丽水市碳青霉烯耐药弗劳地枸橼酸杆菌分子流行病学研究

龚智明; 黄飞武; 赵赟安; 陈娇丽; 赵芯米; 徐建芬; 黄建胜

doi:10.16766/j.cnki.issn.1674-4152.004138

丽水市碳青霉烯耐药弗劳地枸橼酸杆菌分子流行病学研究

doi: 10.16766/j.cnki.issn.1674-4152.004138

温州医科大学附属第五医院丽水市中心医院检验科，浙江丽水 323000

基金项目:

浙江省基础公益研究计划项目 LHDMZ23H190001

浙江省基础公益研究计划项目 LHDMY24H190001

浙江省医药卫生科技项目 2024XY103

丽水市科技计划项目 2023ZDYF16

丽水市科技计划项目 2023GYX43

丽水市科技计划项目 2024SJZC102

详细信息

通讯作者:
黄建胜，E-mail：huangxvy@163.com

中图分类号: R378.2
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出版历程
- 收稿日期: 2024-12-03
- 网络出版日期: 2025-10-31

The molecular epidemiology of carbapenem-resistant Citrobacter freundii in Lishui City

Department of Clinical Laboratory, Lishui Central Hospital, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui, Zhejiang 323000, China

摘要

摘要: 目的分析碳青霉烯耐药弗劳地枸橼酸杆菌(CRCF)的耐药机制和分子流行病学特征，为抗感染诊治提供实验室依据。方法收集丽水市中心医院2016年1月—2022年12月分离的共17株CRCF菌株，采用微量肉汤稀释法检测MIC值，通过PCR法检测碳青霉烯酶基因(bla_KPC-2、bla_NDM-1、bla_IMP-1、bla_VIM-1、bla_OXA-48)及遗传背景；利用接合转化实验分析质粒转移性和质粒类型；采用MLST法确定分型，结合Pubmed数据库菌株数据进行系统发育分析。结果 17株CRCF对复方新诺明的耐药率为100%(17/17)，对头孢他啶、头孢吡肟、氨曲南的耐药率均为76.5%(13/17)，对美罗培南的耐药率为82.4%(14/17)，对亚胺培南的耐药率为64.7%(11/17)，对哌拉西林/他唑巴坦的耐药率为94.1%(16/17)，对LVX的耐药率为58.8%(10/17)，对阿米卡星的耐药率为47.1%(8/17)，对多粘菌素B的耐药率为11.8%(2/17)，对替加环素的耐药率最低，仅为5.9%(1/17)。11株菌携带bla_NDM-1，5株携带bla_KPC-2, 1株携带bla_IMP-4。54.5%(6/11)的bla_NDM-1位于IncX3质粒；bla_KPC-2均位于IncFⅡ质粒。菌株分属于14种ST型，系统发育分析显示散发。结论丽水市CRCF对碳青霉烯类抗生素高度耐药，主要携带bla_NDM-1和bla_KPC-2，呈现散发流行。
- 碳青霉烯耐药弗劳地枸橼酸杆菌 /
- 耐药机制 /
- 耐药性 /
- 多位点序列分型 /
- IncX3质粒
Abstract: Objective To analyze the resistance mechanisms and molecular epidemiological characteristics of carbapenem-resistant Citrobacter freundii (CRCF), providing laboratory evidence for clinical prevention and treatment. Methods CRCF strains collected from January 2016 to December 2022 in Lishui Central Hospital were tested for MIC using the broth microdilution method. PCR was employed to detect carbapenemase genes (bla_KPC-2, bla_NDM-1, bla_IMP-1, bla_VIM-1, bla_OXA-48), and the genetic background of resistance genes. Conjugation and transformation experiments were conducted to analyze plasmid transferability and plasmid typing. MLST typing was combined with CRCF strains from the PubMed database for phylogenetic analysis. Results The resistance rates of 17 CRCF isolates were 100% (17/17) to trimethoprim/sulfamethoxazole; 76.5% (13/17) to ceftazidime, cefepime, and aztreonam; 82.4% (14/17) to meropenem; 64.7% (11/17) to imipenem; 94.1% (16/17) to piperacillin/tazobactam; 58.8% (10/17) to LVX; 47.1% (8/17) to amikacin; 11.8% (2/17) to polymyxin; and the lowest resistance rate was to tigecycline at 5.9% (1/17). Among these strains, 11 carried bla_NDM-1, 5 carried bla_KPC-2, and 1 carried bla_IMP-4. 54.5%(6/11)of bla_NDM-1 were located on IncX3 plasmids, while all of bla_KPC-2 were located on IncFⅡ plasmids. The strains were classified into 14 ST types, with phylogenetic analysis indicating sporadic distribution. Conclusion CRCF in Lishui exhibits high resistance to carbapenem antibiotics, primarily carrying bla_NDM-1 and bla_KPC-2, presenting a sporadic epidemic pattern.
- Carbapenem-resistant Citrate frauddy /
- Mechanisms of resistance /
- Antimicrobial resistance /
- Multi-locus sequence typing /
- IncX3 plasmid

