Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds

A simple disk diffusion test was constructed for detection of IMP-1-type me talla-beta-lactamase-producing gram-negative bacteria. Two Kirby-Bauer disk s containing ceftazidime (CAZ) and a filter disk containing a metallo-beta- lactamase inhibitor were used in this test. Several IMP-1 inhibitors such a s thiol compounds including 2-mercaptopropionic acid, heavy metal salts, an d 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 St andards. The distance between the disks was kept to about 4 to 5 cm, and a filter disk containing a metallo-beta-lactamase inhibitor was placed near o ne of the CAZ disks within a center-to center distance of 1.0 to 2.5 cm, Fo r IMP-1-producing strains, the growth-inhibitory zone between the two disks expanded, while no evident change in the shape of the growth-inhibitory zo ne was observed for CAZ-resistant strains producing serine beta-lactamases such as AmpC or SHV-12. As a result, 2 to 3 mu l of undiluted Zmercaptoprap ionic acid or mercaptoacetic acid able to block IMP-1 activity gave the mos t reproducible and dearest results, and CAZ-resistant strains producing Amp C or extended-spectrum beta-lactamases were distinguishable from IMP-1 prod ucers by this test. A similar observation was made with IMP-1-producing cli nical isolates such as Serratia marrescens, Klebsiella pneumoniae, Escheric hia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundi i, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Acinetobac ter 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.