DETECTION OF EXTENDED-SPECTRUM β-LACTAMASES IN ENTEROBACTERIACEAE

Definition:

Extended-spectrum β-Lactamase (ESBL) is an enzyme that hydrolyzes most penicillin and extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam (Aztreonam).  ESBLs are inhibited by β-Lactamase inhibitors (Clavulanic acid, Sulbactam and Tazobactam).

Aim:

To identify and confirm the ability of the organism against Broad spectrum cephalosporins and monobactam.

Principle:

Resistant bacteria are emerging worldwide as a threat to the favorable outcome of common infections in community and hospital settings. β-lactamase production by several gram-negative organisms is perhaps the most important single mechanism of resistance to penicillins and cephalosporins. In the past it was believed that cephalosporins were relatively immune to attack by β-lactamases. It was surprising to find cephalosporin resistant Klebsiella spp. among the clinical isolates.

The mechanism of this resistance was the production of extended-spectrum β-lactamases (ESBLs). Widespread use of third-generation cephalosporins and aztreonam is believed to be the major cause of the mutations in these enzymes that have led to the emergence of the ESBLs.  These enzymes mediate resistance to cefotaxime, ceftazidime, and other broad-spectrum cephalosporins and to monobactams such as aztreonam, but have no detectable activity against cephamycins and imipenem. Because, of their greatly extended substrate range these enzymes were called extended spectrum β-lactamases.        

Methods for detection of ESBLs in Enterobacteriaceae

ESBLs detection and characterization is recommended or mandatory for infection control purposes. The detection of ESBLs in Enterobacteriaceae is based on non-susceptibility to indicator oxyimino cephalosporins, followed by phenotypic confirmation tests.

ESBL screening methods:

Procedure:

The use of more than one antibiotic for screening improves the sensitivity of identification.

Media:

             For Disk diffusion -MHA

             For Broth or Agar dilution – CAMHB

Incubation: 35±2°C at ambient air

Incubation time: 18-24 hrs

Method	Antibiotic			Result
Broth or Agar dilution	For Klebsiella pneumonia, K. oxytoca and E.coli, Cefpodoxime  4 µl/mL Ceftazidime    1 µl/mL Aztreonam     1 µl/mL Cefotaxime     1 µl/mL Ceftriaxone    1 µl/mL For Proteus mirabilis, Ceftazidime    1 µl/mL Cefpodoxime  1 µl/mL Ceftazidime    1 µl/mL			For E.coli, K. pneumoniae and K. Oxytoca:             MIC ≥ 8 µl/mL for Cefpodoxime  or MIC ≥2 µl/mL for Ceftazidime,    Aztreonam, Cefotaxime or   Ceftriaxone. For Proteus mirabilis,    MIC ≥2 µl/mL for  Ceftazidime,    Cefpodoxime,  Ceftazidime
	*Growth at or above the concentration listed may indicate ESBL production
Disk diffusion	For Klebsiella pneumonia, K. oxytoca and E.coli, Cefotaxime    30 µg Ceftriaxone    30 µg Ceftazidime   30 µg Cefpodoxime, 10 µg Aztreonam, 30 µg For Proteus mirabilis, Ceftazidime   30 µg Cefpodoxime, 10 µg Cefotaxime    30 µg			≤ 27 mm ≤ 25 mm ≤ 22mm ≤ 17 mm ≤ 27 mm    ≤ 22 mm ≤ 22 mm ≤ 27 mm
	*Zones above may indicate ESBL production.

 

ESBL Confirmation test:

It requires use both cefotaxime and ceftazidime, alone and in combination with clavulanic acid.

Media:

           For Disk diffusion -MHA

           For Broth or Agar dilution - CAMHB

Incubation: 35±2°C at ambient air

Incubation time: 18-24 hrs.

Method	Antibiotic	Result
Broth or Agar dilution	Ceftazidime    0.25-128 µl/mL Ceftazidime–clavulanate 0.25/4-128/4 µl/mL and Cefotaxime     0.25-64µl/mL Cefotaxime –clavulanate 0.25/4-64/4µl/mL	A ≥ 3 2 –fold concentration decrease in an MIC for either antimicrobial agent tested in combination with clavulanate vs the MIC of the agent whrn tested alone=ESBL For eg., ceftazidime MIC=8 µl/mL; 16 and ceftazimide – clavulanate MIC=1 µl/mL
Disk diffusion	Ceftazidime   30 µg Ceftazidime –clavulanate 30/10 µg and Cefotaxime    30 µg Cefotaxime –clavulanate 30/10 µg	A 5mm increase of zone diameter of either antimicrobial agent tested alone in combination with clavulanate Vs its zone diameter of the agent when tested alone = ESBL. For eg., ceftazidime is 16 and ceftazimide – clavulanate is 21.

Phenotypic confirmatory test:

There are several phenotypic methods based on the in vitro inhibition of ESBL activity by clavulanic acid is used for ESBL confirmation. The methods are,

Combination disk test (CDT):

The inhibition zone around the cephalosporin (Cefotaxime, Ceftazidime, Cefepime) disk combined with clavulanic acid is compared with the zone around the disk with the cephalosporin alone. The test is positive if the inhibition zone diameter is ≥ 5mm larger with clavulanic acid than cephalosporin alone.

 

Combination disk test (CDT)

Double disk synergy test (DDST):

The test positive result is indicated when the inhibition zone around any of the cephalosporin (Cefotaxime, Ceftazidime, Cefepime) disk are increased in the direction of the disk containing clavulanic acid (amoxicillin- clavulanic acid). The distance between the disk is critical and 20mm centre to centre has been found to be optimal for cephalosporin 30µg disks. However it may be reduced (15mm) or expanded (30mm) for strains with very high or low level of resistance.

Gradient test method:

The test is positive if ≥ 8-fold reduction is observed in the MIC of the cephalosporin combined with clavulanic acid compared with MIC of the cephalosporin alone. The test result is indeterminate if the strip cannot be read due to growth beyond the MIC range of the strip. In all other cases the test result is negative. The ESBL gradient test should be used for confirmation of ESBL production only and is not reliable for determination of the MIC.  

Broth microdilution test: 

Broth microdilution is performed with Mueller-Hinton broth containing serial two-fold dilutions of cefotaxime, ceftazidime and cefipime at concentrations ranging from 0.25 to 512mg/L, withand without clavulanic acid at a fixed concentration of 4mg/L. the test is positive if ≥8-fold reduction is observed in the MIC of the cephalosporin combined with clavulanic acid compared with the MIC of the cephalosporin alone.

Genotypic confirmation:

The genotypic confirmation of ESBL genes, the PCR and ESBL gene sequencing or DNA microarray based method is used. The test results are usually obtained within 24 hrs.

Quality control procedures:

 Appropriate strains are use quality control of ESBL detection tests.

Strain	Mechanism
Escherichia coli ATCC 25922	ESBL- Negative
Klebsiella pneumoniae ATCC 700603	SHV-18 ESBL
Escherichia coli CCUG62975	CTX-M-1 group ESBL and acquired CMY AMPC

Reference:

1.    Gniadowski M. 2008. Evolution of extended-spectrum β-Lactamases by mutation. Clin. Microbiol. Infect, 14; 11-32.

2.    Livermore DM. β-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev.1995;8:557-584.

3. Clinical and Laboratory Standards Institute (CLSI). Perormance Standards for Antimicrobial susceptibility testing; 12^th Informational Supplement. CLSI document M100-S20, Vol 30, No.1.

4.  Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2010.