首次从湖南猪源大肠杆菌中检出mcr-1阳性IncI2(Delta)质粒

doi:10.11843/j.issn.0366-6964.2024.11.043

摘要/Abstract

摘要：

近年来，由于抗生素耐药基因的广泛传播及多重耐药细菌的出现，导致抗生素的使用面临严峻挑战，致使毒性较强的多黏菌素又重新引起业界的重视，为此对多黏菌素耐药情况的研究也变得十分重要。为了调查畜牧场中多黏菌素耐药基因mcr-1的流行情况，本研究选择我国湖南省某猪场240份肠肛拭子样品，培养分离鉴定出含有mcr-1耐药基因的大肠杆菌菌株，选取多黏菌素等10种抗菌药物对分离的菌株进行耐药性试验，对所有含mcr-1耐药基因的大肠杆菌阳性株进行全基因组测序(whole-genome sequencing，WGS)，采用多位点序列分型(multi-locus sequence typing，MLST)技术进行大肠杆菌的遗传多样性分析。研究结果表明，在分离出的172株大肠杆菌中，来自不同个体的9株携带mcr-1基因的大肠杆菌均表现出多重耐药现象；MLST分析共鉴定出ST10、ST196、ST46和ST5229共4种分型和4种血清型(O83:H5, O16:H7, O16:H51, O9:H4)。生物信息学分析显示，所有阳性菌株均未发现与mcr-1基因传播有关的典型可移动遗传元件ISApl1，但均检出可能会导致mcr-1传播的Ⅳ型分泌系统基因。质粒接合转移试验与单倍型的MLST多态性结果表明，mcr-1基因主要以质粒介导的水平传播为主。本研究是国际上首次在猪源细菌中检出常见于人医临床细菌的携带有mcr-1抗性基因的IncI2(Delta)质粒。

关键词: 多黏菌素, mcr-1, IncI2(delta)质粒, 多重耐药, 猪, 大肠杆菌

Abstract:

The rise of antibiotic resistance has brought renewed attention to older drugs like polymyxins, making it important to control the spread of resistance genes. In this study, we focused on colistin-resistant Escherichia coli strains from a pig farm in Hunan, China. Out of 240 samples, 172 E. coli strains were isolated and cultured, PCR was used to identify mcr-1 positive E. coli strains. These strains were tested by ten different antibiotics to check for their drug resistance, and whole-genome sequencing (WGS) was applied to analyze their genetic background. All the nine mcr-1 positive E. coli strains exhibit high levels of multidrug resistance. Four distinct MLST types (ST10, ST196, ST5229, ST46) and 4 serotypes (O83:H5, O16:H7, O16:H51, O9:H4) in the nine strains were identified. Further bioinformatics analyses showed that the ISApl1 element, which is commonly associated with mcr-1 transmission, was not observed in the present study, while the type Ⅳ secretion system genes were detected in all the nine mcr-1 positive E. coli strains. Plasmid conjugation experiments and the presence of polymorphic MLST haplotypes indicated a vertical plasmid-mediated mcr-1 transmission possibility. Notably, our study for the first time uncovered a pig origin IncI2(Delta) plasmid carrying mcr-1 gene in E. coli which is normally presented in the human clinical E. coli strains.

Key words: colistin resistance, mcr-1, plasmid IncI2(delta), multiple drug resistance, pig, Escherichia coli

中图分类号:

S852.612

黄康溦, 周鹏程, 田晨宇, 兰怡, 孙志良. 首次从湖南猪源大肠杆菌中检出mcr-1阳性IncI2(Delta)质粒[J]. 畜牧兽医学报, 2024, 55(11): 5278-5286.

Kangwei HUANG, Pengcheng ZHOU, Chenyu TIAN, Yi LAN, Zhiliang SUN. First Report of Colistin-Resistant Escherichia coli Carrying mcr-1 IncI2(Delta) Plasmids from Pigs in Hunan Province, China[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5278-5286.

