基于ompK36突变快速检测产KPC肺炎克雷伯菌亚胺培南最低抑菌浓度的方法学建立

Establishment of a method for rapid detection of the minimum inhibitory concentration of imipenem in KPC-producing Klebsiella pneumoniae based on ompK36 mutation

目的基于ompK36基因GD突变,建立一种快速检测产肺炎克雷伯菌碳青霉烯酶的肺炎克雷伯菌(KPC-Kp)亚胺培南最低抑菌浓度(MIC)的方法。方法方法学建立。收集丽水市中心医院2011年3月至2019年12月的非重复肺炎克雷伯菌258株,PCR扩增膜孔蛋白o mpK36基因以及碳青霉烯酶基因bla_(KPC)、bla_(NDM)、bla_(IMP)、bla_(OXA-48)并测序确认,微量肉汤稀释法检测菌株亚胺培南MIC值,建立基因型与MIC值的对应模式。基于该模式,设计建立实时荧光聚合酶链反应(RT-PCR)快速检测MIC值的方法。利用丽水市疾控中心2017—2019年收集的159株非重复肺炎克雷伯菌进一步验证。四格表计算敏感度、特异度,Kappa检验比较RT-PCR法与肉汤稀释法结果的一致性。结果258株肺炎克雷伯菌中,109株未携带碳青霉烯酶基因,65株携带ompK36基因GD突变,127株携带bla_(KPC),15株携带bla_(NDM),9株携带bla_(IMP),未检测到bla_(OXA-48)。以肉汤稀释法为标准,肺炎克雷伯菌耐药基因与亚胺培南MIC值存在3种对应模式:4种碳青霉烯酶基因均阴性时,MIC≤1 mg/L,敏感度为100%(107/107),特异度为98.4%(125/127);bla_(KPC)阳性而ompK36基因GD突变阴性时,4 mg/L≤MIC≤16 mg/L,敏感度为88.2%(60/68),特异度为98.8%(164/166);bla_(KPC)和ompK36基因GD突变双阳性时,MIC≥32 mg/L,敏感度为96.6%(57/59),特异度为96.6%(169/175)。RT-PCR法可准确检测bla_(KPC)、bla_(NDM)、bla_(IMP)、bla_(OXA-48)基因;在产酶菌株中o mpK36基因GD突变的RT-PCR结果与测序法100%一致。以丽水市疾控中心来源的159株肺炎克雷伯菌为样本,以肉汤稀释法为参照,RT-PCR检测亚胺培南MIC值在3种模式下敏感度和特异度均大于95%,Kappa值为0.971。结论基于ompK36基因GD突变建立的RT-PCR法有助于快速判断KPC-Kp的亚胺培南MIC值范围。

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 bla_(KPC),bla_(NDM),bla_(IMP)and bla_(OXA-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 bla_(KPC),15 isolates carried bla_(NDM),9 isolates carried bla_(IMP),and bla_(OXA-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 bla_(KPC)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 bla_(KPC)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 bla_(KPC),bla_(NDM),bla_(IMP),bla_(OXA-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.