Rapid Detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring blaVIM-2, blaIMP-1 and blaOXA-23 Genes by Using Loop-Mediated Isothermal Amplification Methods
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Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of blaVIM-2, blaIMP-1, and blaOXA-23, which are critical components for carbapenem resistance. Methods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized blaVIM-2, blaIMP-1, and blaOXA-23 LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. Results: Only one strain of the 100 CRPA isolates harbored blaIMP-1, whereas none of them harbored blaVIM-2. These results indicate that the acquisition of blaVIM-2 or blaIMP-1 may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained blaOXA-23, but none contained blaVIM-2 and blaIMP-1 alleles. Conclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
Abstract
Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of blaVIM-2, blaIMP-1, and blaOXA-23, which are critical components for carbapenem resistance. Methods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized blaVIM-2, blaIMP-1, and blaOXA-23 LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. Results: Only one strain of the 100 CRPA isolates harbored blaIMP-1, whereas none of them harbored blaVIM-2. These results indicate that the acquisition of blaVIM-2 or blaIMP-1 may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained blaOXA-23, but none contained blaVIM-2 and blaIMP-1 alleles. Conclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
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