Saturday, 19 March 2016

Detection of Extended-spectrum beta-lactamases (ESBL)

ESBL Positive

ESBL Negative
Extended-spectrum beta-lactamases (ESBL) are β-lactamases that hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam. Thus ESBLs confer resistance to these antibiotics and related oxyimino-beta lactams. In typical circumstances, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. A broader set of β-lactam antibiotics are susceptible to hydrolysis by these enzymes. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates. Recent publications have reported cases of resistance of ESBL-producing organisms to the Carbapenems, primarily ertapenem.
ESBL test is usually done on any isolate resistant to any 2nd and 3rd generation Cephalosporins. All ESBLs show synergy with Clavulanic acid (β-lactamase inhibitor) which distinguishes them from AmpC and K1. In the test shown below, Cefpodoxime is used because it is an excellent screening antibiotic since all ESBL genotypes show resistance. However, in the presence of Clavulanic acid zones diameter increases by ≥5mm.
The figures above shows a positive and negative ESBL for a Klebsiella pneumoniae isolate on Mueller-Hinton agar. The isolate was homogenised in a saline to get 0.5 McFarland and streaked on the Mueller-Hinton agar to make an even lawn. The agar plates were then incubated at 37oC for 24 hours after placing the antibiotic impregnated discs on the agar. The positive figure shows that there is a zone of inhibition around Cefpodoxime + Clavulanic acid disc (CPD + CV) which is ≥5mm  than the zone around Cefpodoxime (CPD).
The ESBL Negative figure shows that there is no zone difference around CPD and CPD + CV.

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