5.

12 Extended-Spectrum Beta-Lactamase
Testing for Escherichia coli, Klebsiella
pneumoniae, Klebsiella oxytoca, and
Proteus mirabilis

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE
Extended-spectrum beta-lactamases (ESBLs)
are enzymes that hydrolyze or inactivate
extended-spectrum cephalosporins, az-
treonam, and expanded-spectrum penicil-
lins. ESBLs are derived from point muta-
tions in the genes that encode common
beta-lactamases such as TEM-1, TEM-2,
or SHV-1, which confer resistance to pen-
icillins but not to extended-spectrum ceph-
alosporins. The mutations of these com-
mon beta-lactamases extend the spectrum
of beta-lactam antimicrobial agents that
are hydrolyzed to include those with an
oxyimino side chain (such as cefotaxime,
ceftriaxone, and ceftazidime). The oxy-
imino-monobactam aztreonam is also hy-
drolyzed by ESBLs. However, ESBL en-
zymes do not hydrolyze carbapenems and
generally do not hydrolyze cephamycins
(such as cefoxitin and cefotetan). ESBL
activity is blocked by beta-lactamase in-
hibitors such as clavulanic acid, and lab-
oratory tests for confirming ESBL-pro-
ducing bacteria are based on this property.
The phenotypic ESBL confirmatory test
can be performed by disk diffusion or by
a broth microdilution MIC method. Com-
mercially available ESBL Etest strips may
also be used for ESBL confirmatory test-
ing (see “Procedure Notes” below).
The confirmatory disk diffusion test for
ESBLs requires four disk tests: (i) cefta-
zidime alone, (ii) cefotaxime alone, (iii)
ceftazidime plus clavulanic acid, and (iv)
cefotaxime plus clavulanic acid. If the
zone diameter of either ceftazidime or ce-
fotaxime (or both) in the presence of cla-
vulanic acid is ≥5 mm larger than the zone
diameter of the respective agent alone, the
test is considered positive for ESBL pro-
duction.
The confirmatory broth microdilution
MIC test requires four MIC tests: (i) cef-
tazidime alone, (ii) cefotaxime alone, (iii)
ceftazidime plus clavulanic acid, and (iv)
cefotaxime plus clavulanic acid. If the
MIC of either ceftazidime or cefotaxime
(or both) in the presence of clavulanic acid
is three or more twofold dilutions lower
than the MIC of the respective agent alone
(e.g., a difference of ≥8 lg/ml between the
two MIC values), the test is considered
positive for ESBL production.

II. SPECIMEN Prepare inoculum from four or five well-isolated colonies of similar colony mor-
phology.

A. For log-phase-growth inoculum: use colonies grown for 1 or 2 days on nonse-

lective (e.g., BAP or CHOC) or selective (e.g., MAC) medium.
B. For direct colony suspension inoculum: use colonies grown overnight on non-
selective medium.
C. Subculture stock, frozen, or lyophilized isolates two times prior to testing.

ANALYTICAL CONSIDERATIONS

III. QUALITY CONTROL A. QC strains

Include QC information on
reagent container and in
QC records.
1. Klebsiella pneumoniae ATCC 700603
2. Escherichia coli ATCC 25922
B. Acceptable QC – disk diffusion:
E. coli ATCC 25922:
≤2-mm increase in zone diameter for antimicrobial agent tested in combination
with clavulanate versus the zone diameter when tested alone.

doi:10.1128/9781555818814.ch5.12 5.12.1
5.12.2 Antimicrobial Susceptibility Testing

III. QUALITY CONTROL K. pneumoniae ATCC 700603:

(continued) ≥5-mm increase in zone diameter of ceftazidime-clavulanate versus ceftazidime
alone;
≥3-mm increase in zone diameter of cefotaxime-clavulanate versus cefotaxime
alone.
C. Acceptable QC – MIC testing:
E. coli ATCC 25922:
⬍3 twofold concentration decreases in MIC for antimicrobial agent tested in
combination with clavulanate versus the MIC of the agent when tested alone.
K. pneumoniae ATCC 700603:
≥3 twofold concentration decreases in MIC for an antimicrobial agent tested in
combination with clavulanate versus the MIC of the agent when tested alone.
D. Monitoring accuracy
1. Test QC strains by following routine procedure and record results in QC
notebook. Record lot numbers and expiration dates of disks, agar and broth
microdilution trays. See Appendix 5.12–1 and Appendix 5.12–2.
2. Compare to expected results listed above and note any out-of-control result
and document; proceed with corrective action, if necessary.
E. QC testing strategy
1. Disk diffusion method: perform QC testing of disks required for ESBL test
daily or weekly (see procedure 5.1 for daily or weekly testing schedules).
2. Broth microdilution method: perform QC testing of panel containing ESBL
test daily or weekly (see procedure 5.2 for daily or weekly testing schedules).
F. Daily or weekly schedule
1. Weekly QC testing can be performed if ESBL test is performed at least once
a week and criteria for converting daily to weekly QC have been met by the
laboratory. If the test is performed less than once a week and/or the criteria
for converting to weekly QC have not been met, perform QC testing daily.
G. Additional controls for broth microdilution method
1. Growth control well must show 3+ to 4+ growth and purity plate must show
pure growth of organism.
2. Sterility control well should be free of any growth.

