耐碳青霉烯鲍曼不动杆菌临床分离株的流行病学与耐药机制研究

doi:10.3969/j.issn.1673-8640.2015.01.013

摘要/Abstract

摘要：

目的了解耐碳青霉烯类抗菌药物的鲍曼不动杆菌流行病学情况以及耐药机制,帮助临床合理使用抗菌药物,为控制医院感染提供依据。方法收集耐碳青霉烯类抗菌药物的鲍曼不动杆菌110株及敏感株6株,进行来源病区和标本种类的统计分析;应用重复序列聚合酶链反应(REP-PCR)进行基因型分型;使用PCR检测β-内酰胺酶基因(OXA、IMP、VIM等)及外排泵编码基因(adeA、adeB、adeC、adeJ、adeG)的携带情况;采用实时荧光定量PCR对比耐药菌株与敏感菌株的外排泵基因表达量的差异。结果 110株菌株主要来自痰标本,重症监护病房(ICU)分离率最高; 所有菌株REP-PCR电泳图谱分为A~G 7型,110株耐药菌株均为A型,且均被检测到含有OXA-23、OXA-51基因,均未检测到含有OXA-24、OXA-58、IMP-1、IMP-2、VIM-1、VIM-2基因;外排泵编码基因adeA、adeB、adeC、adeJ及adeG的阳性率均为100%;耐药菌株外排泵编码基因adeA、adeB、adeC表达量明显高于敏感菌株。结论耐碳青霉烯类鲍曼不动杆菌的流行型别基本相同,均为A型,应引起临床医护人员的高度重视;β-内酰胺酶基因(OXA-23、OXA-51)的表达以及外排泵基因(adeA、adeB、adeC)的过度表达与其对碳青霉烯类抗菌药物的耐药性有关。

关键词: 鲍曼不动杆菌, β-内酰胺酶, 外排泵, 重复序列聚合酶链反应, 实时荧光定量聚合酶链反应

Abstract:

Objective To understand the drug resistance mechanisms and epidemiology of carbapenem-resistant Acinetobacter baumannii in order to guide the rational use of antibiotics and to provide the reference for the control of nosocomial infections. Methods A total of 116 Acinetobacter baumannii (110 carbapenem-resistant isolates and 6 carbapenem-sensitive isolates) isolated from clinical specimens were classified and analyzed according to endemic areas and specimen types. The isolates were analyzed by repetitive-sequence polymerase chain reaction (REP-PCR) for genotyping. Beta-lactamase genes (OXA, IMP, VIM and soon) and efflux pump-encoding genes (adeA, adeB, adeC, adeJ, adeG) were amplified by PCR. Real-time fluorescence quantitation PCR was performed to compare the expression level of the efflux pump-encoding genes in resistant and sensitive isolates. Results The isolation rate of resistant isolates in intensive care unit (ICU) was the highest-level, and the isolates were mainly isolated from sputum. The genotype types of carbapenem-resistant and -sensitive Acinetobacter baumannii were classified into 7 categories by REP-PCR. All the carbapenem-resistant isolates were type A. OXA-23 and OXA-51 were positive in 110 carbapenem-resistant isolates. However, OXA-24, OXA-58, IMP-1, IMP-2, VIM-1 and VIM-2 could not be detected in all the isolates. The positive rates of the efflux pump-encoding genes (adeA, adeB, adeC, adeJ and adeG) were 100%. The expression level of efflux pump-encoding genes (adeA, adeB, adeC) in resistant isolates was significantly higher than that in sensitive isolates. Conclusions The main popular type of carbapenem-resistant Acinetobacter baumannii is type A. Clinical staff should pay attention to this phenomenon. The expression of OXA-23 and OXA-51 overexpression of adeA, adeB and adeC efflux pump-encoding gene may play an important role in carbapenem-resistant Acinetobacter baumannii.

Key words: Acinetobacter baumannii, beta-lactamase, Efflux pump, Repetitive-sequence polymerase chain reaction, Real-time fluorescence quantitation polymerase chain reaction

中图分类号:

R446.5

张广慧, 曹雪萍, 俞静, 刘瑛, 葛艳. 耐碳青霉烯鲍曼不动杆菌临床分离株的流行病学与耐药机制研究[J]. 检验医学, 2015, 30(1): 53-57.

ZHANG Guanghui, CAO Xueping, YU Jing, LIU Ying, GE Yan. Epidemiology and resistance mechanism study of carbapenem-resistant Acinetobacter baumannii[J]. Laboratory Medicine, 2015, 30(1): 53-57.

