Comparison of nine phenotypic methods for detection of extended-spectrum beta-lactamase production by Enterobacteriaceae

Abstract

The detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is of importance for infection control and epidemiological surveillance. We aimed to compare phenotypic methods available in the routine laboratory and to evaluate two-step strategies using these methods for the detection of ESBL-positive Enterobacteriaceae. Two methods used for routine susceptibility testing (Vitek2 and disk diffusion methods) and seven methods designed for the detection of ESBL production (ESBL Etests, combination disks, double-disk synergy [DDS] methods on Mueller-Hinton [MH] agar and cloxacillin-containing MH agar, and the Cica-Beta test) were tested against 107 strains of Enterobacteriaceae not susceptible to extended-spectrum cephalosporins. All strains were screened for the presence of acquired ESBL-encoding genes by PCR, and the PCR result was considered the gold standard for evaluation of the other test methods. Among the 107 strains, 52 (49%) were ESBL positive. With Vitek2, sensitivities were the highest when using extended cards (73% to 79%), but 25% to 31% of the strains yielded indeterminate results. For the disk diffusion method, sensitivities were the highest (96%) when testing at least cefotaxime, cefepime, and a third compound (ceftazidime, cefpodoxime, or aztreonam). For the specific methods, specificities ranged from 62% (ceftazidime ESBL Etest) to 100% (DDS using a disk spacing of 20 mm). When a method designed for ESBL detection was used on strains considered ESBL negative or with an indeterminate result by a first routine susceptibility method, sensitivities reached 100% for a majority of combinations. In conclusion, two-step strategies using phenotypic methods available in most clinical laboratories may reach a sensitivity of 100% for ESBL detection among a large panel of species, including AmpC producers, providing a sensible choice of tests.

Fig. 1.

Examples of positive double-disk synergy tests between a disk containing clavulanic acid (Cl) and a disk containing aztreonam or an extended-spectrum cephalosporin (3G). The inhibition zone around the 3G disk is enhanced, highly suggesting the production of ESBL.

Fig. 2.

Sensitivity and specificity of tests selected on the basis of the highest sensitivity obtained among each of the nine methods.

Fig. 3.

Assessment of gains from each two-step strategy for all 107 Enterobacteriaceae isolates (panels 1 and 2) and only the group 1 and 2 Enterobacteriaceae (panels 3 and 4) by using likelihood ratio graphs. Symbols: ×, the result with the first routine test, i.e., double-disk synergy test for graphs 1 and 3 or Vitek2 for graphs 2 and 4; ●, double-disk synergy test on MH agar; ○, double-disk synergy test on MH agar plus cloxacillin; ■, ESBL Etest on MH agar; □, ESBL Etest on MH agar plus cloxacillin; ◆, combined disk on MH agar; ◇, combined disk on MH agar plus cloxacillin; ▴, Cica-Beta test.