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图 1 不同国家/地区分离碳青霉烯耐药弗劳地枸橼酸杆菌ST型系统发育树

Figure 1. Phylogenetic tree of ST types of carbapenem-resistant Citrobacter freundii strains isolated from different countries/regions

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图 2 不同国家/地区分离碳青霉烯耐药弗劳地枸橼酸杆菌ST型系统发育树

注：红点标记为丽水市CRCF菌株ST分型。

Figure 2. Phylogenetic tree of ST types of carbapenem-resistant Citrobacter freundii strains isolated from different countries/regions

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图 3 产NDM、IMP菌株的bla_NDM-1、bla_NDM-5、bla_IPM-4周围基因结构

注：箭头方向指示基因编码方向，颜色代表不同基因类型，其中浅蓝色为可移动元件，红色为耐药基因，黄色和绿色为结构基因，紫色为未知基因，黑色为IIc簇内含子基因，粉色为运动蛋白基因。

Figure 3. The genetic structures surrounding bla_NDM-1, bla_NDM-5, bla_IPM-4 in NDM-and IMP-producing strains

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图 4 产KPC菌株的bla_KPC-2周围基因结构

注：箭头方向指示基因编码方向，黑色为耐药基因，白色为可移动元件/嵌合体。

Figure 4. The genetic structure surrounding bla_KPC-2 in KPC-producing strains

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表 1 质粒不相容群分型检测引物

Table 1. Primers for plasmid incompatibility group typing detection

目的基因	引物名称	引物序列	产物大小(bp)	参考序列
IncFⅡ	UTF	TTTACACCTGGCCGCCTGAA	497	pFOS18(KJ653815)
	UTR	GCGCCCAGATGTACCATGAG
IncⅠ	IF	AGATCCGTTTCTGGGAAGAC	574	pKPC-LKEc(KC788405)
	IR	GGTGTTATCCAGACGTAACG
IncR	RF	AAGCTGGATGTCAACAGCGC	775	pSg1-2(CP012428)
	RR	AATGCACGCTGAAGTAGGTG
IncQ	QF	CTATCGACATTCTGGCGAGC	632	pPH18(KU644672)
	QR	GGTCATACCGGAGAGGTATG
IncU	UF	CAGCCATCCATCATGGTCTA	720	pHP2(KU644677)
	UR	CTGGAATAGGTTGATCAGCG
IncX3	X3F	TCGTGAAGACAGTAACGGGA	765	pNDM-HN380(JX104760)
	X3R	CAAATCTAAGCATGTGAGCC
注：质粒不相容群分型检测引物见参考文献报道^[12]。

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表 2 CRCF株临床信息、ST型、质粒类型、携带耐药基因以及碳青霉烯酶类型

Table 2. Clinical information of CRCF strain, ST type, plasmid type, carried resistance genes, and carbapenemase type