图/表 5

表 1

图 1

图 2

图 3

图 4

参考文献 33

1	MURRAY C J L , IKUTA K S , SHARARA F , et al. Global burden of bacterial antimicrobial resistance in 2019:a systematic analysis[J]. Lancet, 2022, 399 (10325): 629- 655. doi: 10.1016/S0140-6736(21)02724-0
2	EL-SAYED AHMED M A E G , ZHONG L L , SHEN C , et al. Colistin and its role in the Era of antibiotic resistance: an extended review (2000-2019)[J]. Emerg Microbes Infections, 2020, 9 (1): 868- 885. doi: 10.1080/22221751.2020.1754133
3	LI Q L , QIAN C R , ZHANG X Y , et al. Colistin resistance and molecular characterization of the genomes of mcr-1-positive Escherichia coli clinical isolates[J]. Front Cell Infect Microbiol, 2022, 12, 854534. doi: 10.3389/fcimb.2022.854534
4	LIU Y Y , WANG Y , WALSH T R , et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study[J]. Lancet Infect Dis, 2016, 16 (2): 161- 168. doi: 10.1016/S1473-3099(15)00424-7
5	LING Z R , YIN W J , SHEN Z Q , et al. Epidemiology of mobile colistin resistance genes mcr-1 to mcr-9[J]. J Antimicrob Chemother, 2020, 75 (11): 3087- 3095. doi: 10.1093/jac/dkaa205
6	SHAFIQ M , HUANG J H , RAHMAN S U , et al. High incidence of multidrug-resistant Escherichia coli coharboring mcr-1 and bla_CTX-M-15 recovered from pigs[J]. Infect Drug Resist, 2019, 12, 2135- 2149. doi: 10.2147/IDR.S209473
7	LUJAN S A , GUOGAS L M , RAGONESE H , et al. Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase[J]. Proc Natl Acad Sci U S A, 2007, 104 (30): 12282- 12287. doi: 10.1073/pnas.0702760104
8	SRIVASTAVA S , SINGH V , KUMAR V , et al. Identification of regulatory elements in 16S rRNA gene of Acinetobacter species isolated from water sample[J]. Bioinformation, 2008, 3 (4): 173- 176. doi: 10.6026/97320630003173
9	MENTASTI M , DAVID S , SANDS K , et al. Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis[J]. J Hosp Infect, 2021, 110, 148- 155. doi: 10.1016/j.jhin.2021.01.010
10	BANKEVICH A , NURK S , ANTIPOV D , et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[J]. J Comput Biol, 2012, 19 (5): 455- 477. doi: 10.1089/cmb.2012.0021
11	ANTONOPOULOS D A , ASSAF R , AZIZ R K , et al. PATRIC as a unique resource for studying antimicrobial resistance[J]. Brief Bioinform, 2019, 20 (4): 1094- 1102. doi: 10.1093/bib/bbx083
12	BORTOLAIA V , KAAS R S , RUPPE E , et al. ResFinder 4.0 for predictions of phenotypes from genotypes[J]. J Antimicrob Chemother, 2020, 75 (12): 3491- 3500. doi: 10.1093/jac/dkaa345
13	MALBERG TETZSCHNER A M , JOHNSON J R , JOHNSTON B D , et al. In silico genotyping of Escherichia coli isolates for extraintestinal virulence genes by use of whole-genome sequencing data[J]. J Clin Microbiol, 2020, 58 (10): e01269- 20.
14	SULLIVAN M J , PETTY N K , BEATSON S A . Easyfig: a genome comparison visualizer[J]. Bioinformatics, 2011, 27 (7): 1009- 1010. doi: 10.1093/bioinformatics/btr039
15	PARTRIDGE S R , ENNE V I , GROHMANN E , et al. Classifying mobile genetic elements and their interactions from sequence data: the importance of existing biological knowledge[J]. Proc Natl Acad Sci U S A, 2021, 118 (35): e2104685118. doi: 10.1073/pnas.2104685118
16	JAIN C , RODRIGUEZ-R L M , PHILLIPPY A M , et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nat Commun, 2018, 9 (1): 5114. doi: 10.1038/s41467-018-07641-9
17	STREPIS N , VOOR IN'T HOLT A F , VOS M C , et al. Genetic analysis of mcr-1-carrying plasmids from gram-negative bacteria in a Dutch tertiary care hospital: evidence for intrapatient and interspecies transmission events[J]. Front Microbiol, 2021, 12, 727435. doi: 10.3389/fmicb.2021.727435
18	SHEN C , ZHONG L L , YANG Y Q , et al. Dynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study[J]. Lancet Microbe, 2020, 1 (1): e34. doi: 10.1016/S2666-5247(20)30005-7
19	WALSH T R , WU Y N . China bans colistin as a feed additive for animals[J]. Lancet Infect Dis, 2016, 16 (10): 1102- 1103. doi: 10.1016/S1473-3099(16)30329-2
20	XIAO X , ZENG F X , LI R C , et al. Subinhibitory concentration of colistin promotes the conjugation frequencies of mcr-1- and bla_NDM-5-positive plasmids[J]. Microbiol Spectr, 2022, 10 (2): e0216021. doi: 10.1128/spectrum.02160-21
21	LUO Q X , WANG Y , XIAO Y H . Prevalence and transmission of mobilized colistin resistance (mcr) gene in bacteria common to animals and humans[J]. Biosaf Health, 2020, 2 (2): 71- 78. doi: 10.1016/j.bsheal.2020.05.001
22	MAAMAR E , ALONSO C A , HAMZAOUI Z , et al. Emergence of plasmid-mediated colistin-resistance in CMY-2-producing Escherichia coli of lineage ST2197 in a Tunisian poultry farm[J]. Int J Food Microbiol, 2018, 269, 60- 63. doi: 10.1016/j.ijfoodmicro.2018.01.017
23	FANG L X , LI X P , LI L , et al. ISEcp1-mediated transposition of chromosome-borne bla_CMY-2 into an endogenous ColE1-like plasmid in Escherichia coli[J]. Infect Drug Resist, 2018, 11, 995- 1005. doi: 10.2147/IDR.S159345
24	SHIGEMURA H , MAEDA T , NAKAYAMA S , et al. Transmission of extended-spectrum cephalosporin-resistant Salmonella harboring a bla_CMY-2-carrying IncA/C₂ plasmid chromosomally integrated by ISEcp1 or IS26 in layer breeding chains in Japan[J]. J Vet Med Sci, 2021, 83 (9): 1345- 1355. doi: 10.1292/jvms.21-0085
25	SNESRUD E , MCGANN P , CHANDLER M . The birth and demise of the ISApl1-mcr-1-ISApl1 composite transposon: the vehicle for transferable colistin resistance[J]. mBio, 2018, 9 (1): e02381- 17.
26	BUCHANAN-WOLLASTON V , PASSIATORE J E , CANNON F . The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants[J]. Nature, 1987, 328 (6126): 172- 175. doi: 10.1038/328172a0
27	CASCALES E , CHRISTIE P J . The versatile bacterial type Ⅳ secretion systems[J]. Nat Rev Microbiol, 2003, 1 (2): 137- 149. doi: 10.1038/nrmicro753
28	CHEETHAM M E , CAPLAN A J . Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function[J]. Cell Stress Chaperones, 1998, 3 (1): 28- 36. doi: 10.1379/1466-1268(1998)003<0028:SFAEOD>2.3.CO;2
29	GUO S Y , TAY M Y F , THU A K , et al. Conjugative IncX1 plasmid harboring colistin resistance gene mcr-5. 1 in Escherichia coli isolated from chicken rice retailed in Singapore[J]. Antimicrob Agents Chemother, 2019, 63 (11): e01043- 19.
30	GOMI R , MATSUDA T , YAMAMOTO M , et al. Molecular characterization of a multidrug-resistant IncF plasmid carrying mcr-3. 1 in an Escherichia coli sequence type 393 strain of wastewater origin[J]. Int J Antimicrobial Agents, 2019, 54 (4): 524- 526. doi: 10.1016/j.ijantimicag.2019.06.024
31	SAIDANI M , MESSADI L , MEFTEH J , et al. Various inc-type plasmids and lineages of Escherichia coli and Klebsiella pneumoniae spreading bla_CTX-M-15, bla_CTX-M-1 and mcr-1 genes in camels in Tunisia[J]. J Global Antimicrob Resist, 2019, 19, 280- 283. doi: 10.1016/j.jgar.2019.05.007
32	WU R J , YI L X , YU L F , et al. Fitness Advantage of mcr-1–bearing IncI2 and IncX4 plasmids in vitro[J]. Front Microbiol, 2018, 9, 331. doi: 10.3389/fmicb.2018.00331
33	GHAZAWI A , STREPIS N , ANES F , et al. First report of colistin-resistant Escherichia coli carrying mcr-1 IncI2(delta) and IncX4 plasmids from camels (Camelus dromedarius) in the gulf region[J]. Antibiotics (Basel), 2024, 13 (3): 227. doi: 10.3390/antibiotics13030227

