产KPC肺炎克雷伯菌检测方法构建与耐药机制

The establishment of detection methods and analysis of drug resistance mechanism of KPC-producing Klebsieila pneumonia

目的探讨产肺炎克雷伯型碳青霉烯酶(KPC)肺炎克雷伯菌检测方法以及肺炎克雷伯菌对碳青霉烯类抗生素的耐药机制、可能的治疗药物。方法选取K-B法证实的耐碳青霉烯肺炎克雷伯菌36株纳入本研究,使用改良Hodge试验、Carba NP试验对菌株进行验证,随后采用PCR扩增、产物琼脂糖凝胶电泳实验,并对电泳阳性条带进行基因测序鉴定。结果 K-B法药敏试验显示36株耐碳氢烯类肺炎克雷伯菌,随后行Hodge验证,33株(91.7%)是产KPC肺炎克雷伯菌,对碳氢烯类耐药而对替加环素及米诺环素仍然敏感。对Hodge试验阳性33株再行Carba NP试验确证,其中27株(87.9%)阳性,6株(12.1%)阴性;对Hodge试验阳性33株行PCR扩增及电泳实验,共有27株出现目标条带(340bp)提示阳性,与Carba NP试验结果相一致。对出现目标条带的27株肺炎克雷伯菌再进行KPC-2耐药基因PCR扩增产物测序,均为KPC-2型耐药基因。结论 K-B法药敏试验加随后改良Hodge试验验证对于筛查、发现KPC肺炎克雷伯菌具有较高的符合度,为发现KPC肺炎克雷伯菌有效筛查方法。K-B法药敏试验证实对KPC肺炎克雷伯菌,替加环素等可能是有效治疗的药物之一。Hodge试验检测的KPC肺炎克雷伯菌,存在12.1%(6/33)的假阳性率。Carba NP试验及PCR扩增检测一致性好,具有确证产KPC肺炎克雷伯菌作用。随后的测序研究结果表明检测出的KPC肺炎克雷伯菌均为KPC-2,该型是引起肺炎克雷伯菌对碳青酶烯酶耐药的主要机制。

Objective To establish the detection methods of Klebsiella pneumoniae carbapenemase and the resistance mechanism of Klebsiella pneumoniae to carbapenem antibiotics,and possible therapeutic drugs. Methods Obtained 36 strains of carbapenem-restant Klebsiella pneumoniae identified by K-B method for the study. Carbapenemase production was confirmed by modified Hodge test and Carba NP test. The resistant genes were detected by PCR. The drug resistance genes of molecular epidemiology characteristics were analyzed by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction. Results A total of 36 strains of Klebsiella pneumonia showed a high level resistance to carbepenems,but susceptibility to the tigecycline. The 33 strains of 36 strains( 91. 7%) with resistance to carbepenems were positive in modified Hodge test,and which 27 strains( 87. 9%)were validated positive by Carba NP experiment,6 strains were negative. The 33 strains positive with modified Hodge test were detected by PCR amplification and electrophoresis images,a total of 27 strains of target strip( 340bp),the target band of the 27 strains of Klebsiella pneumoniae and the KPC-2 gene PCR gene sequencing,whole were KPC-2 type resistance gene,which was consistent with the experimental results of Carba NP. PCR detection and sequence analysis showed that all the positive strains with KPC-2 gene. Conclusion K-B method of drug sensitivity test plus subsequent improved Hodge test to verify the screening,find KPC Klebsiella pneumonia has a high degree of compliance,which is effective screening method to find KPC Klebsiella pneumoniae. The tigecycline may be a valuable drug for KPC-producing Klebsieila pneumonia by the evidence of K-B method. There is a false positive rate of 12. 1%( 6/33) in the detection of Klebsiella pneumoniae carbapenemase in the Hodge test. The Carba NP test and PCR amplification show highly conformity in a definite diagnosis of carbapenem-restant Klebsiella pneumonia strains. The KPC-2 type of carbapenem-restant Klebsiella pneumonia is the main reason to cause the bacterial resistance to carbapenems.