Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods

Extended-spectrum beta -lactamases (ESBLs) are enzymes found in gram-negati ve bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standard s (NCCLS) published methods for screening and confirming the presence of ES BLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance E pidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCL S screening criteria for potential ESBL producers (ceftazidime [CAZ] or cef otaxime [CTX] MICs were greater than or equal to2 mug/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect b y disk diffusion (i.e., an increase in CAZ or CTX zone diameters of greater than or equal to5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by greater t han or equal to3 dilutions). For five isolates, a CA effect could not be de termined initially by broth microdilution because of off scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefe pime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed t o show a CA effect by broth microdilution were greater than or equal to 32 mug/ml, suggesting either the presence of an AmpC-type beta -lactamase or p orin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta -lactamase with a pI of greater than or equal to8.3 suggestive of an AmpC-type beta -lactamase; 6 of the 7 isolate s were shown by PCR to contain both ampC-type and bla(OX)A genes. The IEF p rofiles of the remaining 16 isolates showed a variety of beta -lactamase ba nds, all of which had pls of less than or equal to7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla (CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were id entified initially as presumptive ESBL producers were positive for a CA eff ect, while 5.0% contained beta -lactamases that likely masked the CA effect . The remaining 11.5% of the isolates studied contained beta -lactamases th at did not demonstrate a CA effect. An algorithm based on phenotypic analys es is suggested for evaluation of such isolates.