黏菌素促进mcr-1阳性IncI2质粒在大肠杆菌间的接合转移

doi:10.3864/j.issn.0578-1752.2022.14.015

摘要/Abstract

摘要：

【背景】黏菌素是人医临床治疗多重耐药革兰阴性菌感染的“最后一道防线”药物,其同时作为饲料添加剂及治疗用药长期用于畜禽养殖业。2015年,我国研究人员首次报道了质粒介导的黏菌素耐药基因mcr-1,预示该道防线面临被攻破的风险。然而,杀菌浓度及亚抑菌浓度的黏菌素对mcr-1阳性质粒传播的影响仍未知。【目的】以流行最广的mcr-1阳性IncI2型质粒为研究对象,探究不同浓度黏菌素对该质粒接合转移频率的影响,为黏菌素的科学使用提供理论依据。【方法】使用肉汤法进行了不同浓度黏菌素（0.02-4 μg·mL^-1）作用下携带mcr-1的IncI2型质粒的接合转移试验。通过实时荧光定量PCR（RT-qPCR）及构建的公式计算不同时间点（1-24 h）的接合转移频率,并分析不同浓度黏菌素对IncI2质粒接合转移频率的影响。使用PI染料、活性氧（ROS）检测试剂盒测定不同浓度黏菌素下供体菌和受体菌的细胞膜透过性和ROS产生量。选取了对照组与低中高浓度3个处理组（0.02、1、4 μg·mL^-1）进行了转录组测序,使用Deseq2软件进行基因差异表达分析。采用Prism v8.2.0软件对不同组间接合转移频率、细胞膜透过性及ROS产生量的差异进行统计学分析。【结果】结合本研究建立的接合转移频率计算公式表明黏菌素浓度为4 μg·mL^-1时可在不同时间点提高mcr-1阳性IncI2质粒3-10倍的接合转移频率,而其他浓度则无显著差异。转录组结果显示,与对照组相比,低中高浓度黏菌素均可显著提高IncI2质粒上IV型分泌系统相关基因的表达,其中包括virB1、virB2、virB5以及traC。此外,黏菌素增加了I型菌毛合成基因fim的转录水平。PI染色结果显示当黏菌素浓度为2和4 μg·mL^-1时,可增强供体菌和受体菌的细胞膜透过性,且转录组结果也显示膜相关基因（ompAX、bamDE、lolB、yiaD、csgEF）的转录水平均出现显著上调。但是黏菌素作用后ROS产生量及相关基因的转录水平无显著提高。【结论】揭示了黏菌素可通过增加细菌IV型分泌系统活性、细胞膜透过性和菌毛的生成从而提高mcr-1阳性IncI2质粒在大肠杆菌间的接合转移频率。研究结果提示,杀菌浓度无法完全杀死mcr-1阳性大肠杆菌的同时,还能增加质粒传播速度。因此,畜禽养殖业中黏菌素的治疗使用可能维持mcr-1阳性质粒的存在及传播,此外鉴于黏菌素已批准用于人医临床,该现象有可能引起临床黏菌素治疗mcr-1阳性病原菌的失败。

关键词: 黏菌素, mcr-1, 大肠杆菌, IncI2质粒, 接合转移

Abstract:

