Poly NP试验及MPNP试验快速检测肺炎克雷伯菌多粘菌素类耐药表型的应用评估

目的:探讨快速多粘菌素NP试验（rapid polymyxin NP test,Poly NP test）对多粘菌素类耐药肺炎克雷伯菌的检测价值；进一步评估改良快速多粘菌素NP试验（modified rapid polymyxin Nordmann/Poirel test,MPNP）对产MCR肺炎克雷伯菌的检出价值。方法:本研究共纳入48株菌，包括14株多粘菌素类敏感肺炎克雷伯菌（polymyxin sensitive Klebsiella pneumoniae,PolS-KP），29株多粘菌素类耐药细菌（26株为多粘菌素类耐药肺炎克雷伯菌(polymyxin resistant Klebsiella pneumoniae,PolR-KP)，3株为多粘菌素类天然耐药菌），以及5株非发酵菌。以微量肉汤稀释法的结果为标准，采用Poly NP试验检测多粘菌素类的药敏表型。PCR及测序确定肺炎克雷伯菌的多粘菌素类染色体介导的耐药基因pmrA/pmrB,phoP/phoQ,mgrB和质粒介导的耐药基因mcr-1～mcr-8，采用MPNP试验检测是否为产MCR阳性菌株。结果:29株多粘菌素类耐药菌，3株天然耐药细菌的Poly NP试验均为阳性；26株PolR-KP中，24株为Poly NP试验阳性，2株为阴性；14株PolS-KP均为阴性，且所有结果均在2 h内出现最终颜色变化。该方法判断肺炎克雷伯菌多粘菌素类耐药表型的敏感性和特异性为93.1%和100%。26株PolR-KP中，有4株为MCR阳性，其中3株产mcr-1，1株产mcr-8；22株为染色体介导的耐药，其中12株为双组分系统PmrAB突变，4株为双组分系统PhoPQ突变，6株为mgrB基因改变。4株MCR阳性菌株的MPNP试验表现为阴性结果，其余非MCR株的PolR-KP和PolS-KP，以及多粘菌素类天然耐药菌株的MPNP均表现为阴性结果。MPNP试验不能筛选出MCR阳性的肺炎克雷伯菌。结论:Poly NP试验快速，特异性强，敏感性高，可用于临床多粘菌素类耐药肺炎克雷伯菌的快速检测。MPNP试验对MCR阳性肺炎克雷伯菌检出不佳，仍需进一步探讨适用于肺炎克雷伯菌的MCR快速表型检测方法。

Evaluation of Poly NP test and MPNP test for rapid detection of the polymyxin resistance Klebsiella pneumoniae

AIM: To evaluate the screening capacity of rapid polymyxin NP test (Poly NP test) for the detection of polymyxin B resistance Klebsiella pneumoniae (PolR-KP) and the modified rapid polymyxin Nordmann/Poirel test (MPNP) for the MCR-producing in K.pneumoniae. METHODS: A total of 48 isolates were selected, in which, 14 strains were polymyxin sensitive Klebsiella pneumoniae (PolS-KP), 26 strains were PolR-KP, 3 strains were polymyxin intrinsic resistance and 5 strains of nonfermenters. The rapid polymyxin NP test was used to detect the polymyxin resistance phenotype and the results were compared with the MICs determined by the BMD method, which was taken as the gold standard. The PCR and sequencing were used to screen the polymyxin resistance determinants caused by chromosomal mutations-the two-component phoP/phoQ, pmrA/pmrB and the mgrB gene; and also screen the presence of mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7 and mcr-8. The MCR-producing Klebsiella pneumoniae isolates were detected by MPNP test. RESULTS:All the polymyxin intrinsic resistance strains were positive in Poly NP test, of the 26 isolates PolR-KP, 24 PolR-KP strains were positive in Poly NP test and only 2 strains showed negative result. The sensitivity and specificity of Poly NP test were 93.1% and 100%. Of the 22 isolates which polymyxin resistance was associated with chromosome-encoded mechanisms, 12 isolates had mutations in the pmrA/pmrB two-component system, 4 isolates in the phoP/phoQ two-component system and 6 isolates had different alterations in mgrB gene. 4 strains of MCR-producing K. pneumoniae were detected in PolR-KP and the MPNP test showed lack of inhibitory effect of EDTA on the mcr-positive K. pneumoniae strains. CONCLUSION: The Poly NP test is an easy-to-perform, rapid, sensitive, and specific test and making it a potential useful clinical technique for the detection of PolR-KP, while the MCR-positive K.pneumoniae strains are not identified by the MPNP test. Further methods suitable for rapid detection of the MCR-producing in K.pneumoniae are uegently needed.