土壤环境中四环素抗性基因向病原菌的转移研究

doi:10.16258/j.cnki.1674-5906.2023.11.008

生态环境学报 ›› 2023, Vol. 32 ›› Issue (11): 1978-1987.DOI: 10.16258/j.cnki.1674-5906.2023.11.008

土壤环境中四环素抗性基因向病原菌的转移研究

彭双¹^,²(), 宋丹², 王一明²^,^*(), 林先贵²

1.江苏开放大学环境生态学院，江苏南京 210017
2.中国科学院南京土壤研究所/土壤与农业可持续发展国家重点实验室，江苏南京 210008

收稿日期:2023-02-23 出版日期:2023-11-18 发布日期:2024-01-17
通讯作者: * 王一明。E-mail: ymwang@issas.ac.cn
作者简介:彭双（1986年生），女，副教授，主要研究方向为土壤微生物。E-mail: speng@issas.ac.cn
基金资助:
江苏省自然科学基金面上项目(BK20201110);内蒙古自治区科技重大专项(NMKJXM202009);国家重点研发计划项目(2021YFD1900602);江苏省高校自然科学基金项目(18KJB210002);江苏开放大学预研项目(19-GY-01);江苏高校‘青蓝工程’优秀骨干教师项目

Transfer of Tetracycline Resistance Gene to Human Pathogenic Bacteria in Soil

PENG Shuang¹^,²(), SONG Dan², WANG Yiming²^,^*(), LIN Xiangui²

1. College of Environment and Ecology, Jiangsu Open University, Nanjing 210017, P. R. China
2. State Key Laboratory of Soil and Sustainable Agriculture/Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China

Received:2023-02-23 Online:2023-11-18 Published:2024-01-17

摘要/Abstract

摘要：

抗生素抗性基因（Antibiotic resistance genes，ARGs）已被公认为环境中的新型污染物，水平基因转移是ARGs传播与扩散的主要途径，纯培养体系下ARGs的水平基因转移（Horizontal gene transfer，HGT）已经多有报道，HGT的分子机制也已被充分挖掘。然而，环境中的ARGs在自然条件下是否能够转移到人类致病菌中目前仍然未知。为了探究沙门氏菌是否能够在自然条件下从施肥土壤中获得ARGs并在土壤中长期存活，将含有大量四环素敏感型沙门氏菌的粪肥施入3种土壤，并对土壤进行淹水或不淹水处理，90 d后分离四环素抗性沙门氏菌。结果表明，沙门氏菌未通过HGT获得四环素抗性基因并在土壤中长期存活。从红壤中分离到3株具有四环素抗性的疑似沙门氏菌，经过生理生化和16S测序鉴定确定它们均为产H₂S的大肠杆菌，经HT-qPCR检测，它们均携带多种ARGs。对其中携带ARGs种类最多的T2菌株进行全基因组测序与分析，结果表明T2携带了两个长度分别为40.48 kb的IncX1型和93.31kb的IncY型质粒，15个基因岛，与COG数据库比对得到了3807个注释基因，包含前噬菌体和转座子共197个；经ARDB注释获得34个ARGs，其中包括了2种四环素抗性基因均位于IncY型质粒上。将T2菌体与四环素敏感型E. coli O157:H7菌株共培养，在施加不同浓度梯度四环素的条件下，T2携带的四环素抗性基因没有向O157菌株转移。综上所述，在自然环境中沙门氏菌难以从施肥土壤中获得ARGs并在土壤中长期存活，从土壤环境中分离的抗性大肠杆菌T2所携带的四环素抗性基因也难以在纯培养体系下转移到E. coli O157:H7中。研究结果可为科学评估ARGs在土壤环境中的转移风险提供参考。

关键词: 抗生素抗性基因, 沙门氏菌, 细菌全基因组测序, 基因转移

Abstract:

