Detection of metallo-β-lactamase genes in clinical specimens by a commercial multiplex PCR system
Introduction
Dissemination of Gram-negative bacteria producing metallo-β-lactamases (MβLs) is of great concern. A variety of acquired MβLs, including IMP, VIM, SPM, GIM, and SIM have been described in species of clinical importance (Queenan and Bush, 2007). MβLs exhibit wide hydrolysis spectra affecting virtually all β-lactams including carbapenems. Moreover, MβL-encoding genes are commonly carried by multi-resistant integrons. Therapeutic choices in the respective infections are thus limited. MβLs, mainly of the VIM and IMP types, have been widely diffused among non-fermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii and enterobacteria (mainly Klebsiella pneumoniae) (Walsh et al., 2005, Queenan and Bush, 2007) and in some countries, as in the case of Greece, have reached endemic proportions (Psichogiou et al., 2008). The early and accurate detection of MβL producers would facilitate administration of the appropriate therapy and the implementation of infection control measures, especially in high prevalence settings. Novel detection approaches utilizing molecular methods have been proposed for the timely detection of MβL producers (Cornaglia et al., 2007, Miriagou et al., 2010, Walsh et al., 2005). In this study, a new diagnostic method for the direct detection of MβL genes of the VIM and IMP types in clinical specimens, the hyplex® MBL ID Multiplex PCR-ELISA (AmplexDiagnostics, Gars, Germany), was evaluated. This system involves amplification of blaVIM/blaIMP bacterial DNA by multiplex PCR, reverse hybridisation of the PCR products to oligonucleotide probes specific for blaIMP (IMP-1 to IMP-22) and blaVIM (VIM-1 to VIM-13) genes that have been immobilized on polystyrene ELISA-plates, and detection of the hybridisation complexes by a peroxidase conjugated antibody.
Section snippets
Clinical samples
A total of 326 non-repetitive (one per patient) clinical samples received during September 2007 in the clinical microbiology laboratories of three tertiary care hospitals in Athens, known for their high prevalence of MβL producers (Giakkoupi et al., 2003a, Giakkoupi et al., 2003b, Psichogiou et al., 2008), were included in the study. Samples, including urine (n = 60), pus swabs (n = 91), bronchial secretions/sputum (n = 85), and positive blood cultures (n = 90), each containing at least one
Results and discussion
A total of 73 VIM-positive microorganisms were identified in 326 clinical samples confirming the high prevalence of VIM producers in this setting (Vatopoulos, 2008). Among VIM producers, P. aeruginosa was predominant (n = 42) followed by K. pneumoniae (n = 25) and Acinetobacter spp. (n = 6). VIM-positive isolates of various enterobacterial species were also identified in a sporadic fashion. The higher frequency of VIM producers was observed in respiratory samples (47%). The respective frequency in
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