The purpose of this study was to simultaneously screen for Extended-spectrum β-lactamases (ESBL) and AmpC β-lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC β-lactamases. A total of 272 isolates were screened for ESBL and AmpC β-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime and clavulanate were considered as ESBL producer. Isolates showing reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test. A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli
and 71 (73%) of Klebsiella
spp. The occurrence of AmpC β-lactamases was found to be 8% (22) of the total isolates and the two detection methods for AmpC β-lactamase showed concordant results. Screening for ESBL and AmpC can be simultaneously done by MDDM method and confirmation for AmpC β-lactamase should be carried out routinely in tertiary care hospitals by AmpC disk test, as it is a simple and rapid procedure.