Metallo--lactamase enzymes (ML) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding ML-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (T m ). The real-time PCR assay was able to detect all ML-harboring clinical isolates, and the T m -assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of ML-producing gram-negative bacteria by molecular diagnostic laboratories.Since the first report of acquired metallo--lactamase (ML) in Japan in 1994 (15), genes encoding IMP-and VIMtype enzymes have spread rapidly among Pseudomonas spp. (1,5,10,13,14,16,18,(22)(23)(24), Acinetobacter spp. (3,4,17,21,29), and strains of Enterobacteriaceae (6,8,11,12,20,28). Moreover, new ML types have been described, such as SPM (25), GIM (2), and, more recently, SIM (9).The prevalence of ML-producing gram-negative bacilli has increased in some hospitals, particularly among clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. (21,23,27). Since ML production may confer phenotypic resistance to virtually all clinically available -lactams, the continued spread of ML is a major clinical concern (26). The aim of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding the ML-type enzymes so far described. The ML type identification was based on the characteristic amplicon melting peak.
MATERIALS AND METHODS
ML-harboring clinical isolates and ML-negative control strains.The strains used in this study are listed in Tables 1 and 2. The ML genotypes of the clinical isolates of gram-negative nonfermentative and fermentative bacteria harboring ML were previously characterized by PCR and sequencing. When applicable, these clinical isolates were also previously molecularly typed to ensure that genetically unrelated strains were used. Additionally, several American Type Culture Collection (ATCC; Manassas, VA) reference strains and laboratory strains were used as ML-negative controls (Table 2).DNA preparation. The microorganisms were grown on blood agar plates overnight at 37°C to ensure colony purity. Three or four bacterial colonies were taken from the blood agar plates and suspended in 200 l of DNase/RNase-free distilled water (Invitrogen, CA). Two microliters of this suspension was used as templates for further amplification.Primer design. The currently available reference sequences of the ML-encoding IMP-and VIM-type (http://www.lahey.org/studies/), SPM-1 (AJ492820), GIM-1 (AJ620678), and SIM-1 (AY887066) genes were downloaded from GenBank (National...