菌株 Isolate	血清型 Serotype	来源 Origin	猪舍 Hog house	最小抑菌浓度MIC
菌株 Isolate	血清型 Serotype	来源 Origin	猪舍 Hog house	CTX(2)	CAZ(4)	MEM(8)	GEN(2)	CIP(0.5)	AMK(8)	CL(2)	TCY(4)^*	FLR(16)^*	SXT(4)
23cg187	O83:H5	NP	NH 1	＞32	＞32	＞8	＞64	＞2	＞64	＞16	＞8	8	8
23cg189	O16:H7	NP	NH 1	＞32	＞32	4	＞64	2	＞64	＞16	8	2	4
23cg201	O16:H7	NP	NH 4	＞32	＞32	4	＞64	＞2	＞64	＞16	＞8	8	8
23cg214	O16:H51	FP	FH 2	＞32	＞32	0.25	＞64	＞2	＞64	＞16	8	4	4
23cg215	O16:H51	FP	FH 2	＞32	＞32	4	＞64	＞2	＞64	＞16	＞8	4	4
23cg223	O9:H4	FP	FH 6	＞32	32	1	＞64	＞2	＞64	＞16	＞8	2	2
23cg225	O16:H51	FP	FH 6	＞32	＞32	4	＞64	＞2	＞64	＞16	＞8	4	4
23cg226	O16:H51	FP	FH 6	＞32	＞32	8	＞64	＞2	＞64	＞16	＞8	4	4
23cg230	O9:H4	FP	FH 6	＞32	＞32	8	＞64	＞2	＞64	＞16	＞8	16	16

