呼和浩特地区腹泻患者艰难梭菌毒素基因多位点序列分型研究

doi:10.3969/j.issn.1673-8640.2021.11.008

摘要/Abstract

摘要：

目的了解呼和浩特地区住院腹泻患者艰难梭菌产毒情况和产毒艰难梭菌多位点序列分型（MLST）情况。方法收集2018年7月—2019年12月内蒙古医科大学附属医院成人腹泻患者的粪便样本326份,进行艰难梭菌培养和鉴定,采用聚合酶链反应（PCR）扩增艰难梭菌毒素基因（tcdA和tcdB）和二元毒素基因（cdtA和cdtB）,以明确是否为产毒艰难梭菌;对产毒艰难梭菌进行MLST。结果共培养出艰难梭菌46株,其中35株为产毒艰难梭菌,毒素基因tcdA（-）、tcdB（+）型3株,tcdA（+）、tcdB（+）型32株,二元毒素基因检测均为阴性;产毒菌株包括13个不同ST型别（2、3、14、33、35、39、42、48、52、54、55、109、512）,其中ST54型是最主要的型别,占22.9%（8/35）,其次是ST2型和ST55型,各占14.3%（5/35）,未检出核糖体027型（ST1型）和核糖体078型（ST11型）高产毒株。结论呼和浩特地区成人腹泻患者感染的产毒艰难梭菌毒素以tcdA（+）、tcdB（+）为主,未检出产二元毒素的高产毒株。艰难梭菌基因型别较多（13种）,ST54型、ST2型、ST55型为居前3位的ST型别。

关键词: 艰难梭菌, 毒素类型, 多位点序列分型, 临床分离株

Abstract:

Objective To understand the clinical toxin status of clinical isolates of Clostridium difficile in patients with acquired diarrhea in Hohhot and the status of multilocus sequence typing（MLST） of Clostridium difficile. Methods The 326 fecal specimens of adult diarrhea patients in the Affiliated Hospital of Inner Mongolia Medical University were collected from July 2018 to December 2019 for the cultivation and identification of Clostridium difficile. The toxin genes（tcdA and tcdB） and binary toxin genes（cdtA and cdtB） of cultivated Clostridium difficile were amplified by polymerase chain reaction（PCR）to determine producing toxin. The MLST of Clostridium difficile producing toxin was performed. Results Totally,46 isolates of Clostridium difficile were cultivated,of which 35 isolates were Clostridium difficile producing toxin,among them 3 isolates were toxin gene tcdA（-） and tcdB（+） type,and 32 isolates were tcdA（+） and tcdB（+） type. However,binary toxin genes were all negative. The producing toxin isolates came from 13 different ST types（ST2,3,14,33,35,39,42,48,52,54,55,109 and 512）,ST54 was the most common type（22.9%,8/35）,followed by ST2（14.3%,5/35） and ST55（14.3%,5/35）,and no high-yielding isolate of ribosome type 027 （ST1）and high-yielding isolate of ribosome type 078 （ST11）were determined. Conclusions The producing toxin Clostridium difficile with tcdA（+） and tcdB（+） mainly is isolated from adult diarrhea patients in Hohhot,and no high-yielding isolates producing binary toxin are determined. The genotypes of Clostridium difficile are relatively more（13 types）,and ST54,ST2 and ST55 are the top 3 types.

Key words: Clostridium difficile, Toxin type, Multilocus sequence typing

中图分类号:

R378.8

王艳艳, 王俊瑞, 郑文琪, 申慧敏, 吕莹莹, 郭素芳. 呼和浩特地区腹泻患者艰难梭菌毒素基因多位点序列分型研究[J]. 检验医学, 2021, 36(11): 1131-1134.

WANG Yanyan, WANG Junrui, ZHENG Wenqi, SHEN Huimin, LÜ Yingying, GUO Sufang. Detection of Clostridium difficile toxin genes and diagnosis of multilocus sequence typing in patients with diarrhea in Hohhot[J]. Laboratory Medicine, 2021, 36(11): 1131-1134.