图/表 6

参考文献 16

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引物	引物序列	预期长度 (bp)	参考文献
OXA-23	P1 5'-GATCGGATTGGAGAACCAGA-3'	501	[4]
	P2 5'-ATTTCTGACCGCATTTCCAT-3'
OXA-24	P1 5'-GGTTAGTTGGCCCCCTTAAA-3'	246	[4]
	P2 5'-AGTTGAGCGAAAAGGGGATT-3'
OXA-51	P1 5'-TAATGCTTTGATCGGCCTTG-3'	353	[4]
	P2 5'-TGGATTGCACTTCATCTTGG-3'
OXA-58	P1 5'-AAGTATTGGGGCTTGTGCTG-3'	599	[4]
	P2 5'-CCCCTCTGCGCTCTACATAC-3'
IMP-1	P1 5'-ACCGCAGCAGAGTCTTTGCC-3'	587	[5]
	P2 5'-ACAACCAGTTTTGCCTTACC-3'
IMP-2	P1 5'-GTTTTATGTGTATGCTTCC-3'	678	[5]
	P2 5'-AGCCTGTTCCCATGTAC-3'
VIM-1	P1 5'-AGTGGTGAGTATCCGACAG-3'	261	[5]
	P2 5'-ATGAAAGTGCGTGGAGAC-3'
VIM-2	P1 5'-ATGTTCAAACTTTTGAGTAAG-3'	801	[5]
	P2 5'-CTACTCAACGACTGAGCG-3'
adeA	P1 5'- GGGTGTGATTCAAAAGAAGTCGC-3'	169	[6]
	P2 5'-GAACCTTTTCAATGATACCTCCG-3'
adeB	P1 5'-AAAGACTTCAAAGAGCGGACTA-3'	623	[7]
	P2 5'-TCACGCATTGCTTCACCC-3'
adeC	P1 5'-ACAATCGTATCTCGTGGACTC-3'	361	[6]
	P2 5'-TAGAAACTGGGTTATTGGGGT-3'
adeJ	P1 5'-CCTATTGCACAATATCCAACGA-3'	119	[8]
	P2 5'-AGGATAAGTCGCAGCAATCG-3'
adeG	P1 5'-GTCCTGAAATGGTCGTTCGT-3'	143	[8]
	P2 5'-AGCTTCTGCTTGGCTAGATGA-3'
rpoB	P1 5'-TTCATCTAGCCAAGCAGAAG-3'	200	[9]
	P2 5'-CCTGCTAATGGTAGGGTTAAG-3'

引物	引物序列	预期长度 (bp)	参考文献
OXA-23	P1 5'-GATCGGATTGGAGAACCAGA-3'	501	[4]
	P2 5'-ATTTCTGACCGCATTTCCAT-3'
OXA-24	P1 5'-GGTTAGTTGGCCCCCTTAAA-3'	246	[4]
	P2 5'-AGTTGAGCGAAAAGGGGATT-3'
OXA-51	P1 5'-TAATGCTTTGATCGGCCTTG-3'	353	[4]
	P2 5'-TGGATTGCACTTCATCTTGG-3'
OXA-58	P1 5'-AAGTATTGGGGCTTGTGCTG-3'	599	[4]
	P2 5'-CCCCTCTGCGCTCTACATAC-3'
IMP-1	P1 5'-ACCGCAGCAGAGTCTTTGCC-3'	587	[5]
	P2 5'-ACAACCAGTTTTGCCTTACC-3'
IMP-2	P1 5'-GTTTTATGTGTATGCTTCC-3'	678	[5]
	P2 5'-AGCCTGTTCCCATGTAC-3'
VIM-1	P1 5'-AGTGGTGAGTATCCGACAG-3'	261	[5]
	P2 5'-ATGAAAGTGCGTGGAGAC-3'
VIM-2	P1 5'-ATGTTCAAACTTTTGAGTAAG-3'	801	[5]
	P2 5'-CTACTCAACGACTGAGCG-3'
adeA	P1 5'- GGGTGTGATTCAAAAGAAGTCGC-3'	169	[6]
	P2 5'-GAACCTTTTCAATGATACCTCCG-3'
adeB	P1 5'-AAAGACTTCAAAGAGCGGACTA-3'	623	[7]
	P2 5'-TCACGCATTGCTTCACCC-3'
adeC	P1 5'-ACAATCGTATCTCGTGGACTC-3'	361	[6]
	P2 5'-TAGAAACTGGGTTATTGGGGT-3'
adeJ	P1 5'-CCTATTGCACAATATCCAACGA-3'	119	[8]
	P2 5'-AGGATAAGTCGCAGCAATCG-3'
adeG	P1 5'-GTCCTGAAATGGTCGTTCGT-3'	143	[8]
	P2 5'-AGCTTCTGCTTGGCTAGATGA-3'
rpoB	P1 5'-TTCATCTAGCCAAGCAGAAG-3'	200	[9]
	P2 5'-CCTGCTAATGGTAGGGTTAAG-3'

按标本种类划分		按病区划分
来源	菌株数	病区	菌株数
痰液	77	ICU	49
胸、腹腔引流液	11	心胸外科病区	14
全血(包括静脉穿刺)	9	脑外科病区	13
脑脊液	4	干部病区	10
咽拭子	4	呼吸科病区	9
创面分泌物	3	急诊病区	7
气管插管	2	其它科室	8

按标本种类划分		按病区划分
来源	菌株数	病区	菌株数
痰液	77	ICU	49
胸、腹腔引流液	11	心胸外科病区	14
全血(包括静脉穿刺)	9	脑外科病区	13
脑脊液	4	干部病区	10
咽拭子	4	呼吸科病区	9
创面分泌物	3	急诊病区	7
气管插管	2	其它科室	8