Antibiotic resistance genes (ARGs) have been recognized as new pollutants in the environment. Horizontal gene transfer (HGT) is the main way of ARGs transmission and diffusion. The horizontal transfer of ARGs in pure culture system has been reported, and the molecular mechanism of HGT has also been fully explored. However, whether ARGs in the environment can be captured by human pathogenic bacteria under natural conditions is still unknown. In order to investigate whether Salmonella can obtain ARGs from manure-amended soil under natural conditions and survive in soil for a long time, this study applied manure containing a large amount of tetracycline sensitive Salmonella to three types of soil, and treated the soils with or without flooding. After 90 days, tetracycline resistant Salmonella was isolated using selective medium containing tetracycline. The results showed that Salmonella in these soils did not obtain tetracycline resistance gene and survived in the soil for more than 90 days. Three suspected Salmonella strains resistant to tetracycline were isolated from the red clay soil, but they were identified as H₂S producing Escherichia coli by physiological and biochemical analysis as well as 16S sequencing. They all carried multiple ARGs detected by HT-qPCR. Among the three strains, T2 carried the most ARGs, so the whole genome of T2 was sequenced and analyzed. The results showed that T2 carried an IncX1 type plasmid with a length of 40.48 kb and an IncY type plasmid with a length of 93.31 kb, 15 gene islands, and 3807 injection-release genes which were annotated by COG database, including 197 prophages and transposons. A total of 34 ARGs were obtained through the ARDB annotation, including two tetracycline resistance genes located on the IncY type plasmid. By co-culturing T2 with tetracycline sensitive E. coli O157:H7 and applying different concentrations of tetracycline, the tetracycline resistance gene carried by T2 did not transfer to O157 strain. To sum up, it is rare for Salmonella to obtain ARGs from soil or manure in natural environment, and it is also difficult for the resistant E. coli T2 isolated from soil environment to transfer its tetracycline resistance genes to E. coli O157:H7 in pure culture system. These results can provide reference for scientific assessment of the transfer risk of ARGs in soil environment.

Key words: Antibiotic resistance gene, Salmonella, Whole bacterial genome sequencing, Horizontal gene transfer

中图分类号:

X171.5

彭双, 宋丹, 王一明, 林先贵. 土壤环境中四环素抗性基因向病原菌的转移研究[J]. 生态环境学报, 2023, 32(11): 1978-1987.

PENG Shuang, SONG Dan, WANG Yiming, LIN Xiangui. Transfer of Tetracycline Resistance Gene to Human Pathogenic Bacteria in Soil[J]. Ecology and Environment, 2023, 32(11): 1978-1987.

图/表 7

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生化管/培养基名称	沙门氏菌CICC 21484	T1	T2	T3	大肠杆菌ATCC25922
色氨酸肉汤	黄色环 (−)	黄色环 (−)	黄色环 (−)	黄色环 (−)	玫红色环 (+)
尿素酶	淡红色变黄色 (−)	变黄 (−)	变黄 (−)	变黄 (−)	变黄 (−)
氰化钾对照	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)	生长 (+)
氰化钾	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)
赖氨酸脱羧酶肉汤	维持紫色 (+)	紫色 (+)	紫色 (+)	紫色 (+)	紫色 (+)
氨基酸脱羧酶对照	紫色变黄色	黄色	黄色	黄色	黄色
甘露醇	由蓝绿色变黄色 (+)	黄色 (+)	黄色 (+)	黄色 (+)	黄色 (+)
山梨醇	由蓝绿色变黄色 (+)	黄色 (+)	黄色 (+)	蓝绿色 (−)	黄色 (+)
水杨苷	维持蓝绿色,不变色 (−)	不变色 (−)	不变色 (−)	黄色 (+)	不变色 (−)
丙二酸盐	维持淡绿色,不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)
邻硝基酚−半乳糖苷	保持无色,不变色 (−)	变黄色 (+)	变黄色 (+)	变黄色 (+)	黄色 (+)
卫矛醇半固体	蓝绿色变为黄色 (+)	变黄色 (+)	变黄色 (+)	不变色 (−)	不变色 (−)
西蒙氏枸橼酸盐	绿色变为蓝色 (+)	不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)
吲哚试验	黄色环 (−)	红色环 (+)	红色环 (+)	红色环 (+)	红色环 (+)
HE琼脂	蓝绿色菌落,黑色中心	+	+	+	黄色菌落
沙门氏菌显色培养基	紫色菌落	乳白色	乳白色	浅绿色	蓝色
SS琼脂	黑色菌落	+	+	+	桃红色
亚硫酸铋琼脂 (BS)	黑色或灰绿色菌落, 有金属光泽	+	+	+	不生长
麦康凯琼脂 (MAC)	无色至浅橙色, 透明或半透明, 菌落中心有时为暗色	+	+	+	桃红色菌落, 周围有胆盐沉淀环
三铁糖琼脂	黑色菌落	+	+	+	黄色