图/表 4

参考文献 23

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引物名称	基因序列（5'→3'）	片段长度/bp
tcdA	F-AGATTCCTATATTTACATGACAATAT	369
	R-GTATCAGGCATAAAGTAATATACTTT
tcdB	F-TGATGAAGATACAGCAGAAGC	688
	R-TGATTCTCCCTCAAAATTCTC
cdtA	F-TGAACCTGGAAAAGGTGATG	389
	R-AGGATTATTTACTGGACCATTTG
cdtB	F-CTTAATGCAAGTAAATACTGAG	512
	R-AACGGATCTCTTGCTTCAGTC
adk	F-TTACTTGGACCTCCAGGTFF	635
	R-TTTCCACTTCCTAAGGCTGC
atpA	F-TGATGATTTAAGTAAACAAGCTG	674
	R-AATCATGAGTGAAGTCTTCTCC
dxr	F-GCTACTTTCCTTCTCTG	525
	R-CCAACTCTTTGTGCATAAA
glyA	F-ATAGCTGATGAGGTTGGAGC	625
	R-TTCTAGCCTTAGATTCTTCATC
recA	F-CAGTAATGAAATTGGGAGAAGC	705
	R-ATTCAGCTTGCTTAAATGGTG
sodA	F-CCAGTTGTCAATGTATTCATTTC	585
	R-ATAACTTCATTTGCTTTTACACC
tpi	F-ATGAGAAAACCTATAATTGCAG	640
	R-TTGAAGGTTTAACACTTCCACC

引物名称	基因序列（5'→3'）	片段长度/bp
tcdA	F-AGATTCCTATATTTACATGACAATAT	369
	R-GTATCAGGCATAAAGTAATATACTTT
tcdB	F-TGATGAAGATACAGCAGAAGC	688
	R-TGATTCTCCCTCAAAATTCTC
cdtA	F-TGAACCTGGAAAAGGTGATG	389
	R-AGGATTATTTACTGGACCATTTG
cdtB	F-CTTAATGCAAGTAAATACTGAG	512
	R-AACGGATCTCTTGCTTCAGTC
adk	F-TTACTTGGACCTCCAGGTFF	635
	R-TTTCCACTTCCTAAGGCTGC
atpA	F-TGATGATTTAAGTAAACAAGCTG	674
	R-AATCATGAGTGAAGTCTTCTCC
dxr	F-GCTACTTTCCTTCTCTG	525
	R-CCAACTCTTTGTGCATAAA
glyA	F-ATAGCTGATGAGGTTGGAGC	625
	R-TTCTAGCCTTAGATTCTTCATC
recA	F-CAGTAATGAAATTGGGAGAAGC	705
	R-ATTCAGCTTGCTTAAATGGTG
sodA	F-CCAGTTGTCAATGTATTCATTTC	585
	R-ATAACTTCATTTGCTTTTACACC
tpi	F-ATGAGAAAACCTATAATTGCAG	640
	R-TTGAAGGTTTAACACTTCCACC

MLST	ST型别	Tcd基因	菌株数/株
adk-atpA-dxr-glyA-recA-sodA-tpi	ST型别	Tcd基因	菌株数/株
1-4-7-1-1-3-3	54	A+B+	8
1-1-6-6-1-12-12	55	A+B+	5
1-1-2-1-5-3-1	2	A+B+	5
1-1-2-1-1-1-1	3	A+B+	3
1-1-2-1-1-7-1	42	A-B+	1
	1	A+B+	2
2-5-8-1-1-3-6	35	A+B+	3
1-1-2-6-1-12-12	512	A+B+	2
1-1-2-1-1-5-1	48	A+B+	1
3-12-10-18-6-18-15	109	A+B+	1
3-7-10-8-7-2-10	39	A-B+	1
1-1-2-6-1-12-1	52	A-B+	1
1-1-2-1-6-5-3	33	A+B+	1
1-1-2-1-5-5-3	14	A+B+	1

MLST	ST型别	Tcd基因	菌株数/株
adk-atpA-dxr-glyA-recA-sodA-tpi	ST型别	Tcd基因	菌株数/株
1-4-7-1-1-3-3	54	A+B+	8
1-1-6-6-1-12-12	55	A+B+	5
1-1-2-1-5-3-1	2	A+B+	5
1-1-2-1-1-1-1	3	A+B+	3
1-1-2-1-1-7-1	42	A-B+	1
	1	A+B+	2
2-5-8-1-1-3-6	35	A+B+	3
1-1-2-6-1-12-12	512	A+B+	2
1-1-2-1-1-5-1	48	A+B+	1
3-12-10-18-6-18-15	109	A+B+	1
3-7-10-8-7-2-10	39	A-B+	1
1-1-2-6-1-12-1	52	A-B+	1
1-1-2-1-6-5-3	33	A+B+	1
1-1-2-1-5-5-3	14	A+B+	1