1. Methods for detection β-lactamases Sarah Alharbi

2. The most commonly used class of agents to treat bacterial infections They consist of four major groups: penicillins, cephalosporins, monobactams and Carbapenems B-lactam antibiotics Figure.1 Structure of B-lactam ring

3. Beta- lactamases Hydrolyze b-lactam antibiotics using two techniques: 1)A metallo –based mechanism of action 2)A serine based mechanism of action They are calssified into 4 main classes: 1)class B βlactamses exhibits metallo-based mechanism of action 2) Class A,C and D contianing active –site serine enzymes

4. Example, clauvulinc acid, sulbactam, and tazobactam - Clavulanic acid does not have any antibacterial properties of its own, - Irreversibly binding to the β-lactamase molecules B lactamase inhibitor

5. Methods for B lactamase detection 1)Direct tests for B lactamase activity ( most use chromogenic cephalosporins “nitrocefin test”) for staphylococci, fastidious gram negative species and gram negative bacilli 2)Microbilogical test for B lactamase activities ( cloverleaf method, Masuda double disk method). Very sensitive 3)ESBLs tests ( double disc, combined disc method, VITEK ESBL cards) 1)PCR (B lactamases can be detected accurately through gene or protein sequencing)

6. B – lactamase detection ( chromogenic cephalosporinase test/ nitrocefin test. Nitrocefin is a chromogenic cephalosporin that changes from yellow to red on hydrolysis.

7. Detection of B lactamase in Enterbacteriacea 1) ESBL -Resistance to ceftazidime or cefpodoxime implies extended-spectrum β-lactamase - (ESBL) production in Escherichia coli and Klebsiella spp. -Broad spectrum : these enzymes are inhibited by clauvalinc acid ESBLs -Resistance to ceftazidime or cefpodoxime implies extended-spectrum β-lactamase - (ESBL) production in Escherichia coli and Klebsiella spp. -Broad spectrum : these enzymes are inhibited by clauvalinc acid ESBLs 2) Metallo based B lactamase

8. 8 Detection of ESBL producing strains ( double disc test ESBL production can be detected by disk diffusion using combinations of antibiotics and cluvalinate. Enhanced zone of inhibition after adding clauvalnite is evidence of ESBL negativepositive

9. Detection of Metallo-β-lactamase (MBL) production in Enterobacteriaceae- The Imipenem-EDTA double-Disk Synergy Test - catalyze the hydrolysis of a broad range of β-lactam drugs including carbapenems. - Carbapenems are broad –spectrum B lactam antibiotics with reliable coverage against all bacterial pathogens except mycobacteria, cell wall deficient organisms, some infrequent non-fermenters aeromonads. Carpanemes are often used a last resort antibiotics for chemotherapy of infectious diseases caused by multi drug resistant Gram –negative bacilli Resistance against carpanemems conferred by metallo B lactamases (MBL) is an emerging problem MBLS require zinc, for enzyme activity and as a result can be inhibited by (ethylenediamine tetra acetic acid) EDTA.

10. When EDTA is added to a bacterial colony capable of producing MBLs, resistance to carbapenems is attenuated. Measure the reduction to detect the presence of MBLs The Impenem-EDTA double –disk synergy test is the one of the tests designed to detect this reduction