IV. PROCEDURE – DISK Refer to procedure 5.1 for additional details of performing the disk diffusion test
DIFFUSION METHOD and materials required. Primary materials and steps specific to the ESBL test are
listed below.
Include QC information on
reagent container and in A. Materials
QC records. 1. Media and reagents
a. Mueller-Hinton agar (MHA). Store at 2 to 8⬚C.
b. Antimicrobial agent disks. Store with desiccant at –14 to 8⬚C.
(1) Ceftazidime, 30 lg
(2) Ceftazidime-clavulanic acid, 30/10 lg
(3) Cefotaxime, 30 lg
(4) Cefotaxime-clavulanic acid, 30/10 lg
c. Nutrient broth (e.g., Mueller-Hinton, TSB) or 0.9% saline (3.0- to 5.0-ml
aliquots). Store at 2 to 30⬚C.
2. Supplies
a. Sterile cotton-tipped swabs
b. 0.5 McFarland turbidity standard or photometric device
3. Equipment
a. Vortex mixer
b. Ambient-air incubator (34 to 35⬚C)
ESBL Testing for E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis
VI. PROCEDURE – DISK
DIFFUSION METHOD (continued)
V. PROCEDURE – BROTH
MICRODILUTION MIC METHOD
Include QC information on
reagent container and in
QC records.
5.12.3
B. Procedure
1. Use either direct colony suspension or log-phase method of inoculum prep-
aration to prepare a 0.5 McFarland standardized suspension of test bacteria.
2. Within 15 min after turbidity adjustment, inoculate plate and allow to stand
for 3 to 15 min before applying the following disks to agar surface:
Ceftazidime, 30 lg
Ceftazidime-clavulanic acid, 30/10 lg
Cefotaxime, 30 lg
Cefotaxime-clavulanic acid, 30/10 lg
3. Invert plates and incubate at 35 Ⳳ 2⬚C in an ambient-air incubator for 16 to
18 h.
4. Reading and interpreting results
a. Measure zones as described for testing rapidly growing nonfastidious
bacteria using reflected light.
b. Positive for ESBL production: a ≥5-mm increase in the zone diameter
for either ceftazidime or cefotaxime tested in combination with clavulanic
acid versus its zone diameter when tested alone
c. Negative for ESBL production: a ⬍5-mm increase in the zone diameter
for either ceftazidime or cefotaxime tested in combination with clavulanic
acid versus its zone diameter when tested alone
d. Indeterminate for ESBL production: a decrease in the zone diameter for
either ceftazidime or cefotaxime tested in combination with clavulanic
acid versus its zone diameter when tested alone; or, no change in the disk
zones with the addition of clavulanic acid
Refer to procedure 5.21 for additional details on preparation of broth microdilution
MIC trays and procedure 5.2 for details of the performance of broth microdilution
MIC tests. Primary materials and steps specific to the ESBL test are listed below.
A. Materials
1. Media and reagents
a. Cation-adjusted Mueller-Hinton broth (CAMHB) containing antimicro-
bial agents listed below, dispensed in a microdilution plate. Store at
ⳮ70⬚C.
(1) Ceftazidime, 0.25 to 128 lg/ml
(2) Ceftazidime-clavulanic acid, 0.25/4 to 128/4 lg/ml
(3) Cefotaxime, 0.25 to 64 lg/ml
(4) Cefotaxime-clavulanic acid, 0.25/4 to 64/4 lg/ml
b. Nutrient broth (e.g., Mueller-Hinton, TSB) or 0.9% saline (3.0- to 5.0-
ml aliquots). Store at 2 to 30⬚C.
c. Water diluent dispensed in aliquots of 40 ml in screw-cap tubes. Store at
25⬚C.
2. Supplies
a. Sterile disposable plastic multipronged inoculator sets (include inoculum
reservoir)
b. 0.5 McFarland turbidity standard or photometric device
3. Equipment
a. Vortex mixer
b. Ambient-air incubator (34 to 35⬚C)
B. Procedure
1. Use either direct colony suspension or log-phase method of inoculum prep-
aration to prepare a 0.5 McFarland standardized suspension of test bacteria.
5.12.4
V. PROCEDURE – BROTH
MICRODILUTION MIC METHOD
(continued)
POSTANALYTICAL CONSIDERATIONS
VI. REPORTING RESULTS
VII. PROCEDURE NOTES A. In 2010, the CLSI published revised Enterobacteriaceae breakpoints for several
Antimicrobial Susceptibility Testing
2. Within 15 min after turbidity adjustment, dilute standardized suspension in
water so that after inoculation each well contains approximately 5 ⳯ 105
CFU/ml or 5 ⳯ 104 CFU in each well (plastic inoculator prong device de-
livers 0.01 ml/pin).
3. Inoculate wells.
4. Perform purity check by subculturing inoculum suspension onto a BAP and
incubate overnight.
5. Place inoculated tray in an enclosed container (plastic bag or container) and
incubate at 35 Ⳳ 2⬚C in an ambient-air incubator for 16 to 20 h.
6. Reading and interpreting results
a. Read MICs as described for testing rapidly growing nonfastidious bac-
teria.
b. Positive for ESBL production: three or more twofold dilution decreases
in an MIC (e.g., three or more doubling dilutions, or a difference of ≥8
lg/ml between the two MIC values) for either ceftazidime or cefotaxime
tested in combination with clavulanic acid versus its MIC when tested
alone
c. Negative for ESBL production: fewer than three twofold dilution de-
creases in an MIC (e.g., fewer than three doubling dilutions, or a differ-
ence of ⬍8 lg/ml between the two MIC values) for either ceftazidime or
cefotaxime tested in combination with clavulanic acid versus its MIC
when tested alone
d. Indeterminate for ESBL production: an increase in the MIC for either
ceftazidime or cefotaxime tested in combination with clavulanic acid ver-
sus its MIC when tested alone; or, no change in the MIC with the addition
of clavulanic acid
1. If the ESBL confirmatory test is positive, report as in the following example:
“This E. coli is positive for ESBL production.”
If using the current CLSI interpretive criteria (1), do not edit any susceptible
or intermediate MIC or zone diameter interpretations to resistant for the
penicillins, cephalosporins (excluding the cephamycins such as cefoxitin or
cefotetan), or aztreonam.
2. If the ESBL confirmatory test is negative, report as in the following example:
“This E. coli is negative for ESBL production.”
beta-lactam drugs, including extended-spectrum cephalosporins and aztreonam
that are typically hydrolyzed by ESBLs (2). Breakpoints were changed for ce-
fotaxime, ceftizoxime, ceftazidime, ceftriaxone, and aztreonam. The different
ESBL enzymes hydrolyze beta-lactam drugs at different rates; as such, some
ESBL-producing isolates may test susceptible to one of these cephalosporins
(and/or aztreonam) but resistant to another (3). The CLSI states that routine
screening and confirmatory testing for ESBLs is no longer necessary when using
the current cephalosporin and aztreonam breakpoints and it is not necessary to
edit susceptible or intermediate results for cephalosporins, aztreonam, or peni-
cillins to resistant if an isolate tests positive for ESBL production using the
current CLSI breakpoints. However, testing for ESBLs may still be helpful for
epidemiologic or infection control purposes.
ESBL Testing for E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis 5.12.5

