Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide

Strains of Enterobacteriaceae producing an extended spectrum P-lactamase have become a concern in medical bacteriology as regards both antimicrobial treatment and infection control in hospitals. Extended-spectrum P-lactamase (ESBL) detection tests should accurately discriminate between bacteria producing these enzymes and those with other mechanisms of resistance to beta-lactams, e.g., broadspectrum beta-lactamases, inhibitor-resistant beta-lactamases and cephalosporinase overproduction. Several phenotypic detection tests, based on the synergy between a third-generation cephalosporin and clavulanate, have been designed: the double-disk synergy test (DDST), ESBL Etests, and the combination disk method. These tests often need to be refined in order for them to detect an ESBL in some bacterial strains, such as those that also overproduce a cephalosporinase. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The use of cefepime, a fourth-generation cephalosporin that is less rapidly inactivated by cephalosporinase than by ESBL, improves the detection of synergy with clavulanate when there is simultaneous stable hyperproduction of a cephalosporinase; alternatively, the cephalosporinase can be inactivated by performing phenotypic tests on a cloxacillin-containing agar. Some beta-lactamases can hydrolyse both third-generation cephalosporins and carbapenems, such as the metallo-beta-lactamases, which are not inhibited by clavulanate, but rather by EDTA. The production of an ESBL masked by a metallo-beta-lactamase can be detected by means of double inhibition by EDTA and clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly inhibited by clavulanate and not inhibited by EDTA, their detection is difficult in the routine laboratory.