【Background】 Colistin is a last line antibiotic for the treatment of clinical infections caused by multi-drug resistant Gram-negative bacteria, and it has also been extensively used in animal industry as a feed additive and therapeutic drug. In 2015, Chinese researchers discovered the plasmid-mediated colistin resistance gene mcr-1, indicating that this last defense line is at risk of being breached. However, the effects of bactericidal concentration and sub-inhibitory concentration of colistin on the transmission of mcr-1-positive plasmid is still unknown. 【Objective】 This study used the most prevalent mcr-1-positive plasmid IncI2 as an object to explore the influence on the conjugative transfer frequency under different colistin concentrations. 【Method】 The conjugation experiment under different colistin concentrations (0.02-4 μg·mL^-1) was carried out by the broth method. Real time quantitative PCR and the constructed formula were used to calculate the conjugative transfer frequency at different timepoints (1-24 h) and also different colistin concentrations. The cell membrane permeability and ROS production of donor and recipient bacteria under different colistin concentrations were detected by using PI dye and reactive oxygen species (ROS) detection kit, respectively. Colistin negative group and the three treatment groups (0.02, 1, and 4 μg·mL^-1) were subjected to RNA sequencing as control, low, medium and high concentration groups, respectively, and the gene differential expression was analyzed by Deseq2 software. All statistical analysis were conducted by Prism v8.2.0 software. 【Result】 A formula was established to calculate the conjugative frequency in this study, and it was found that the bactericidal concentration (4 μg·mL^-1) of colistin significantly increased the conjugative transfer frequency of mcr-1-positive IncI2 plasmid by 3-10 times at different timepoints, whilst no significant difference on other concentrations. Transcriptome results showed that when compared with control group, the expression of genes related to type IV secretion system (T4SS) in IncI2 plasmid, including virB1, virB2, virB5 and traC, were significantly increased in all colistin concentrations groups. In addition, the expressional level of type I fimbrium biosynthesis genes were significantly increased in all colistin groups. PI staining results showed that 2 and 4μg·mL^-1 colistin could elevate the cell membrane permeability in donor and recipient bacteria, and the coincidently transcriptome results showed that the expressional levels of membrane-related genes, including ompAX, bamDE, lolB, yiaD, csgEF, were significantly up-regulated. However, ROS production and expressional level of related genes were not significantly increased after colistin treatment. 【Conclusion】 This study revealed that colistin promoted the conjugative transfer frequency of mcr-1-positive IncI2 plasmid between E. coli by increasing the activity of bacterial T4SS, cell membrane permeability and pilus formation, suggesting the bactericidal concentration of colistin could increase the plasmid transmission in all survived mcr-1-positive E. coli. Therefore, the therapeutic use of colistin in animals might maintain the existence and transmission of mcr-1-positive plasmids. In addition, since colistin has been approved for clinical use in human medicine, this phenomenon could lead to the failure on colistin treatment for mcr-1-positive pathogens.

Key words: colistin, mcr-1, Escherichia coli, IncI2 plasmid, conjugation

王雪杨,蒋君瑶,杨璐,邵东延,吴聪明,沈建忠,沈应博,汪洋. 黏菌素促进mcr-1阳性IncI2质粒在大肠杆菌间的接合转移[J]. 中国农业科学, 2022, 55(14): 2862-2874.

WANG XueYang,JIANG JunYao,YANG Lu,SHAO DongYan,WU CongMing,SHEN JianZhong,SHEN YingBo,WANG Yang. Colistin Promotes mcr-1-positive IncI2 Plasmid Conjugation Between Escherichia coli[J]. Scientia Agricultura Sinica, 2022, 55(14): 2862-2874.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.14.015

