Multiplex PCR for rapid detection of genes encoding classA carbapenemases

Abstract number: P1864

Hong S.G., Hong S.K., Huh J.Y., Kang M.S.

Objectives: Class A carbapenemases (CACs) include the SME, IMI/NMC-A, SFC, KPC, and some type of GES families. The genes for SME, IMI/NMC-A, SFC enzymes are all chromosomally located except IMI-2 and the genes for KPC and GES enzymes are carried on plasmid. KPC producers have induced severe treatment problems in hospitals around New York and have also been reported in Europe, South America and China. Therefore, the confirmation of type of CACs is important to ensure optimal therapy and to prevent their spread. This study was to develop a multiplex PCR assay to detect and differentiate CAC genes in a single reaction.

Methods: The strains comprised 11 CAC-producers (1 SME-producing Serratia marcescens, 2 IMI/NMC-producing Enterobacter cloaceae, 2 KPC-producing Enterobacteriaceae and 6 GES-producing Klebsiella pneumoniae), 8 metallo-b-lactamase (MBL) producers (2 IMP-producing Pseudomonas aeruginosa, 3 VIM-producing P. aeruginosa, 1 IMP-producing Acinetobacter baumannii, 1 SIM-producing A. baumannii and 1 VIM-producing Alcaligenes feacalis), and 5 non-carbapenemase-producing Enterobacteriaceae. Four primer pairs were designed to amplify fragments of 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The PCR were done for the detection of CAC genes with above strains.

Results: The multiplex PCR detected all the genes for 4 CAC families that could be differentiated by the fragments size according to the gene types. All non-CAC producers did not show PCR product bands.

Conclusion: This multiplex PCR appears to be a simple and useful approach to detecting and distinguishing CAC genes in carbapenem resistant strains that show negative results to the detecting test for MBL producers. Therefore, this method should be helpful for characterization of CACs and in controlling the spread of pathogens producing these enzymes.