VII. PROCEDURE NOTES B. At least 650 different types of ESBL enzymes have been described. Use of more
(continued) than one screening antimicrobial agent increases the likelihood of ESBL detec-
tion.
C. The genes for ESBL production are typically located on plasmids, and genes
for resistance to other antimicrobial agents are often found on the same plasmid.
Consequently, isolates that produce ESBLs often (but not always) demonstrate
resistance to other classes of antimicrobial agents (e.g., aminoglycosides, fluoro-
quinolones, or trimethoprim-sulfamethoxazole).
D. ESBLs do not hydrolyze carbapenems, and ESBL-producing E. coli and Kleb-
siella spp. remain susceptible to these antimicrobial agents.
E. Etest (agar gradient diffusion method) may be used for ESBL confirmatory
testing. Laboratories should follow the manufacturer’s guidelines for Etest.
There is a single strip configured with a cephalosporin on one end and the
cephalosporin with clavulanic acid on the other end. Two strips are available:
(i) ceftazidime (TZ) and ceftazidime plus clavulanic acid (TZ/TZL) and (ii)
cefotaxime (CT) and cefotaxime plus clavulanic acid (CT/CTL). The test is
performed as described in the Etest procedure 5.3 and the suspected presence
of an ESBL is confirmed using both TZ/TZL and CT/CTL Etests. A “phantom”
zone or deformation of the inhibition ellipse is indicative of ESBL production,
regardless of the MICs (refer to Etest ESBL package insert for photo). When
MIC values are above the test range, the result is indeterminate.

