耐碳青霉烯类抗菌药物肠杆菌科细菌的耐药基因分析

doi:10.3969/j.issn.1673-8640.2017.011.010

摘要/Abstract

摘要：

目的分析郑州大学第一附属医院耐碳青霉烯类抗菌药物肠杆菌科细菌耐药基因的携带和分布情况。方法采用Vitek 2 Compact全自动微生物分析系统筛选2014年6月—2015年12月临床分离的大肠埃希菌和肺炎克雷伯菌,根据美国临床实验室标准化协会（CLSI）2013年文件判读,排除重复分离菌株,选出对亚胺培南/美罗培南耐药菌株共29株;采用聚合酶链反应（PCR）对KPC、NDM、VIM、SPM、GIM、IMP、OXA23、OXA24、OXA51和OXA58等相关基因进行检测。结果在分离的29株耐药菌株中共检出KPC阳性11株,其中肺炎克雷伯菌10株、大肠埃希菌1株;检出NDM阳性7株,其中肺炎克雷伯菌3株、大肠埃希菌4株,并有2株检测出变异基因NDM-5。结论郑州大学第一附属医院耐碳青霉烯类抗菌药物肠杆菌科细菌的主要耐药机制为携带KPC和NDM型碳青霉烯酶基因。

关键词: 碳青霉烯类抗菌药物, 肠杆菌科细菌, KPC, NDM-5, 三维结构

Abstract:

Objective To investigate the drug resistance genes in carbapenem-resistant Enterobacteriaceae isolated from the First Affiliated Hospital of Zhengzhou University. Methods The clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from June 2014 to December 2015,and they were screened by Vitek 2 Compact automatic bacterium identification system,according to the Clinical and Laboratory Standards Institute（CLSI） 2013. A total of 29 isolates being resistant to imipenem/meropenem were sorted out except repeated isolates. The encoding genes of KPC,NDM,VIM,SPM,GIM,IMP,OXA23,OXA24,OXA51 and OXA58 were analyzed by polymerase chain reaction（PCR）. Results Among the 29 isolates,there were 11 isolates of KPC positive（10 isolates of Klebsiella pneumoniae and 1 isolate of Escherichia coli）. A total of 7 isolates were NDM positive（3 isolates of Klebsiella pneumoniae and 4 isolates of Escherichia coli）,and 2 isolates were identified as variant gene NDM-5. Conclusions Resistance to carbapenems is mainly due to the production of KPC and NDM in Enterobacteriaceae.

Key words: Carbapenems, Enterobacteriaceae, KPC, NDM-5, Three-dimensional structure

中图分类号:

R446.5

何建业, 曹在秋, 代志峰. 耐碳青霉烯类抗菌药物肠杆菌科细菌的耐药基因分析[J]. 检验医学, 2017, 32(11): 985-989.

HE Jianye, CAO Zaiqiu, DAI Zhifeng. Drug resistance genes in carbapenem-resistant Enterobacteriaceae[J]. Laboratory Medicine, 2017, 32(11): 985-989.

图/表 2

参考文献 17

[1]	NORDMANN P,DORTET L, POIREL L.Carbapenem resistance in Enterobacteriaceae:here is the storm![J]. Trends in Mol Med,2012,18(5):263-272.
[2]	PASTERAN F,MEO A,GOMEZ S,et al.Emergence of genetically related NDM-1-producing Providencia rettgeri strains in Argentina[J]. J Glob Antimicrob Resist,2014,2(4):344-345.
[3]	KHAJURIA A,PRAHARAJ A K,KUMAR M,et al.Emergence of NDM-1 in a clinical isolate of Pantoea agglomerans from India[J]. J Glob Antimicrob Resist,2014,2(4):340-341.
[4]	QIN S,ZHOU M,ZHANG Q,et al.First identification of NDM-4-producing Escherichia coli ST410 in China[J]. Emerg Microbes Infect,2016,5(11):e118.
[5]	COSTA PDE O,ATTA E H,SILVA A R.Predictors of 7- and 30-day mortality in pediatric intensive care unit patients with cancer and hematologic malignancy infected with Gram-negative bacteria[J]. Braz J Infect Dis,2014,18(6):591-599.
[6]	NORDMANN P,NAAS T,POIREL L.Global spread of carbapenemase-producing Enterobacteriaceae[J]. Emerg Infect Dis,2011,17(10):1791-1798.
[7]	WAN Q Q,YE Q F,YUAN H.Multidrug-resistant Gram-negative bacteria in solid organ transplant recipients with bacteremias[J]. Eur J Clin Microbiol Infect Dis,2015,34(3):431-437.
[8]	DJAHMI N,DUNYACH-REMY C,PANTEL A,et al.Epidemiology of carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean countries[J]. Biomed Res Int,2014,2014:305784.
[9]	LI H,ZHANG J,LIU Y,et al.Molecular characteristics of carbapenemase-producing Enterobacteriaceae in China from 2008 to 2011:predominance of KPC-2 enzyme[J]. Diagn Microbiol Infect Dis,2014,78(1):63-65.
[10]	SCHUURMANS J M,VAN HIJUM S A,PIET J R,et al. Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains[J]. Plasmid,2014,72:1-8.
[11]	JACQUIER H,MARCADE G,RAFFOUX E,et al.In vivo selection of a complex mutant TEM(CMT) from an inhibitor-resistant TEM(IRT) during ceftazidime therapy[J]. J Antimicrob Chemother,2013,68(12):2792-2796.
[12]	RAHMAN M,SHUKLA S K,PRASAD K N,et al.Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1,NDM-5,NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India[J]. Int J Antimicrob Agents,2014,44(1):30-37.
[13]	HORNSEY M,PHEE L,WAREHAM D W.A novel variant,NDM-5,of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom[J]. Antimicrob Agents Chemother,2011,55(12):5952-5954.
[14]	GHATAK S,SINGHA A,SEN A,et al.Detection of New Delhi metallo-beta-lactamase and extended-spectrum beta-lactamase genes in Escherichia coli isolated from mastitic milk samples[J]. Transbound Emerg Dis,2013,60(5):385-389.
[15]	SASSI A,LOUCIF L,GUPTA S K,et al.NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria[J]. Antimicrob Agents Chemother,2014,58(9):5606-5608.
[16]	HAMMERUM A M,HANSEN F,OLESEN B,et al.Investigation of a possible outbreak of NDM-5-producing ST16 Klebsiella pneumoniae among patients in Denmark with no history of recent travel using whole-genome sequencing[J]. J Glob Antimicrob Resist,2015,3(3):219-221.
[17]	TELLO A,AUSTIN B,TELFER T C.Selective pressure of antibiotic pollution on bacteria of importance to public health[J]. Environ Health Perspect,2012,120(8):1100-1106.

