Double disk synergy test (DDST) is a phenotypic test that is used to confirm the production of ESBL in test bacterial isolates suspected of producing ESBL in the screening test. DDST should be performed as a standard disk diffusion assay on Mueller-Hinton (MH) agar (Oxoid, UK) plates in line with the Clinical Laboratory Standard Institute (CLSI) criteria.

In carrying out DDST for ESBL phenotypic confirmation, sterile swab sticks should be dipped into bacterial suspension(s) standardized to 0.5 McFarland turbidity standards, and then spread on MH agar plates.

Antibiotic disks of amoxycillin-clavulanic acid (20/10µg) should be placed at the center of the MH agar plate, and antibiotic disks containing cefotaxime (30µg) and ceftazidime (30µg) should each be placed at a distance of 15 mm (center to enter) from the central disc, amoxycillin-clavulanic acid 20/10µg.

The MH agar plates should be incubated at 37oC for 18-24hrs. ESBL production should be inferred phenotypically when the zones of inhibition of the cephalosporins (cefotaxime 30µg and ceftazidime 30µg) is expanded by the amoxycillin-clavulanic acid disk (20/10µg).

A ≥5mm increase in the inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxycillin-clavulanic acid versus its zone when tested alone confirms ESBL production phenotypically


ESBLs (extended spectrum beta-lactamases) are plasmid – mediated beta – lactamases capable of hydrolyzing many beta – lactam antibiotics including third – generation cephalosporins and monobactams, but are yet inhibited by clavulanic acid, a beta – lactamase inhibitor. Since the discovery of the ESBLs in the 1980’s, over 100 different enzymes have been described, and they have become a worldwide health problem affecting many countries of the world with varying prevalence.

ESBLs arise by mutations in genes for common plasmid – mediated beta – lactamases (especially TEM and SHV enzymes) that alter the amino acid configuration of the enzyme near its active site to increase the affinity and hydrolytic ability of the beta – lactamases for oxyimino cephalosporins. ESBLs are detected mostly in Enterobacteriaceae especially Klebsiella pneumoniae, Escherichia coli, and Klebsiella oxytoca. They often contain resistance determinants for other classes of antibiotics like the aminoglycosides, sulfonamides, fluoroquinolones which are readily transmissible from one strain of organism to another and between different species of Gram – negative bacteria.

Apart from the TEM- and SHV- type ESBLs, other classes of ESBLs have also been described including CTX – M type ESBLs, PER- and OXA- type ESBLs, mutations that expand the spectrum of the earlier beta – lactamases (TEM and SHV) usually increase the sensitivity of ESBLs to beta – lactamase inhibitors like clavulanic acid, tazobactam and sulbactam. ESBL – producing organisms are not new, having first been reported in the 1980’s from Germany, and they are now found worldwide in both the community hospital settings. Resistance of Gram – negative bacteria to third – and fourth – generation cephalosporins is mediated by ESBLs, and they are an important cause of treatment failure in patients receiving cephalosporins because these agents are hydrolyzed in vivo by ESBLs when used for therapy especially in patients harbouring ESBL – producing bacteria.


Peter C. Ejikeugwu, Nkechukwu M.I., Ugwu C.M., Iroha I.R and Esimone C.O (2012). Extended-spectrum β-lactamase-producing Escherichia coli isolates from suspected community acquired urinary tract infections. European Journal of Scientific Research.  84(4):565-571.


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