生化管/培养基名称	沙门氏菌CICC 21484	T1	T2	T3	大肠杆菌ATCC25922
色氨酸肉汤	黄色环 (−)	黄色环 (−)	黄色环 (−)	黄色环 (−)	玫红色环 (+)
尿素酶	淡红色变黄色 (−)	变黄 (−)	变黄 (−)	变黄 (−)	变黄 (−)
氰化钾对照	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)	生长 (+)
氰化钾	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)	不生长 (−)
赖氨酸脱羧酶肉汤	维持紫色 (+)	紫色 (+)	紫色 (+)	紫色 (+)	紫色 (+)
氨基酸脱羧酶对照	紫色变黄色	黄色	黄色	黄色	黄色
甘露醇	由蓝绿色变黄色 (+)	黄色 (+)	黄色 (+)	黄色 (+)	黄色 (+)
山梨醇	由蓝绿色变黄色 (+)	黄色 (+)	黄色 (+)	蓝绿色 (−)	黄色 (+)
水杨苷	维持蓝绿色,不变色 (−)	不变色 (−)	不变色 (−)	黄色 (+)	不变色 (−)
丙二酸盐	维持淡绿色,不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)
邻硝基酚−半乳糖苷	保持无色,不变色 (−)	变黄色 (+)	变黄色 (+)	变黄色 (+)	黄色 (+)
卫矛醇半固体	蓝绿色变为黄色 (+)	变黄色 (+)	变黄色 (+)	不变色 (−)	不变色 (−)
西蒙氏枸橼酸盐	绿色变为蓝色 (+)	不变色 (−)	不变色 (−)	不变色 (−)	不变色 (−)
吲哚试验	黄色环 (−)	红色环 (+)	红色环 (+)	红色环 (+)	红色环 (+)
HE琼脂	蓝绿色菌落,黑色中心	+	+	+	黄色菌落
沙门氏菌显色培养基	紫色菌落	乳白色	乳白色	浅绿色	蓝色
SS琼脂	黑色菌落	+	+	+	桃红色
亚硫酸铋琼脂 (BS)	黑色或灰绿色菌落, 有金属光泽	+	+	+	不生长
麦康凯琼脂 (MAC)	无色至浅橙色, 透明或半透明, 菌落中心有时为暗色	+	+	+	桃红色菌落, 周围有胆盐沉淀环
三铁糖琼脂	黑色菌落	+	+	+	黄色

菌株名称	长度	相似度	相似序列
T1	1701	1498/1503 (99%)	Escherichia coli strain SCDC-1 16S ribosomal RNA gene, partial sequence(HM576813.1)
T2	1696	1500/1504 (99%)	Escherichia coli strain C3, complete genome(CP010119.1)
T3	1694	1502/1504 (99%)	Escherichia coli strain ECCHD184 chromosome, complete genome(CP033250.1)
沙门氏菌 CICC 21484	1702	1504/1505 (99%)	Salmonella enterica subsp. enterica serovar Typhimurium strain 22792, complete genome(CP017621.1)

菌株名称	长度	相似度	相似序列
T1	1701	1498/1503 (99%)	Escherichia coli strain SCDC-1 16S ribosomal RNA gene, partial sequence(HM576813.1)
T2	1696	1500/1504 (99%)	Escherichia coli strain C3, complete genome(CP010119.1)
T3	1694	1502/1504 (99%)	Escherichia coli strain ECCHD184 chromosome, complete genome(CP033250.1)
沙门氏菌 CICC 21484	1702	1504/1505 (99%)	Salmonella enterica subsp. enterica serovar Typhimurium strain 22792, complete genome(CP017621.1)

