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Laboratory Detection of Extended-Spectrum
-Lactamases
(ESBLs)What are extended-spectrum -lactamases?
ESBLs are enzymes that mediate resistance to extended-spectrum
(third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and
monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and
cefotetan) or carbapenems (e.g., meropenem or imipenem).
Why should clinical laboratory personnel be concerned about detecting these enzymes?
The presence of an ESBL-producing organism in a clinical
infection can result in treatment failure if one of the above classes of drugs is used.
ESBLs can be difficult to detect because they have different levels of activity against
various cephalosporins. Thus, the choice of which antimicrobial agents to test is
critical. For example, one enzyme may actively hydrolyze ceftazidime, resulting in
ceftazidime minimum inhibitory concentrations (MICs) of 256 g/ml, but have
poor activity on cefotaxime, producing MICs of only 4 g/ml. If an ESBL is detected, all
penicillins, cephalosporins, and aztreonam should be reported as resistant, even if in
vitro test results indicate susceptibility (1).
How can clinical laboratory personnel screen for ESBL production in bacteria?
The National Committee for Clinical Laboratory Standards (NCCLS)
has developed broth microdilution and disk diffusion screening tests using selected
antimicrobial agents (1). Each Klebsiella pneumoniae, K. oxytoca, or Escherichia
coli isolate should be considered a potential ESBL-producer if the test results are as
follows:
Disk diffusion |
MICs |
cefpodoxime < 22 mm |
cefpodoxime > 2 g/ml |
ceftazidime < 22 mm |
ceftazidime > 2 g/ml |
aztreonam < 27 mm |
aztreonam > 2 g/ml |
cefotaxime < 27 mm |
cefotaxime > 2 g/ml |
ceftriaxone < 25 mm |
ceftriaxone > 2 g/ml |
The sensitivity of screening for ESBLs in
enteric organisms can vary depending on which antimicrobial agents are tested. The use of
more than one of the five antimicrobial agents suggested for screening will improve the
sensitivity of detection. Cefpodoxime and ceftazidime show the highest sensitivity for
ESBL detection.
How can clinical laboratory personnel confirm ESBL production?
NCCLS recommends performing phenotypic confirmation of
potential ESBL-producing isolates of K. pneumoniae, K. oxytoca, or E.
coli by testing both cefotaxime and ceftazidime, alone and in combination with
clavulanic acid (1). Testing can be performed by the broth microdilution method or by disk
diffusion. For MIC testing, a decrease of > 3 doubling dilutions in an MIC for
either cefotaxime or ceftazidime tested in combination with 4 g/ml clavulanic
acid, versus its MIC when tested alone, confirms an ESBL-producing organism. For disk
diffusion testing, a > 5 mm increase in a zone diameter for either antimicrobial
agent tested in combination with clavulanic acid versus its zone when tested alone
confirms an ESBL-producing organism.
How should laboratory personnel test for cefotaxime and ceftazidime
in combination with clavulanic acid?
NCCLS suggests making disks by adding 10 l of a 1000 g/ml stock
solution of clavulanic acid to cefotaxime and ceftazidime disks each day of testing (1).
In the future, commercial manufacturers of antimicrobial disks may produce disks
containing cefotaxime and ceftazidime with clavulanic acid. Until commercial disks are
available, SmithKline Beecham can provide clinical laboratories with clavulanic acid
powder for routine testing of clinical isolates (please contact Karen Dobbs [U.K.]
44-1-279-627-562, fax 44-1-279-627-567. Alternatively, contact Rosaria Fajardo [U.S.]
1-610-270-6854, fax 1-610-270-6798. In your request, provide your name, address, telephone
and fax numbers, and the name of the compound required).
What quality control organisms should laboratories use for ESBL
testing?
K. pneumoniae ATCC 700603 (positive control) and E.
coli ATCC 25922 (negative control) should be used for quality control of ESBL tests
(1).
Can an ESBL be present in an isolate of K. pneumoniae that is resistant to
ceftazidime and/or cefotaxime, but demonstrates no clavulanic acid effect in the
phenotypic confirmatory test?
Yes. The phenotypic confirmatory test does not detect all ESBLs.
Some organisms with ESBLs contain other -lactamases that can mask ESBL production in the phenotypic test,
resulting in a false-negative test. These -lactamases include AmpCs and inhibitor-resistant TEMs (IRTs).
Hyper-production of TEM and/or SHV -lactamases in organisms with ESBLs also may cause false-negative
phenotypic confirmatory test results. Currently, detection of organisms with multiple -lactamases that may
interfere with the phenotypic confirmatory test can only be accomplished using isoelectric
focusing and DNA sequencing, methods that are not usually available in clinical
laboratories.
How should cephalosporin and penicillin results be reported?
If an isolate is confirmed as an ESBL-producer by the
NCCLS-recommended phenotypic confirmatory test procedure, all penicillins, cephalosporins,
and aztreonam should be reported as resistant. This list does not include the cephamycins
(cefotetan and cefoxitin), which should be reported according to their routine test
results. If an isolate is not confirmed as an ESBL-producer, current recommendations
suggest reporting results as for routine testing. Do not change interpretations of
penicillins, cephalosporins, and aztreonam for isolates not confirmed as ESBLs.
Do isolates other than K. pneumoniae, K. oxytoca, or E.
coli produce ESBLs?
Yes. Other isolates of Enterobacteriaceae, such as Salmonella
species and Proteus mirabilis, and isolates of Pseudomonas aeruginosa
produce ESBLs. However, at this time, methods for screening and phenotypic confirmatory
testing of these isolates have not been determined by NCCLS.
- National Committee for Clinical Laboratory Standards. 1999. Performance standards
for antimicrobial susceptibility testing. NCCLS approved standard M100-S9. National
Committee for Clinical Laboratory Standards, Wayne, PA.
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