目的基因	引物名称	引物序列（5'~3'）	片段大小（bp）	退火温度（℃）
KPC	KPC-F	ATGTCACTGTATCGCCGTCT	893	55
	KPC-R	TTTTCAGAGCCTTACTGCCC
NDM	NDM-F	CACCTCATGTTTGAATTCGCC	984	58
	NDM-R	CTCTGTCACATCGAAATCGC
VIM	VIM-F	GTCTATTTGACCGCGTC	774	60
	VIM-R	CTACTCAACGACTGAGCG
IMP	IMP-F	TCACATTTCCATAGCGACAG	450	50
	IMP-R	AGTGGTACTTTTTTTGCTTTCAT
SPM	SPM-F	CTGCTTGGATTCATGGGCGC	784	55
	SPM-R	CCTTTTCCGCGACCTTGATC
GIM	GIM-F	TTGCCAGCTTTAGCTCAGGGTC	713	60
	GIM-R	TAATCAGCCGACGCTTCAGCGG
OXA-23 Group	OXA-23 Group-F	GGACATAATCAGGTGATTCA	670	55
	OXA-23 Group-R	TAGATGCCGGCATTTCTGAC
OXA-24 Group	OXA-24 Group-F	TAGTTTCTCTCAGTGCATGT	780	55
	OXA-24 Group-R	TTCCAAGATTTTCTAGCGAC
OXA-51 Group	OXA-51 Group-F	TTATTTCAGCCTGCTCACCT	720	55
	OXA-51 Group-R	TCGAACAGAGCTAGGTATTC
OXA-58 Group	OXA-58 Group-F	TAAGTATTGGGGCTTGTGCT	740	55
	OXA-58 Group-R	GACAATTGTTTACGTAGAGC

目的基因	引物名称	引物序列（5'~3'）	片段大小（bp）	退火温度（℃）
KPC	KPC-F	ATGTCACTGTATCGCCGTCT	893	55
	KPC-R	TTTTCAGAGCCTTACTGCCC
NDM	NDM-F	CACCTCATGTTTGAATTCGCC	984	58
	NDM-R	CTCTGTCACATCGAAATCGC
VIM	VIM-F	GTCTATTTGACCGCGTC	774	60
	VIM-R	CTACTCAACGACTGAGCG
IMP	IMP-F	TCACATTTCCATAGCGACAG	450	50
	IMP-R	AGTGGTACTTTTTTTGCTTTCAT
SPM	SPM-F	CTGCTTGGATTCATGGGCGC	784	55
	SPM-R	CCTTTTCCGCGACCTTGATC
GIM	GIM-F	TTGCCAGCTTTAGCTCAGGGTC	713	60
	GIM-R	TAATCAGCCGACGCTTCAGCGG
OXA-23 Group	OXA-23 Group-F	GGACATAATCAGGTGATTCA	670	55
	OXA-23 Group-R	TAGATGCCGGCATTTCTGAC
OXA-24 Group	OXA-24 Group-F	TAGTTTCTCTCAGTGCATGT	780	55
	OXA-24 Group-R	TTCCAAGATTTTCTAGCGAC
OXA-51 Group	OXA-51 Group-F	TTATTTCAGCCTGCTCACCT	720	55
	OXA-51 Group-R	TCGAACAGAGCTAGGTATTC
OXA-58 Group	OXA-58 Group-F	TAAGTATTGGGGCTTGTGCT	740	55
	OXA-58 Group-R	GACAATTGTTTACGTAGAGC