Convenient Test for Screening Metallo-β-Lactamase-Producing Gram-Negative Bacteria by Using Thiol Compounds

Abstract

ABSTRACT A simple disk diffusion test was constructed for detection of IMP-1-type metallo-β-lactamase-producing gram-negative bacteria. Two Kirby-Bauer disks containing ceftazidime (CAZ) and a filter disk containing a metallo-β-lactamase inhibitor were used in this test. Several IMP-1 inhibitors such as thiol compounds including 2-mercaptopropionic acid, heavy metal salts, and EDTA were evaluated for this test. Two CAZ disks were placed on a Mueller-Hinton agar plate on which a bacterial suspension was spread according to the method recommended by the National Committee for Clinical Laboratory Standards. The distance between the disks was kept to about 4 to 5 cm, and a filter disk containing a metallo-β-lactamase inhibitor was placed near one of the CAZ disks within a center-to-center distance of 1.0 to 2.5 cm. For IMP-1-producing strains, the growth-inhibitory zone between the two disks expanded, while no evident change in the shape of the growth-inhibitory zone was observed for CAZ-resistant strains producing serine β-lactamases such as AmpC or SHV-12. As a result, 2 to 3 μl of undiluted 2-mercaptopropionic acid or mercaptoacetic acid able to block IMP-1 activity gave the most reproducible and clearest results, and CAZ-resistant strains producing AmpC or extended-spectrum β-lactamases were distinguishable from IMP-1 producers by this test. A similar observation was made with IMP-1-producing clinical isolates such as Serratia marcescens , Klebsiella pneumoniae , Escherichia coli , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Proteus vulgaris , Pseudomonas aeruginosa , Pseudomonas putida , Acinetobacter spp., and Alcaligenes xylosoxidans . The specificity and sensitivity of this test were comparable to those of PCR analysis using bla IMP -specific primers. Therefore, this convenient test would be valuable for daily use in clinical laboratories.