https://www.chinaagrisci.com/CN/Y2022/V55/I14/2862

图/表 8

表1

图1

图2

图3

图4

图5

图6

图7

参考文献 32

[1]	郝海红, 程古月, 戴梦红, 王旭, 王玉莲, 黄玲利, 刘振利, 袁宗辉. 对动物饲料中禁用抗菌促生长剂的反思. 中国农业科学, 2015, 48(3): 594-603.
	HAO H H, CHENG G Y, DAI M H, WANG X, WANG Y L, HUANG L L, LIU Z L, YUAN Z H. Rethinking the Withdrawal of Antimicrobial Growth Promotants in Animal Feed. Scientia Agricultura Sinica, 2015, 48(3): 594-603. (in Chinese)
[2]	SHEN Y B, ZHANG R, SCHWARZ S, WU C M, SHEN J Z, WALSH TR, WANG Y. Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes. Environmental Microbiology, 2020, 22(7): 2469-2484. doi: 10.1111/1462-2920.14961
[3]	OLAITAN A O, MORAND S, ROLAIN J M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Frontier in Microbiology, 2014, 5: 643.
[4]	宗劲, 徐凯进, 黄莹, 刘超群, 吴淼星, 韩东. 质粒介导黏菌素耐药机制研究进展. 中国微生态学杂志, 2021, 33(01): 120-125.
	ZONG J, XU K J, HUANG Y, LIU C Q, WU M X, HAN D. Progress in research on plasmid-mediated colistin resistance. Chinese Journal of Microecology, 2021, 33(01): 120-125. (in Chinese)
[5]	LIU Y Y, WANG Y, WALSH T R, YI L X, ZHANG R, SPENCER J, DOI Y, TIAN G B, DONG B L, HUANG X H, YU L F, GU D X, REN H W, CHEN X, J LV L C, HE D D, ZHOU H W, LIANG Z S, LIU J H, SHEN J Z. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet Infectious Diseases, 2016, 16(2): 161-168. doi: 10.1016/S1473-3099(15)00424-7
[6]	SUN J, ZHANG H M, LIU Y H, FENG Y J. Towards Understanding MCR-like Colistin Resistance. Trends in Microbiology, 2018, 26(9): 794-808. doi: 10.1016/j.tim.2018.02.006
[7]	WANG R B, VAN D L, SHAW L P, BRADLEY P, WANG Q, WANG X J, JIN L Y, ZHANG Q, LIU Y Q, RIEUX A, DORAI-SCHNEIDERS T, WEINERT L A, IQBAL Z, DIDELOT X, WANG H, BALLOUX F. The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nature Communications, 2018, 9(1): 1179. doi: 10.1038/s41467-018-03205-z
[8]	WANG Y, XU C Y, ZHANG R, CHEN Y Q, SHEN Y B, HU F P, LIU D J, LU J Y, GUO Y, XIA X, JIANG J Y, WANG X Y, FU Y L, YANG L, WANG J Y, LI J, CAI C, YIN D D, CHE J, FAN R, WANG Y Q, QING Y, LI Y, LIAO K, CHEN H, ZOU M X, LIANG L, TANG J, SHEN Z Q, WANG S L, YANG X R, WU C M, XU S X, WALSH T R, SHEN J Z. Changes in colistin resistance and mcr-1 abundance in Escherichia coli of animal and human origins following the ban of colistin-positive additives in China: an epidemiological comparative study. The Lancet Infectious Diseases, 2020, 20(10): 1161-1171. doi: 10.1016/S1473-3099(20)30149-3
[9]	ANDERSSON D I, HUGHES D. Microbiological effects of sublethal levels of antibiotics. Nature Review Microbiology, 2014, 12(7): 465-478. doi: 10.1038/nrmicro3270
[10]	LUNDSTROM S V, OSTMAN M, BENGTSSON-PALME J, RUTGERSSON C, THOUDAL M, SIRCAR T, BLANCK H, ERIKSSON K M, TYSKLIND M, FLACH C F, LARSSON D G J. Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms. Science of the Total Environment, 2016, 553: 587-595. doi: 10.1016/j.scitotenv.2016.02.103
[11]	MARKOU N, MARKANTONIS S L, DIMITRAKIS E, PANIDIS D, BOUTZOUKA E, KARATZAS S, RAFAILIDIS P, APOSTOLAKOS H, BALTOPOULOS G. Colistin serum concentrations after intravenous administration in critically ill patients with serious multidrug-resistant, gram-negative bacilli infections: a prospective, open-label, uncontrolled study. Clinical Therapeutics, 2008, 30(1): 143-151. doi: 10.1016/j.clinthera.2008.01.015
[12]	MOHAMED A F, KARAISKOS I, PLACHOURAS D, KARVANEN M, PONTIKIS K, JANSSON B, PAPADOMICHELAKIS E, ANTONIADOU A, GIAMARELLOU H, ARMAGANIDIS A, CARS O, FRIBERG L E. Application of a loading dose of colistin methanesulfonate in critically ill patients: population pharmacokinetics, protein binding, and prediction of bacterial kill. Antimicrobial Agents and Chemotherapy, 2012, 56(8): 4241-4249. doi: 10.1128/AAC.06426-11
[13]	The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, Version 11.0. http://www.eucast.org. 2021.