检测到的基因	T1	T2	T3
共有ARGs	aadA、aadA1、aadA2、acrA、acrB、acrF、acrR、ampC、aphA1、bacA、blaCMY2、blaCTX-M、blaKPC、blaOXY、blaSHV、cmlA1、cmlA5、dfrA12、floR、fox5、mepA、oprD、qacH、rarD、tetA、tetM、tetPB、tetR、tetZ、tolC、vanC、vanTC、mdtE/yhiU、mdtF、yceE/mdtG、yceL/mdtH、yidY/mdtL
共有MGEs	intl1、IS1111、pBS228-IncP-1α、tnpA-03/ IS6、tnpA-05/IS26
特有ARGs	qacE∆1	blaCMY、blaPAO、blaSFO cphA、tetW、vanHB、vanXA	aadA5、blaDHA、blaPAO blaSFO、cphA、tetO
特有MGEs	tnpA-07/ISEcp1 intI3	incW_trwAB、orf37-IS26、tnpA-01/Tn21、 tnpA-02/IS4tnpA-06/IS1216、pNI105map-F	incW_trwAB、orf37-IS26、tnpA-01/Tn21、 tnpA-02/IS4、tnpA-06/IS1216

土壤环境中四环素抗性基因向病原菌的转移研究

Transfer of Tetracycline Resistance Gene to Human Pathogenic Bacteria in Soil

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质粒名称	质粒长度/bp	相似序列编号	覆盖率/%	相似度/%	相似序列描述
质粒1 (IncX1)	40481	NZ_CP018104.1	19.43	99.71	Escherichia coli strain MRSN352231 plasmid pMR0716_tem1, complete sequence
		NZ_CP026492.1	27.18	99.85	Escherichia coli strain HS13-1 plasmid pHS13-1-IncHI2, complete sequence
		NZ_CP010155.1	99.19	99.97	Escherichia coli strain D9 plasmid C, complete genome
		NZ_CP011062.1	21.71	99.68	Escherichia coli str. Sanji plasmid pSJ_255, complete sequence
		NZ_CP022451.1	22.22	99.66	Salmonella enterica subsp. enterica serovar Indiana strain D90 plasmid pD90-1, complete sequence
质粒2 (IncY)	93314	NZ_CP018104.1	16.32	99.93	Escherichia coli strain MRSN352231 plasmid pMR0716_tem1, complete sequence
		NZ_CP026492.1	33.89	99.86	Escherichia coli strain HS13-1 plasmid pHS13-1-IncHI2, complete sequence
		NZ_CP021937.1	19.26	99.66	Escherichia coli strain AR_0055 plasmid unitig_3j3rc_linear, complete sequence
		NZ_CP025740.1	27.04	97.44	Escherichia coli strain Ec40 plasmid unnamed
		NZ_CP017632.1	35.36	99.96	Escherichia coli SLK172 plasmid pSLK172-1, complete sequence

基因组序列	ARDB (相似度大于98%)	CARD (相似度大于91%)
染色体	emrD、mdtF、mdtE、acrB、bacA、tolC、arnA、bcr、mdtK、mdfA、macB、mdtG、mdtH、acrA、acrB、ksga、mdtM、bl1_ec、mdtN、mdtO、mdtP、mdtL	tolC、bacA、patA、acrS、acrE、mdtL、adeG、cpxA、cpxR、mdtP、mdtO、mdtN、PmrB、PmrC、adiY、CRP、mdtM、leuO、adeG、kdpE、marA、marR、emrD、cysB、H-NS、mfd、mdtH、mdtG、msbA、mdfA、gadE、mdtE、mdtF、gadW、gadX、PmrE、mdtA、mdtC、mdtC、mdtD、baeS、baeR、YojI、GlpT、PmrF、arnA、evgS、adeG、emrR、emrA、emrB、alaS
质粒1 (IncX1)	aph3ia、qnrS、cml_e3	APH(3’)-Ia、QnrS2、floR
质粒2 (IncY)	bl2b_tem1、tetA、tetM、dfra12、 ant2ia、cml_e1、ant3ia、ermE，sul3	sul3、qacH、aadA、aadA17 dfrA12、tetO、TEM-1