耐碳青霉烯类大肠埃希菌临床分离情况及碳青霉烯酶基因研究

doi:10.16150/j.1671-2870.2019.06.010

摘要/Abstract

摘要：

目的：研究碳青霉烯酶基因,探讨临床分离的大肠埃希菌碳青霉烯类抗生素的耐药基因分布情况,为相关抗感染治疗提供依据。方法：收集2009年3月至2017年10月期间,我院住院患者临床标本中分离到的非重复耐碳青霉烯类大肠埃希菌菌株;使用Microflex^TMMALDI-TOF MS进行菌种复核;用Vitek-2 Compact全自动微生物分析仪联合纸片扩散法进行药物敏感性试验;采用碳青霉烯类抗菌药物灭活试验（modified carbapenem inactivation method,mCIM）快速筛查大肠埃希菌的产碳青霉烯酶表型;采用耐药基因常规PCR及测序的方法检测常见碳青霉烯酶基因;另收集所有患者的临床资料,分析其临床特征。结果：2009年3月至2017年12月,我院住院患者临床标本中分离到的非重复耐碳青霉烯类大肠埃希菌共84株。药物敏感性（药敏）试验结果显示,只有氨基糖苷类抗生素中阿米卡星的耐药率<50%,其他各种药物的耐药率都非常高,达到80%~100%。mCIM检测共筛选出71株产碳青霉烯酶菌株,而耐药基因PCR及测序的结果则显示,有65株携带bla_NDM,以bla_NDM-5（64.6%,42/65）和bla_NDM-1（24.6%,16/65）为主,其中2株同时携带bla_NDM-1和bla_MCR-1。另有6株携带bla_kpc-2,未检测到bla_GES、bla_IMP、bla_VIM、bla_OXA等碳青霉烯酶基因。检测到产碳青霉烯酶菌株来自23个科室的17种标本,其中尿液占30.95%（26/84）和痰占21.43%（18/84）;标本检出产碳青霉烯酶的患者则以儿科最多（66.7%,56/84）。儿童及婴幼儿中分离的大肠埃希菌中产酶型占96.4%（54/56）,且以产酶型别bla_NDM-5最多（63.0%,34/54）;而老年患者中分离的非产酶型大肠埃希菌占62.5%（10/16）; 在这些患者中,54%（45/84）的患者在住院期间曾接受过手术,33%（28/84）的患者曾接受留置深静脉导管等侵入性医疗操作。结论：我院临床患者分离到的耐碳青霉烯类大肠埃希菌呈多重耐药,产碳青霉烯酶的类型为NDM和KPC 2种,其中NDM-5检出率高达50%（42/84）;来自儿科病房尤其是儿重症病房的标本,耐碳青霉烯类大肠埃希菌分离率最高。

关键词: 大肠埃希菌, 碳青霉烯酶, 耐药基因

Abstract:

Objective: To study the carbapenem resistance genes of clinically isolated carbapenem-resistant Escherichia coli strains. Methods: Non-repetitive carbapenem-resistant Escherichia coli strains isolated from clinical specimens of inpatients in our hospital from March 2009 to October 2017 were collected. The strains were firstly confirmed by Microflex^TM MALDI-TOF MS, then drug sensitivity of isolates was tested by K-B method combined with Vitek-2 Compact Automatic Microbiological Analyzer. Rapid screening of carbapenemase-producing Escherichia coli was performed using the modified carbapenem inactivation method (mCIM). Commonly seen carbapenemase was detected by regular PCR and DNA sequencing. The clinical data of patients were analyzed. Result: From March 2009 to December 2017, a total of 84 non-repetitive carbapenem-resistant Escherichia coli strains were isolated. Drug resistance test showed that carbapenem-resistant Escherichia coli had a very high resistance rate (up to 80%-100%) for most of the commonly used drugs except amikacin, whose drug resistance rate was less than 50%. A total of 71 carbapenemase-producing strains were identified by mCIM. The PCR and DNA sequencing results for drug resistance genes showed that there were 65 strains carrying bla_NDM, with bla_NDM-5 (64.6%, 42/65) and bla_NDM-1(24.6%, 16/65) as the main types, and of them 2 strains carried both bla_NDM-1 and bla_MCR-1; 6s trains carriedblakpc-2; while no bla_GES, bla_IMP, bla_VIM, bla_OXA were detected. Altogether 17 types of clinical specimens and 23 departments were concerned, urine samples accounted for 30.95% (26/84) and sputum samples accounted for 21.43% (18/84). Carbapenemase-producing strains were mainly isolated from pediatric patients (66.7%, 56/84). Most of the strains isolated from children and infants were enzyme-producing (96.4%, 54/56), of which bla_NDM-5 accounted for 63.0%(34/54). Most of the strains isolated from elderly patients were non-enzyme-producing (62.5%, 10/16). Besides, 54% (45/84) of these patients underwent surgery, while 33% (28/84) of them were treated with indwelling deep vein catheter and invasive medical procedures. Conclusions: The carbapenem-resistant Escherichia coli strains isolated from patients in our hospital mostly are multi-drug resistant. NDM and KPC are the main carbapenemase, and NDM-5 is the major type with a detection rate as high as 50%(42/84). The isolated rate of carbapenem-resistant Escherichia coli in pediatric wards is high, especially in critical care wards.

