Evaluation of a Double-Disk Synergy Test with a Common Metallo-^|^beta;-Lactamase Inhibitor, Mercaptoacetate, for Detecting NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii

Abstract

New Delhi metallo-b-lactamase (NDM)-1 carbapenemase-producing bacteria are resistant to antibiotics of the carbapenem family, which are used as a last resort for the treatment of infectious diseases caused by drugresistant bacteria. Therefore, the emergence of these bacteria presents a serious public health issue. This is particularly true given that NDM-1 carbapenemase has been detected in many clinical isolates worldwide since it was first identified in clinical isolates of Klebsiella pneumoniae and Escherichia coli from a patient hospitalized in New Delhi (1). The NDM-1 carbapenemase gene has been predominantly identified in Enterobacteriaceae, but it can also occur in non-fermenters (2). Hence, it is necessary to monitor the emergence and spread of NDM-1 producers with a convenient and effective screening method.NDM-1 carbapenemase is a class B zinc metallo-blactamase (MBL) (3). A variety of techniques have already been developed to detect MBL producers (4), including a disk-based synergy test, the sodium mercaptoacetate (SMA) test, which is conventionally used in Japanese clinical microbiology laboratories (5). This test uses a Kirby-Bauer (KB) disk containing a b-lactam antibiotic (ceftazidime [CAZ] recommended) and a disk containing SMA, an MBL inhibitor that can bind to the MBL active site through interactions with zinc ions (6). Although this test works well for the detection of IMPand VIM-type MBL producers, which are the predominant MBL types in Japan (7), preliminary results have indicated that it may fail to detect NDM-1 producers when using the SMA test with the CAZ disk according to general recommendations (8). Hence, the present study aimed at improving the SMA test by replacing the CAZ disk for detecting NDM-1 producers among Enterobacteriaceae and Acinetobacter baumannii.A collection of 15 NDM-1-positive bacterial isolates (5 E. coli, 4 K. pneumoniae, 1 Enterobacter cloacae, 1 Citrobacter freundii, and 4 A. baumannii), obtained from hospitals in Vietnam in 2010, and 1 NDM-1-positive K. pneumoniae strain (MRY10-722) isolated in a hospital in Japan in 2010, were used in this study. These isolates were identified using the API 20E and Vitek2 systems (bioMerieux, Marcy l'Etoile, France). Identification of A. baumannii isolates was further confirmed by rpoB gene sequencing (9). The NDM-1 gene was detected using PCR analysis with specific primers as described previously (10).A total of 16 isolates were subjected to the SMA test. The inhibitory effect of CAZ was compared with that of the carbapenems, imipenem (IPM) and meropenem (MPM). Suspensions of the bacterial isolates were adjusted and spread on Mueller-Hinton agar plates according to the protocol recommended by the Clinical and Laboratory Standards Institute guidelines (11). The KB disks containing b-lactam antibiotics (CAZ [30 mg] or IPM [10 mg] or MPM [10 mg]) (Eiken Chemical Co., Ltd., Tokyo, Japan) were placed on the plates, and disks containing SMA (3 mg) (Eiken Chemical) were placed close to 1 b-lactam disk as shown in Fig. 1. The center-t...

Antimicrobial Profile and Phenotypic Metallo-β-Lactamase Detection of Acinetobacter baumannii Isolated From Clinical and Environmental Specimens

“…More recently, new methods for phenotypic detecting carbapenemase producing isolates have been described based on expansion of inhibition zone [19,20]. They have used other materials like mercaptoacetate and dipicolinic acid which shows statistical and practical benefits.…”