菌株	科室分布	标本类型	ST型	质粒类型	耐药基因	mCIM	eCIM	碳青霉烯酶类型
CRCF01	神经内科	痰液	ST129	IncFⅡ	bla_KPC-2	阳性	阴性	丝氨酸碳青霉烯酶
CRCF02	心内科	痰液	ST937	IncFⅡ	bla_KPC-2	阳性	阴性	丝氨酸碳青霉烯酶
CRCF03	康复科	尿液	ST299	IncN1	bla_IMP4	阳性	阳性	金属β-内酰胺酶
CRCF04	消化内科	胆汁	ST91	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF05	泌尿外科	尿液	ST116	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF06	泌尿外科	尿液	ST116	IncFⅡ	bla_KPC-2	阳性	阴性	丝氨酸碳青霉烯酶
CRCF07	血液内科	痰液	ST103	IncFⅡ	bla_KPC-2	阳性	阴性	丝氨酸碳青霉烯酶
CRCF08	介入科	脓液	ST396	-	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF09	泌尿外科	尿液	ST344	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF10	康复医学科	尿液	ST63	IncF	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF11	妇产科	尿液	ST22	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF12	肝胆胰外科	胆汁	ST91	-	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF13	呼吸内科	痰液	ST22	-	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF14	血液内科	全血	ST532	IncFⅡ	bla_KPC-2	阳性	阴性	丝氨酸碳青霉烯酶
CRCF15	心胸外科	脓液	ST186	-	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF16	康复医学科	尿液	ST589	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
CRCF17	重症医学科	肛拭子	ST552	IncX3	bla_NDM-1	阳性	阳性	金属β-内酰胺酶
注：-表示多次转化、接合未能分离到碳青霉烯耐药质粒，故不能确定质粒分型。

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表 3 CRCF菌株药敏检测结果(MIC, μg/mL)

Table 3. Antimicrobial susceptibility test results of CRCF strains (MIC, μg/mL)

菌株	CAZ	FEP	ATM	IPM	MEM	TZP	AMK	POL	TGC	LVX	SXT
CRCF01	8	2	64	2	1	128/4	≤4	0.5	0.25	4	≥64/1 216
CRCF02	8	4	64	4	4	256/4	≤4	0.5	1	0.25	≥64/1 216
CRCF03	≥256	≥256	≥256	2	4	≥1 024/4	≥1 024	0.5	32	16	≥64/1 216
CRCF04	≥256	≥256	≥256	≥128	≥128	≥1 024/4	≥1 024	32	0.25	32	≥64/1 216
CRCF05	≥256	≥256	≥256	16	32	≥1 024/4	≥1 024	2	0.25	64	≥64/1 216
CRCF06	16	64	≥256	≥64	16	128/4	≥1 024	0.5	0.5	1	≥64/1 216
CRCF07	4	4	32	2	2	128/4	≥1 024	0.5	0.25	4	≥64/1 216
CRCF08	≥256	≥256	≥256	≥64	≥64	≥1 024/4	≤4	0.5	0.5	1	≥64/1 216
CRCF09	≥256	32	≤1	16	16	256/4	8	2	0.25	1	≥64/1 216
CRCF10	≥256	64	4	8	8	≥1 024/4	≥1 024	2	0.25	4	≥64/1 216
CRCF11	≥256	≥256	32	4	4	256/4	≤4	0.5	0.25	2	≥64/1 216
CRCF12	≥256	64	128	16	32	512/4	≤4	0.5	0.25	16	≥64/1 216
CRCF13	≥256	16	≤1	4	4	256/4	≤4	0.5	0.25	8	≥64/1 216
CRCF14	8	8	128	2	4	256/4	≤4	0.5	1	0.5	≥64/1 216
CRCF15	≥256	128	32	16	≥64	≥1 024/4	≤4	0.5	0.5	1	≥64/1 216
CRCF16	≥256	32	≥256	0.5	2	4/4	32	≥64	0.125	1	≥64/1 216
CRCF17	≥256	32	≤1	2	4	512/4	≥1 024	0.5	0.25	2	≥64/1 216
敏感MIC	≤4	≤2	≤4	≤1	≤1	≤8/4	≤4		≤2	≤0.5	≤2/38
中介MIC	8	4~8	8	2	2		8	2	4	1	4/76
耐药MIC	≥16	≥16	≥16	≥4	≥4	≥32/4	≥16	≥4	≥8	≥2	≥8/152

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