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ESBL

LABORATORY DETECTION OF EXTENDED-SPECTRUM Β-LACTAMASES (ESBLS)

Extended-spectrum ß-lactamase (ESBL) producing organisms are a major problem in the area of infectious disease. 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). In addition, ESBL-producing organisms frequently show cross-resistance to many other classes of antibiotics; including aminoglycosides and fluoroquinolones thus treatment of these infections is often a therapeutic challenge. The frequency of ESBL-producing organisms differs significantly in accordance with geographic location. Published data from Pakistan supports an urgent need for regular screening and surveillance for these organisms in this region. Detection of ESBL is a major challenge for the clinical microbiology laboratory as its presence in bacterial cells does not always produce phenotypic resistance resulting in some ESBL isolates appearing susceptible to third-generation cephalosporin in vitro. However treatment of these isolates with third-generation cephalosporins is frequently ineffective. If an ESBL is detected, all penicillins, cephalosporins, and aztreonam should be reported as resistant, even if in vitro test results indicate susceptibility.

SCREENING AND CONFIRMATORY TESTS FOR ESBL DETECTION IN E.COLI AND KLEBSIELLA SPP.

SCREENING TESTS:

Media: Muellar Hinton agar (MHA)

Inoculum and Incubation requirement: According to standard disk diffusion

If the zone sizes of following antibiotics are above the mentioned zone size then perform confirmatory tests for ESBL production.

Cefotaxime (30μg): ≤27mm

Ceftriaxone (30μg): ≤25mm

Aztreonam (30μg): ≤27mm

Ceftazidime (30μg): ≤22mm

Cefpodoxime (10μg): ≤17mm

Use of more than one of the above agents is recommended to increase the sensitivity of detection.

QC recommendations: Either of the following strains should be used for routine QC (daily or weekly)

E.coli ATCC 25922 (Control limits)

Cefotaxime (30μg): 29-35 mm

Ceftriaxone (30μg): 29-35 mm

Aztreonam (30μg): 28-36 mm

Ceftazidime (30μg): 25-32 mm

Cefpodoxime (10μg): 23-28 mm

K.pneumoniae ATCC 700603 (Control limits)

Cefotaxime (30μg): 17-25 mm

Ceftriaxone (30μg): 16-24 mm

Aztreonam (30μg): 9-17 mm

Ceftazidime (30μg): 10-18 mm

Cefpodoxime (10μg): 9-16 mm

CONFIRMATORY TESTS:

Media: Muellar Hinton agar (MHA)

Inoculum and Incubation requirement: According to standard disk diffusion

Confirmatory testing requires use of both of the following methods.

1. Ceftazidime (30μg)

Ceftazidime-clavulanic acid* (30/10μg)

2.Cefotaxime (30μg)

Cefotaxime-clavulanic acid* (30/10μg)

An ESBL producing organism would have ≥5mm increase in zone diameter for either antimicrobial agent tested in combination with clavulanic acid vs. when tested alone.

QC recommendations: Both of the following strains should be used for routine QC (daily or weekly)

E.coli ATCC 25922: ≤2mm increase in zone diameter for antibiotic tested alone vs. its zone when tested in combination with clavulanic acid.

K.pneumoniae ATCC 700603: ≥5mm increase in ceftazidime-clavulanic acid zone diameter and ≥3mm increase in cefotaxime-clavulanic acid zone diameter.

*Preparation of ceftazidime-clavulanic acid (30/10μg) and cefotaxime-clavulanic acid* (30/10μg) disks: Use a stock solution of clavulanic acid at 1000 μg/mL (either freshly prepared or taken from small aliquots that have been frozen at -700C). Add 10 μL of clavulanic acid to ceftazidime 30μg and cefotaxime 30μg disks. Use a micropipette to apply the 10 μL of the stock solution to the disks within one hour before they are applied to the plates. This would allow about 30 minutes for the clavulanic acid to absorb and the disks to be dry enough for application. Use disks immediately after preparation or discard; do not store.

Reference:

National Committee for Clinical Laboratory Standards. 2007. Performance standards for antimicrobial susceptibility testing. NCCLS approved standard M100-S17. National Committee for Clinical Laboratory Standards, Wayne, PA

 
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