ESBL Testing - Disk Method

DISK METHOD: Screening and Confirmatory Tests for ESBLs in Klebsiella pneumoniae , K. oxytoca , and Escherichia coli and P. mirabilis a

For Use With M2-A9 - Disk Diffusion

Extended-Spectrum-Beta-Lactamases (ESBLs) are newly described enzymes that arise by mutations in genes for common plasmid-mediated-beta-lactamases such as TEM-1, TEM-2 and SHV-1. ESBLs may confer resistance to penicillins, cephalosporins, and aztreonam in clinical isolates of Klebsiella pneumoniae , Klebsiella oxytoca , Escherichia coli , and Proteus mirabilis , and a few other genera of the family Enterobacteriaceae that are usually susceptible to these agents. Some of these strains will show zones of inhibition smaller than the normal susceptible population but below the standard breakpoints for certain extended-spectrum cephalosporins or aztreonam; such strains may be screened for potential ESBL production by using the screening breakpoints listed in the table below. Other strains may test intermediate or resistant by standard breakpoints to one or more of these agents. In all strains with ESBLs, the zone diameters for one or more of the extended-spectrum cephalosporins or aztreonam should increase in the presence of clavulanic acid. For all ESBL-producing strains, the test interpretation should be reported as resistant for all penicillins, cephalosporins, and aztreonam.

Method Initial Screen Test Phenotypic Confirmatory Test
Medium Mueller Hinton Agar Mueller Hinton Agar
Antimicrobial Disk Concentration For K . pneumoniae , K . oxytoca , and E . coli :
Cefpodoxime  10ug or
ceftazidime  30ug or
aztreonam   30ug or
cefotaxime   30ug or
ceftriaxone   30ug

For P . mirabilis a :
Cefpodoxime   10ug or
ceftazidime  30ug or
cefotaxime   30ug

(The use of more than one antimicrobial agent for screening improves the sensitivity of detection.)

Ceftazidime   30ug
Ceftazidime/clavulanic acid a 30/10ug
Cefotaxime   30ug
Cefotaxime/clavulanic acid a 30/10ug

(Confirmatory testing requires use of both cefotaxime and ceftazidime, alone and in combination with clavulanic acid.)

Inoculum Standard disk diffusion
Standard disk diffusion
Incubation Conditions
Incubation Length
Results For K . pneumoniae , K . oxytoca , and E . coli :
Cefpodoxime zone < 17mm
Ceftazidime zone < 22mm
Aztreonam zone < 27mm
Cefotaxime zone < 27mm
Ceftriaxone zone < 25mm

For P . mirabilis a :
Cefpodoxime zone < 22mm
Ceftazidime zone < 22mm
Cefotaxime zone < 27mm
Zones above may indicate ESBL production

A > 5mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone = ESBL
(e.g., ceftazidime zone = 16mm; ceftazidime/clavulanic acid zone = 21mm)
QC Recommendations When testing ESBL-screening antimicrobial agents, K . pneumoniae ATCC ® 700603 is provided for quality assessment (e.g.,
training, competency, or test evaluation). Either strain, K . pneumoniae ATCC ® 700603 or E. coli ATCC ® 25922, may then be used for routine QC (e.g., weekly or daily).
E . coli ATCC ® 25922 (see control limits in Table 3)
Klebsiella pneumoniae ATCC ® 700603:
Cefpodoxime zone   9-16mm
Ceftazidime zone     10-18mm
Aztreonam zone      9-17mm
Cefotaxime zone     17-25mm
Ceftriaxone zone     16-24mm

When performing the ESBL confirmatory tests, K . pneumoniae ATCC ® 700603 and E . coli ATCC ® 25922 should be tested routinely (e.g., weekly or daily).

E. coli ATCC ® 25922: < 2mm increase in zone diameter for antimicrobial agent tested alone versus its zone when tested in combination with clavulanic acid.

Klebsiella pneumoniae ATCC ® 700603:
> 5mm increase in ceftazidime/clavulanic acid zone diameter;
> 3mm increase in cefotaxime/clavulanic acid zone diameter.


a. Screening of Proteus mirabilis for ESBL production is recommended only when it is deemed clinically relevant (e.g., a bacteremic isolate).


1. Performance Standards for Antimicrobial Disk Susceptibility Tests; Eighteenth Informational Supplement , M100, Appendix A. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

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