ESBL Detection

Detection of extended spectrum beta lactamase in gram negative pathogenic bacteria

1. ESBL Detection Dr. Samira Fattah PhD in Medical Bacteriology College of Health Sciences-HMU

2. ESBL Extended Spectrum Beta Lactamases (ESBL) are β-lactamases capable of conferring bacterial resistance to the penicillins; first-, second- and third-generation cephalosporins; and aztreonam (but not the cephamycins “like Cefoxitin” or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by β-lactamase inhibitors such as clavulanic acid This can limit treatment options for ESBL-producing pathogens Early diagnosis followed by appropriate treatment is essential to decrease both mortality and morbidity rates of diseases caused by such pathogenic bacteria.

3. Confirmatory Tests for ESBL Production Double-disk synergy test Combined disk test ESBL E tests Automated method Polymerase chain reaction (PCR) – TEM, SHV, STX-M and OXA genes Sequencing

4. Double-disk synergy test Principle ESBL produce enzyme that destroy beta-lactam antibiotic. That enzyme is inhibited by acid clavulanic and restore the activity of the beta-lactam antibiotic. In the synergy test, what happened is that the clavulanic acid in Amoxicillin-clavulanic acid inhibit beta-lactamase and the Cephalosporin become active on the bacteria and the diameter of inhibition becomes wider.

5. Double-disk synergy test Requirements : Mueller-Hinton agar 0.5 McFarland standard. Ceftazidime CAZ disk (30 µg) or Cefpodoxime FEP (30 μ g) Cefotaxime CTX disk (30 µg) Amoxicillin-clavulanate disk (AMC, 20+10 µg) Incubator

6. Double-disk synergy test Method Prepare MHA Prepare 0.5 McFarland standard suspension of enterobacteriaceae isolates using direct colony suspension. Make a lawn culture of bacteria in agar plates paired discs of CAZ (30 µg) and CTX (30 µg) were each positioned at distances of 20 mm ( centre to centre ) from amoxicillin-clavulanate disc (AMC, 20+10 µg). Plates were read after overnight incubation at 35°C.

7. Double-disk synergy test Result and interpretation The test is considered as positive when a decreased susceptibility to cefotaxime is combined with a clear-cut enhancement of the inhibition zone of cefotaxime in front of the clavulanate-containing disk, often resulting in a characteristic shape-zone referred to as ‘champagne-cork’ or ‘keyhole’.

8. Double-disk synergy test for extended spectrum beta-lactamases in A. baumannii , A (positive result), B (negative result). AMC-Amoxycillin + Clavulanate, CTX-Cefotaxime, FEP-Cefepime (A clear extension of the edge (Synergy) of the FEP inhibition zone towards the disc containing clavulanate).