A New Multiplex PCR Method for Rapid Detection of Genes Encoding VIM and IMP Types of Metallo Beta Lactamases

Abstract

Objective: This study describes a multiplex polymerase chain reaction (PCR) assay protocol for rapid detection of genes encoding VIM and IMP types of metallo beta lactamases (MBL). This assay is also used for evaluate the presence and types of MBL among Pseudomonas aeruginosa isolates in a large university hospital in Istanbul. Material and Methods: bla IMP1-22 and blaVIM1-12 prototype sequences were downloaded from GenBank and aligned using ClustalW multiple alignment program in Bioedit software package. Following optimisation using different concentrations and combination of primers, the 1.2 VIM/IMP ratio for primers were determined and used throughout the screening assays. The multiplex PCR protocol was used to screen 51 P.aeruginosa isolates resistant or intermediate resistant to imipenem. Minimum inhibitory concentrations (MIC) of imipenem, meropenem, ceftazidime and cefepime were determined by the agar dilution method. Control strains comprised of P.aeruginosa strains producing IMP-1, IMP-2, IMP13, VIM-1, VIM-2, VIM-4 and a Klebsiella pneumoniae strain producing VIM-5. Results: The multiplex PCR method was able to detect all the control strains producing VIM and IMP types of MBL genes. No isolate was found to be positive for bla VIM and bla IMP genes among 51 P.aeruginosa isolates with the multiplex PCR method. MIC50 values for P.aeruginosa isolates of imipenem, meropenem, ceftazidime and cefepime were 32 mu g/ml, 32 mu g/ml, 128 mu g/ml, 64 mu g/ml, respectively. Conclusion: The multiplex PCR assay described in this study could be helpful for monitoring the epidemiology of VIM and IMP types of MBL in different clinical settings.