[1]	田晶晶, 王晓庆, 李棉燕, 王海玲, 吴启钿, 王立贤, 张龙超, 赵福平. 北京黑猪全基因组ROH检测和选择信号分析[J]. 畜牧兽医学报, 2024, 55(9): 3833-3842.
[2]	陈栋, 周文譞, 赵真坚, 申琦, 余杨, 崔晟頔, 王俊戈, 陈子旸, 禹世欣, 陈佳苗, 王翔枫, 吴平先, 郭宗义, 王金勇, 唐国庆. 基于计算机视觉技术的猪肌内脂肪含量和眼肌面积测定系统的研发[J]. 畜牧兽医学报, 2024, 55(9): 3843-3852.
[3]	陈南珠, 李俊良, 余大为, 周心仪, 王晶, 邹惠影, 杜卫华. 猪MKRN3基因的印记表达和DNA甲基化状态分析[J]. 畜牧兽医学报, 2024, 55(9): 3853-3863.
[4]	杨柏高, 龙熙, 张亮, 徐皆欢, 戴建军, 赵学明, 潘红梅. 基于Smart-seq2探究玻璃化冷冻对猪孤雌激活囊胚基因表达的影响[J]. 畜牧兽医学报, 2024, 55(9): 3936-3946.
[5]	任聪, 张虎, 王钰明, 解竞静, 萨仁娜, 赵峰. 仿生消化法估测生长猪饲料有效能的准确性及可加性研究[J]. 畜牧兽医学报, 2024, 55(9): 3988-4000.
[6]	高力国, 申翰钦, 陈诒全, 陈胜, 蔺文成, 陈峰. 猪轮状病毒重组VP6*蛋白的原核表达及间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2024, 55(9): 4021-4028.
[7]	朋璐, 张衡, 庞思琪, 乔竹林, 张小芬, 谭臣, 宋云峰, 周锐, 黎璐. 利用大蜡螟幼虫和小鼠感染模型筛选猪链球菌血清2、3和9型三价灭活疫苗候选菌株[J]. 畜牧兽医学报, 2024, 55(9): 4077-4090.
[8]	付艺乾, 梁东阁, 王铭洋, 潘佳佳, 杨彦宾, 曾磊, 康相涛. 干扰素调节因子敲减细胞系的构建及其对猪伪狂犬病病毒增殖的影响[J]. 畜牧兽医学报, 2024, 55(9): 4100-4109.
[9]	王佳宁, 张自强, 王帅帅, 刘玉梅. 兔源大肠杆菌、奇异变形杆菌和球虫混合感染及药物敏感性分析[J]. 畜牧兽医学报, 2024, 55(9): 4204-4212.
[10]	彭宁, 梁雅旭, 龙菲, 余东明, 钟翔. 白藜芦醇对轮状病毒感染猪肠上皮细胞IPEC-J2的抑制效应[J]. 畜牧兽医学报, 2024, 55(9): 4213-4225.
[11]	冯露, 田宏, 郑海学, 石正旺, 罗俊聪, 张晓阳, 尉娟娟, 周静, 廖焕程, 王婉莹. 基于酶促重组酶扩增的非洲猪瘟病毒检测方法[J]. 畜牧兽医学报, 2024, 55(9): 4226-4231.
[12]	夏振涛, 王楠, 王婉洁, 周期律, 黄雷, 牟玉莲. pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析[J]. 畜牧兽医学报, 2024, 55(8): 3395-3407.
[13]	王怡, 高娟, 胡悦旻, 杨跃飞, 范博钧, 鞠辉明. 短期血清饥饿胁迫对猪骨骼肌卫星细胞代谢及自噬发生的影响[J]. 畜牧兽医学报, 2024, 55(8): 3408-3417.
[14]	杨程, 刘野, 程宁, 王凯月, 李欣蕾, 孙久英, 韩俊平, 李文军, 王欢欢, 邵笑, 程雪娇, 孙英峰. 一株PRRSV-2谱系1.8与1.5重组毒株的基因组特征分析[J]. 畜牧兽医学报, 2024, 55(8): 3570-3578.
[15]	李跃, 张长春, 刘光裕, 高梦源, 符超俊, 邢家宝, 徐思佳, 邝麒元, 刘静, 高校鹏, 王衡, 龚浪, 张桂红, 孙彦阔. 宏转录组测序技术在一起仔猪病毒性腹泻疾病诊断中的运用及分析[J]. 畜牧兽医学报, 2024, 55(8): 3579-3589.