[14]	BETHKE J H, DAVIDOVICH A, CHENG L, LOPATKIN A J, SONG W, THADEN J T, FOWLER V G, J., XIAO M F, YOU L C. Environmental and genetic determinants of plasmid mobility in pathogenic Escherichia coli. Science Advances, 2020, 6(4): eaax3173.
[15]	WAN Z, VARSHAVSKY J, TEEGALA S, MCLAWRENCE J, GODDARD NL. Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR. Biophysical Journal, 2011, 101(1): 237-244. doi: 10.1016/j.bpj.2011.04.054
[16]	FU Y L, LIU D J, SONG H W, LIU Z H, JIANG H Y, WANG Y. Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene tet(X) Variants from Bacterial, Fecal, and Environmental Samples. Antimicrobial Agents and Chemotherapy, 2020, 64(4): e02292-19.
[17]	LI J Y, SHI X M, YIN W J, WANG Y, SHEN Z Q, DING S Y, WANG S L. A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples. Frontier in Microbiology, 2017, 8: 2078.
[18]	CHEN S F, ZHOU Y Q, CHEN Y R, GU J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i890.
[19]	TJADEN B. A computational system for identifying operons based on RNA-seq data. Methods, 2020, 176: 62-70. doi: 10.1016/j.ymeth.2019.03.026
[20]	KOPYLOVA E, NOE L, TOUZET H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics, 2012, 28(24): 3211-7. doi: 10.1093/bioinformatics/bts611
[21]	KIM D, LANGMEAD B, SALZBERG S L. HISAT: a fast spliced aligner with low memory requirements. Nature Methods, 2015, 12(4): 357-360. doi: 10.1038/nmeth.3317
[22]	LIAO Y, SMYTH G K, SHI W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2014, 30(7): 923-930. doi: 10.1093/bioinformatics/btt656
[23]	LOVE M I, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014, 15(12): 550. doi: 10.1186/s13059-014-0550-8
[24]	CABEZON E, ROZADA R J, PENA A, DE LA CRUZ F, ARECHAGA I. Towards an integrated model of bacterial conjugation. FEMS Microbiology Reviews, 2015, 39(1): 81-95.
[25]	RENDON M A, SALDANA Z, ERDEM A L, MONTEIRO-NETO V, VAZQUEZ A, KAPER J B, PUENTE J L, GIRON J A. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. Proceedings of the National Academy of Sciences (USA), 2007, 104(25): 10637-10642.
[26]	THOMAS C M, NIELSEN K M. Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nature Review Microbiology, 2005, 3(9): 711-721. doi: 10.1038/nrmicro1234
[27]	GUO M T, YUAN Q B, YANG J. Distinguishing effects of ultraviolet exposure and chlorination on the horizontal transfer of antibiotic resistance genes in municipal wastewater. Environmental Science Technology, 2015, 49(9): 5771-5778. doi: 10.1021/acs.est.5b00644
[28]	QIU Z G, YU Y M, CHEN Z L, JIN M, YANG D, ZHAO Z G, WANG J, F SHEN Z Q, WANG X W, QIAN D, HUANG A H, ZHANG B C, LI J W. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera. Proceedings of the National Academy of Sciences (USA), 2012, 109(13): 4944-4949.
[29]	BEABER J W, HOCHHUT B, WALDOR M K. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature, 2004, 427(6969): 72-74. doi: 10.1038/nature02241
[30]	JUTKINA J, RUTGERSSON C, FLACH C F, JOAKIM L D G. An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance. Science of the Total Environment, 2016, 548-549: 131-138. doi: 10.1016/j.scitotenv.2016.01.044
[31]	KOCH-WESER J, SIDEL V W, FEDERMAN E B, KANAREK P, FINER D C, EATON A E. Adverse effects of sodium colistimethate. Manifestations and specific reaction rates during 317 courses of therapy. Annals of Internal Medicine, 1970, 72(6): 857-868. doi: 10.7326/0003-4819-72-6-857
[32]	AVALOS V I, HOSSEINI V, KOLLMANNSBERGER P, MEIER S, WEBER S S, ARNOLDINI M, ACKERMANN M, VOGEL V. How type1 fimbriae help Escherichia coli to evade extracellular antibiotics. Scientific Reports, 2016, 6: 18109.

基因 Gene	引物序列（5′-3′） Primer sequence	标准曲线 Standard curve	片段大小 Size	退火温度 Temperature
807	F: GAGCATCCACCTCAAAGCAG	Y=-3.328X+38.293	181 bp	60℃
807	R: CGACCCAGATTCTTTCAACC	Y=-3.328X+38.293	181 bp	60℃
mcr-1	F: AAAGACGCGGTACAAGCAAC	Y=-3.3521X+39.177	213 bp	60℃
mcr-1	R: GCTGAACATACACGGCACAG	Y=-3.3521X+39.177	213 bp	60℃
16S	F: CGGTGAATACGTTCYCGG	Y= -3.545X+ 41.182	143 bp	60℃
16S	R: GGWTACCTTGTTACGACTT	Y= -3.545X+ 41.182	143 bp	60℃