Key words: Escherichia coli, Carbapenem, Resistance genes

中图分类号:

R378.21

刁文晶, 皇甫昱婵, 朱威南, 刘婧娴, 刘瑛, 沈立松. 耐碳青霉烯类大肠埃希菌临床分离情况及碳青霉烯酶基因研究[J]. 诊断学理论与实践, 2019, 18(06): 655-661.

DIAO Wenjing, HUANGFU Yuchan, ZHU Weinan, LIU Jingxian, LIU Ying, SHEN Lisong. Study on carbapenem resistance genes within isolated carbapenem-resistant Escherichia coli strains[J]. Journal of Diagnostics Concepts & Practice, 2019, 18(06): 655-661.

图/表 2

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耐药基因	引物序列（5′→3′）	扩增片段长度（bp）	参考文献
KPC	F:GCTACACCTAGCTCCACCTTC R:ACAGTGGTTGGTAATCCATGC	989	[26]
IMP	F: CATGGTTTGGTGGTTCTTGT R: ATAATTTGGCGGACTTTGGC	488	[27]
VIM	F: ATTGGTCTATTTGACCGCGTC R: TGCTACTCAACGACTGAGCG	780	[27]
GES	F:AGCGACAATGGGGCTACTAAC R:GTGTAATAACTTGACCGACAGAGG	465	本实验室设计
OXA	F:ACACAATACATATCAACTTCGC R:AGTGTGTTTAGAATGGTGATC	813	[28]
NDM	F:GAGCACCGCATTAGCCGCTG R:GCTATCGGGGGCGGAATGG	727	本实验室设计
MCR	F:CGGTCAGTCCGTTTGTTC R:CTTGGTCGGTCTGTAGGG	310	本实验室设计

药物名称	产NDM酶（n=65）	产KPC酶（n=6）	不产酶株（n=13）	P值
亚胺培南	92.31%	66.67%	100%	0.524
美罗培南	90.77%	66.67%	100%	0.448
厄他培南	100%	100%	100%	-
氨苄西林	100%	100%	100%	-
阿米卡星	24.62%	33.33%	0.77%	0.948
头孢美唑	89.23%	100%	76.92%	0.375
头孢曲松	100%	100%	100%	-
头孢吡肟	100%	100%	92.31%	0.337
头孢呋辛钠	100%	100%	100%	-
头孢噻肟	100%	100%	100%	-
妥布霉素	50.77%	66.67%	38.46%	0.365
头孢哌酮/舒巴坦	98.46%	100%	100%	1
氨苄西林/舒巴坦	100%	100%	100%	-
氨曲南	98.46%	100%	92.31%	0.706
环丙沙星	87.69%	100%	76.92%	0.476
头孢替坦	98.46%	100%	92.31%	0.706
头孢唑林	98.46%	100%	92.31%	0.706
庆大霉素	70.77%	66.67%	53.85%	0.393
左氧氟沙星	89.23%	83.33%	76.92%	0.476
哌拉西林	98.46%	100%	100%	1
复方新诺明	56.92%	66.67%	76.92%	0.193
头孢他啶	100%	100%	100%	-
哌拉西林/他唑巴坦	98.46%	100%	61.54%	0