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Methods for detection β-lactamases
Sarah Alharbi
B-lactam antibiotics
The most commonly used class of agents to treat bacterial infections
They consist of four major groups: penicillins, cephalosporins, monobactams and
Carbapenems
Figure.1 Structure of B-lactam ring
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
B lactamase inhibitor
Example, clauvulinc acid, sulbactam, and tazobactam
- Clavulanic acid does not have any antibacterial properties of its own,
- Irreversibly binding to the β-lactamase molecules
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)
B – lactamase detection ( chromogenic cephalosporinase test/ nitrocefin test.
Nitrocefin is a chromogenic cephalosporin that changes from yellow to red
on hydrolysis .
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
2) Metallo based B lactamase
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
negative
positive
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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.
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