ABSTRACT
Objective:To establish a rapid method to detect the minimum inhibitory concentration (MIC) of imipenem in Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) based on ompK36 gene′s GD mutation. Methods:This was a methodological evaluation study. A total of 258 isolates of Klebsiella pneumoniae were collected from Lishui Municipal Central Hospital from March 2011 to December 2019. Porin gene ompK36 and carbapenemase genes blaKPC, blaNDM, blaIMP and blaOXA-48 were amplified by PCR and confirmed by sequencing. The MIC was detected and confirmed by microbroth dilution susceptibility test, and the corresponding patterns of genotype and MIC were constructed. Based on the patterns, a method for rapid detection of imipenem MIC by real-time fluorescence PCR (RT-PCR) was designed and established. The 159 isolates of non-repetitive Klebsiella pneumoniae collected by Lishui Disease Prevention and Control Center (CDC) from 2017 to 2019 were used for further verification. The sensitivity and specificity were calculated by fourfold table. Kappa test was used to compare the consistency between RT-PCR and microbroth dilution susceptibility test. Results:Among 258 isolates, 109 isolates did not carry carbapenemase gene, 65 isolates carried ompK36 gene GD mutation, 127 isolates carried blaKPC, 15 isolates carried blaNDM, 9 isolates carried blaIMP, and blaOXA-48 was not detected. With mircobroth dilution susceptibility test as the standard, there were 3 corresponding patterns between the drug resistance gene and the imipenem MIC of Kp: when all the 4 carbapenemase genes were negative, MIC≤1 mg/L, the sensitivity was 100% (107/107) and the specificity was 98.4% (125/127); when blaKPC was positive and ompK36 gene GD mutation was negative, 4 mg/L≤MIC≤16 mg/L, the sensitivity was 88.2% (60/68) and the specificity was 98.8% (164/166); when blaKPC and ompK36 gene GD mutation were both positive, MIC≥32 mg/L, the sensitivity was 96.6% (57/59) and the specificity was 96.6% (169/175). RT-PCR detected blaKPC, blaNDM, blaIMP, blaOXA-48 genes accurately.The RT-PCR results of ompK36 gene GD mutation in the KPC-producing isolates were 100% consistent with the sequencing results. In the 159 isolates from Lishui CDC, the sensitivity and specificity of imipenem MIC detected by RT-PCR were higher than 95% in all 3 patterns with mircobroth dilution susceptibility test as the standard, and Kappa value was 0.971. Conclusion:The RT-PCR based on ompK36 gene GD mutation was helpful to quickly determine the MIC range of imipenem in KPC-Kp.