VIII. LIMITATIONS A. Indeterminate results: Indeterminate results on ESBL tests may indicate the
presence of a resistance mechanism other than or in addition to an ESBL, such
as an AmpC with or without a porin loss. Occasionally, the MICs may be too
high or the zone diameters too low on the confirmatory test for accurate inter-
pretation. In such situations, it may be appropriate to use a different method-
ology for ESBL testing and/or to assess for other resistance mechanisms, such
as AmpC. Currently, there are no CLSI standardized methods for detection of
ampC enzymes.
B. Some organisms that produce ESBLs express other beta-lactamases or resistance
mechanisms (e.g., porin alterations) that can mask ESBL production in the con-
firmatory test, resulting in a false-negative result.
C. Other members of Enterobacteriaceae and other Gram-negative bacilli can pro-
duce ESBLs. However, practical clinical laboratory methods for confirmation
of ESBL production in these isolates have not been standardized.

REFERENCES 1. CLSI. 2015. Performance Standards for An-
timicrobial Susceptibility Testing: Twenty-
Fifth Informational Supplement. CLSI docu-
ment M100-S25. CLSI: Wayne, PA.
2. CLSI. 2010. Performance Standards for An-
timicrobial Susceptibility Testing: Twentieth
Informational Supplement. CLSI document
M100-S20. CLSI: Wayne, PA.
3. Wang P, Hu F, Xiong Z, Ye X, Zhu D, Wang
YF, Wang M. 2011. Susceptibility of ex-
tended-spectrum-beta-lactamase-producing
Enterobacteriaceae according to the new
CLSI breakpoints. J Clin Microbiol 49:3127–
3131.
5.12.6 Antimicrobial Susceptibility Testing

APPENDIX 5.12–1 ESBL Disk Diffusion Test QC

Test QC Strain Exp MHA Agar Exp Zone In Control

Tech Lot #
Date Antimicrobial Disk, Content Date Lot # Date (mm) (Y/N)a
E. coli ATCC 25922
Cefotaxime, 30 lg
Cefotaxime-clavulanate, 30/10 lg
Ceftazidime, 30 lg
Ceftazidime-clavulanate, 30/10 lg
K. pneumoniae ATCC 700603
Cefotaxime, 30 lg
Cefotaxime-clavulanate, 30/10 lg
Ceftazidime, 30 lg
Ceftazidime-clavulanate, 30/10 lg
E. coli ATCC 25922
Cefotaxime, 30 lg
Cefotaxime-clavulanate, 30/10 lg
Ceftazidime, 30 lg
Ceftazidime-clavulanate, 30/10 lg
K. pneumoniae ATCC 700603
Cefotaxime, 30 lg
Cefotaxime-clavulanate, 30/10 lg
Ceftazidime, 30 lg
Ceftazidime-clavulanate, 30/10 lg
a
Acceptable QC:
E. coli ATCC 25922:
≤2-mm increase in zone diameter for antimicrobial agent tested in combination with clavulanate versus the zone diameter when tested alone
K. pneumoniae ATCC 700603:
≥5-mm increase in zone diameter of ceftazidime-clavulanate versus ceftazidime alone
≥3-mm increase in zone diameter of cefotaxime-clavulanate versus cefotaxime alone
ESBL Testing for E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis 5.12.7

APPENDIX 5.12–2 ESBL MIC Test QC

Test QC Strain Panel Exp MIC In Control

Tech
Date Antimicrobial Agent Lot # Date (lg/ml) (Y/N)a
E. coli ATCC 25922
Cefotaxime
Cefotaxime-clavulanate
Ceftazidime
Ceftazidime-clavulanate
K. pneumoniae ATCC 700603
Cefotaxime
Cefotaxime-clavulanate
Ceftazidime
Ceftazidime-clavulanate
E. coli ATCC 25922
Cefotaxime
Cefotaxime-clavulanate
Ceftazidime
Ceftazidime-clavulanate
K. pneumoniae ATCC 700603
Cefotaxime
Cefotaxime-clavulanate
Ceftazidime
Ceftazidime-clavulanate
a
Acceptable QC:
E. coli ATCC 25922:
⬍3 twofold concentration decreases in MIC for antimicrobial agent tested in combination with cla-
vulanate versus the MIC of the agent when tested alone
K. pneumoniae ATCC 700603:
≥3 twofold concentration decreases in MIC for an antimicrobial agent tested in combination with
clavulanate versus the MIC of the agent when tested alone