LLC, Raritan, New Jersey 08869

Received 9 July 2003/ Returned for modification 12 September 2003/ Accepted 15 October 2003

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum ß-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 ß-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 ß-lactamases gave consistent MIC results with inocula of 10⁵ and 10⁶ 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 ß-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.

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