耐碳青霉烯类药物肺炎克雷伯菌β-内酰胺酶与膜孔蛋白编码基因以及KPC—ISKpn6连锁检测

Detection of beta-lactamase, porin-coding genes and linkage of KPC-ISKpn6 of the carbapenems-re- sistant Klebsiella pneumoniae

目的探讨耐碳青霉烯类药物肺炎克雷伯菌的耐药机制。方法对南京地区两家三甲综合性医院的耐碳青霉烯抗菌药物肺炎克雷伯菌临床分离株，采用PCR方法对其40种β-内酰胺酶基因、膜孔蛋白编码基因及KPC-ISKr，n6连锁进行检测，PCR阳性产物进行测序，测序结果BLAST对比分析。结果24株耐碳青霉烯类药物肺炎克雷伯菌的A类β-内酰胺酶编码基因TEM-1及SHV的阳性检出率为100％（24／24）、KPC-2的阳性检出率为95．8％（23／24）、LAP-2的阳性检出率为45．8％（11／24），C类β-内酰胺酶编码基因DHA的阳性检出率为4．2％（1／24），KPC—ISKpn6连锁检测阳性率为95．8％（23／24），膜孔蛋白编码基因ompK35与ompK36的突变率分别为95．8％（23／24）及100％（24／24）。结论本组肺炎克雷伯菌β-内酰胺酶TEM-1、SHV、KPC-2、LAP-2的携带率较高，其中KPC-2的高携带率及膜孔蛋白编码基因ompK35、ompK36的高突变率是本组肺炎克雷伯菌对碳青霉烯类抗菌药物耐药的主要机制；插入序列ISKpn6可能参与了KPC-2基因的介导。

Objective To investigate the resistance-mechanism of the carbapenems-resistant K/eb- siella pneumoniae isolated from clinical. Methods The clinical isolates of carbapenems-resistant Klebsiella pneumoniae from top three comprehensive hospitals of Nanjing area were examined by 40 beta-lactamase, pot- in-coding genes and linkage of KPC-ISKpn6 using PCR method, the PCR positive results were picked out for sequencing and sequencing BLAST search for comparison analysis. Results Twenty-four strains of carbapen- ems-resistant Klebsiella pneumoniae were detected, the positive rate of A beta-lactamase TEM-1 and SHV was 100% （24/24） ,KPC-2 and LAP-2 was 95.8% （23/24） ,45.8% （11/24） respectively, and C beta-lacta- mase DHA was 4. 2% （1/24）. Meanwhile, the positive detection rates of KPC-ISKpn6 linkage was 95. 8% （23/24） ,and the mutation rate of porin-coding genes ompK35 and ompK36 were up to 95.8% （23/24） and 100% （24/24）. Conclusion High incidence of beta-lactamase TEM-1, SHV, KPC-2 and LAP-2 was found in the group of Klebsiella pneumoniae isolates, and carbapenems-resistant of which was primarily due to the high carrying rate of KPC-2 and the high mutation rate of porin-coding genes ompK35 and ompK36. The In- sertion sequence ISKpn6 may be involved in the KPC-2 gene mediated-expression.