NDM-1 positive bacteria have been found not only in clinical specimens, but also in drinking water and seepage in New Delhi [10]. The first case of a NDM-1 producing E. coli (NDM-1 Dok01) infection in Japan was reported in 2010 [11]. This organism was isolated from the blood culture of a patient who had been hospitalized in India. The complete sequence of the NDM-1-bearing plasmid was also reported (GenBank accession number AP012208) [12]. Rapid detection of MBL-producing strains, including NDM-1 producers, is necessary to

prevent their dissemination and associated nosocomial infections. Researchers have developed several phenotypic methods to detect MBL production. These tests include DDSTs using 2-mercaptoacetic acid or Metformin clinical trial EDTA, combined disk tests with dipicolinic acid or EDTA, Etest MBL (BioMérieux,

Durham, NC, USA) and the modified CH5424802 concentration Hodge test [13-17]. The DDSTs using SMA with CAZ or IPM disks are simple methods and commonly used in clinical laboratories in Japan. However, the growth-inhibitory zone does not enhance sufficiently when the DDST using SMA with CAZ is performed for NDM-1 Dok01. In addition, with IPM disks, the results are equivocal because the enhancement of the zone of inhibition is only 4 mm, which researchers have interpreted as negative [11]. Aoki et al. reported that calcium disodium EDTA, a metal–EDTA complex that incorporates calcium ions into EDTA, is an effective

inhibitor of MBL [18]. The purpose of this study was to evaluate the efficacy of detection of MBL, including NDM-1, of DDSTs using seven kinds of metal-EDTA complexes. NDM-1 Dok01 was isolated at Dokkyo Medical University Hospital. K. pneumoniae ATCC BAA-2146 was used as a quality control strain that produces NDM-1. Strains evaluated were stock cultures of known MBL-producing strains of 46 P. aeruginosa, 7 A. baumannii, 5 P. putida, 3 E. coli, 2 Achromobacter xylosoxidans, 2 E. cloacae, 2 Serratia marcescens, 2 K. pneumoniae, 1 K. oxytoca, 1 Citrobacter freundii, 1 Pseudomonas spp., and 1 Acinetobacter spp. Non-MBL producing strains of 7 K. pneumoniae, 1 K. oxytoca, 6 E. coli, 3 C. freundii, 4 P. aeruginosa, and 4 A. baumannii were also evaluated. Minimum inhibitory PLEKHM2 concentrations were determined by the broth microdilution method, which was performed on Dry Plate Eiken DPD1 (Eiken Chemical, Tokyo, Japan) according to the manufacturer’s instructions. Sodium mercaptoacetic acid and seven types of metal-EDTA complexes were used as MBL inhibitors. Metallo-β-lactamase SMA Eiken (SMA disk; Eiken Chemical) contains 3 mg of SMA. Ca-EDTA, Mg-EDTA, Co-EDTA, Cu-EDTA, Mn-EDTA, Fe-EDTA and Zn-EDTA were purchased from Dojindo Laboratories (Dojindo Laboratories, Kumamoto, Japan). These seven metal-EDTA complexes were dissolved in water at concentrations that provided maximum solubility.