Unreliable Extended-Spectrum β-Lactamase Detection in the Presence of Plasmid-Mediated AmpC in Escherichia coli Clinical Isolates

Abstract

The emergence of extended-spectrum ␤-lactamase (ESBL) and plasmid-mediated AmpC (pAmpC) enzymes in Escherichia coli raises concern regarding accurate laboratory detection and interpretation of susceptibility testing results. Twenty-six cefpodoxime ESBL screen-positive, cefoxitin-resistant E. coli clinical isolates were subjected to clavulanate ESBL confirmatory testing employing disk augmentation, Etest, and the BD Phoenix NMC/ID-132 panel. Phenotypic pAmpC production was assessed by boronic acid disk augmentation. ESBL and pAmpC genes were detected by gene amplification and sequencing. ESBL genes (SHV and/or CTX-M-type genes) were detected in only 7/26 ESBL screen-positive isolates. Of 23 aminophenylboronic acid screen-positive isolates, pAmpC genes were detected in 20 (CMY-2 or FOX-5 genes). High incidences of false-positive ESBL confirmatory results were observed for both clavulanate disk augmentation (9/19) and BD Phoenix (5/19). All were associated with the presence of pAmpC genes with or without TEM-1. Etest performed poorly, as the majority of interpretations were nondeterminable. In addition, false-negative ESBL confirmatory results were observed in isolates possessing concomitant ESBL and pAmpC genes for Etest (four of five), BD Phoenix (three of five), and disk augmentation (one of five). The results indicate poor performance of currently employed ESBL confirmatory methods in the setting of concomitant pAmpC. Some isolates with pAmpC and ESBL genes fell within the susceptible category to extended-spectrum cephalosporins, raising concern over currently employed breakpoints.Ambler class A extended-spectrum ␤-lactamase (ESBL) genes in Escherichia coli are well documented. Possible ESBL production has been reported to occur in up to 9% of European E. coli isolates (15). In addition, chromosomal Ambler class C AmpC genes have been mobilized and are now being disseminated on plasmids, reminiscent of the early dissemination and evolution of ESBLs (11). Increasingly, reports document the detection of plasmid-mediated AmpC resistance (pAmpC) in E. coli (4,6,12). Data from the SENTRY antimicrobial surveillance program for North America show that 19/65 ESBL screen-positive E. coli isolates harbored pAmpC (5).The ESBL hydrolytic spectrum includes the oxyimino-cephalosporins and monobactams but not 7-␣-methoxy-cephalosporins (cephamycins) and is inhibited by clavulanate, sulbactam, and tazobactam. The broader spectrum of the AmpC enzymes includes the cephamycins, and AmpC enzymes are not inhibited by clavulanate, sulbactam, or tazobactam. The Clinical and Laboratory Standards Institute (CLSI) recommends that antimicrobial susceptibility testing include screening for ESBL production in E. coli, employing cefpodoxime, ceftazidime, aztreonam, cefotaxime, or ceftriaxone, followed by phenotypic confirmation with clavulanate (3).ESBL screening results are reviewed to select for those organisms that need phenotypic ESBL confirmation, as recommended by CLSI, and results are issued with the aim of preventing inappropriate u...

Rapid molecular detection of extended-spectrum β-lactamase gene variants with a novel ligation-mediated real-time PCR

“…Discrepancy testing of CDT-positive/ LM-PCR-negative isolates confirmed all six LM-PCRnegative results. In three of the six isolates, an AmpC gene was detected (two Enterobacter cloacae and one Escherichia coli combined with a non-ESBL TEM), a b-lactamase hyperproducer with similar patterns of resistance to extendedspectrum cephalosporins such as ESBL that is able to generate false-positive results in phenotypic ESBL tests by producing an increase in the inhibition zone with the combination cephalosporin-clavulanate disc Robberts et al, 2009). …”

“…Wu et al (2001) found that decreased outermembrane permeability, due to loss of outer-membrane protein K35 (OmpK35), and the hydrolytic effect of TEM-1 can increase the MIC of cefotaxim slightly. The addition of clavulanic acid inhibits TEM-1 b-lactamase production and reduces the MIC, causing augmentation of the inhibition zone diameter, resulting in a false-positive ESBL test result (Beceiro et al, 2011;Robberts et al, 2009). …”

Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

“…It is also of note that although the CLSI ESBL confirmatory test showed false-positive results among four non-ESBL-producing isolates, the modified test was negative among all non-ESBL producers of the study. Accordingly, previous surveys have shown that the CLSI ESBL confirmatory test may give a few false-positive results among non-ESBL-, AmpC-producing Enterobacteriaceae, while a modification of the test using BA did not give any false-positive results in this bacterial population (16,38). In addition, among isolates coproducing AmpCs or carbapenems, the proposed modified test provided considerably higher increases in the inhibitory zone diameters around disks containing CAZ or CTX, allowing an easy and straightforward interpretation of the phenotypic test.…”

Extended-Spectrum-β-Lactamase, AmpC, and Carbapenemase Issues

“…Inconclusive cefotaxime-and ceftazidime-based confirmatory tests should be reported as such (and not as ESBL negative) to avoid the risk of reporting false susceptibility to a more potent cephalosporin such as cefepime. Etest confirmatory strips are convenient but expensive and yield more inconclusive results than CLSI tests due to a more restricted concentration range (11,40). Until proven otherwise, confirmed ESBL-producing isolates should be reported as resistant to all penicillins, cephalosporins, and aztreonam (8) to avoid therapy with antibiotics that may be clinically ineffective (6,20,32,38).…”