温州口岸截获蜱体内微生物群落结构、抗生素抗性基因及毒力因子的宏基因组分析

许雪莲, 韩阿祥, 叶诗晴, 关万春, 楼永良

doi:10.11853/j.issn.1003.8280.2021.06.021

中国媒介生物学及控制杂志 >

2021 , Vol. 32 >Issue 6: 763 - 771

DOI: https://doi.org/10.11853/j.issn.1003.8280.2021.06.021

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温州口岸截获蜱体内微生物群落结构、抗生素抗性基因及毒力因子的宏基因组分析

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温州医科大学检验医学院生命科学学院, 检验医学教育部重点实验室, 温州市环境卫生微生物检验重点实验室, 浙江温州 325035

许雪莲,女,硕士,检验师,从事微生物与微生物检验研究,E-mail:1940841700@qq.com

收稿日期: 2021-05-11

网络出版日期: 2021-12-15

基金资助

国家传染病防治重大专项（2018ZX10201001-009）

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Metagenomic analysis of microbial community structure, antibiotic resistance genes, and virulence factors of ticks captured at Wenzhou port, Zhejiang province, China

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Wenzhou Key Laboratory of Sanitary Microbiology, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China

Received date: 2021-05-11

Online published: 2021-12-15

Supported by

Supported by the National Science and Technology Major Project (No. 2018ZX10201001-009)

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摘要

目的以宏基因组学为技术手段，了解温州口岸在进口南非盐湿牛皮上检疫发现的无色扇头蜱体内微生物群落结构特征，及抗生素抗性基因（ARGs）、毒力因子携带情况。方法将截获的蜱随机分成5份，经HiPure Bacterial DNA Kits提取DNA后，采用高通量测序的方法，分析其微生物群落结构、致病菌、抗生素抗性基因及毒力因子。结果 5份样本微生物组成相似。在门水平上，均以厚壁菌门、变形菌门为主要菌门。其中厚壁菌门丰度最高，占全部门的66.00%以上。在属水平上，均以链球菌属（>55.30%）、芽孢杆菌属（>11.20%）为优势菌属。在全部蜱样本中均检测到变形链球菌、蜡样芽孢杆菌、肠沙门菌、肺炎链球菌等常见条件致病菌，占注释总水平的71.00%以上。抗性基因分析发现蜱样本含有Acr，bacA，mdtK，arnA，mdtH等16种抗性基因，其中Acr丰度最高（30.19%），其次是bacA（20.75%）。这些抗性基因分别介导对大环内酯类、氨基糖苷类、β-内酰胺酶、甘氨酰环素、吖啶黄素等多种抗菌药物的耐药。细菌毒力因子数据库（VFDB）注释分析发现蜱样本共有3 923个毒力基因，主要与黏附（22.66%）、分泌系统（13.97%）、侵袭（13.56%）等毒力因子相关。结论无色扇头蜱体内病原体种类较多，检出肠沙门菌等危害较大的致病菌，同时携带大量抗生素抗性基因及毒力因子，因此相关部门及一线从业人员应注意防控跨境蜱传疾病传入，防止对我国公共卫生造成影响。

关键词： 抗生素抗性基因; 微生物群落结构; 宏基因组学; 蜱; 毒力因子

本文引用格式

许雪莲, 韩阿祥, 叶诗晴, 关万春, 楼永良 . 温州口岸截获蜱体内微生物群落结构、抗生素抗性基因及毒力因子的宏基因组分析[J]. 中国媒介生物学及控制杂志, 2021 , 32(6) : 763 -771 . DOI: 10.11853/j.issn.1003.8280.2021.06.021

Abstract

Objective To investigate the microbial community structure, antibiotic resistance genes (ARGs), and virulence factors by the metagenomic technique in ticks captured from wet-salted cow hides imported from South Africa at a port in Wenzhou, Zhejiang province, China. Methods We randomly divided the captured ticks into 5 groups, extracted DNA using HiPure Bacterial DNA Kits, and used high-throughput sequencing to analyze the microbial community structure, pathogenic bacteria, ARGs, and virulence factors of the ticks. Results The 5 samples had similar microbial community structure. At the phylum level, Firmicutes and Proteobacteria were the dominant phyla, and Firmicutes was the most abundant, accounting for more than 66.00% of the total phyla. At the genus level, Streptococcus (>55.30%) and Bacillus (>11.20%) were the dominant genera. All the tick samples were positive for St. mutans, Ba. cereus, Salmonella enterica, and St. pneumoniae, accounting for more than 71.00% of the total species annotated. Sixteen ARGs were identified in the tick samples, including Acr, bacA, mdtK, arnA, and mdtH, and Acr had the highest abundance (30.19%), followed by bacA (20.75%). These ARGs mediated resistance to antibiotics such as macrolides, aminoglycosides, β-lactamases, glycylcycline, and acriflavine. A total of 3 923 virulence genes were found in the tick samples through annotation with the virulence factor database, which were mainly related to adherence (22.66%), secretion system (13.97%), and invasion (13.56%). Conclusion The imported ticks carry various species of pathogens, including highly harmful pathogens such as Sa. enterica, and harbor numerous ARGs and virulence factors. Relevant authorities and frontline workers should pay attention to preventing imported tick-borne diseases and taking effective treatment measures to avoid affecting public health in China.

Key words： Antibiotic resistance gene; Microbial community structure; Metagenomics; Tick; Virulence factor

参考文献

[1] Shi JM,Hu ZH,Deng F,et al. Tick-borne viruses[J]. Virol Sin,2018,33(1):21-43. DOI:10.1007/s12250-018-0019-0.
[2] Koedel U,Fingerle V,Pfister HW. Lyme neuroborreliosis-epidemiology,diagnosis and management[J]. Nat Rev Neurol,2015,11(8):446-456. DOI:10.1038/nrneurol.2015.121.
[3] Ismail N,McBride JW. Tick-borne emerging infections:ehrlichiosis and anaplasmosis[J]. Clin Lab Med,2017,37(2):317-340. DOI:10.1016/j.cll.2017.01.006.
[4] Chmielewski T,Tylewska-Wierzbanowska S. Q fever at the turn of the century[J]. Pol J Microbiol,2012,61(2):81-93. DOI:10.33073/pjm-2012-011.
[5] Zeng PB,Yang ZD,Bakkour S,et al. Development and validation of a real-time reverse transcriptase PCR assay for sensitive detection of SFTSV[J]. J Med Virol,2017,89(7):1131-1138. DOI:10.1002/jmv.24760.
[6] 赵丹云,孙毅,陈卫军,等. 天津口岸首次截获随人入境外来蜱的形态学及分子鉴定[J]. 中国国境卫生检疫杂志,2016,39(4):256-259. DOI:10.16408/j.1004-9770.2016.04.009. Zhao DY,Sun Y,Chen WJ,et al. Morphology and molecular identification of an imported tick with entry passenger intercepted at Tianjin port[J]. Chin J Front Health Quar,2016,39(4):256-259. DOI:10.16408/j.1004-9770.2016.04.009.
[7] 余招锋,徐慧,陈鹏程,等. 一批泰国进境板材中截获的囊形扇头蜱的鉴定和检疫[J]. 中国动物检疫,2017,34(2):58-60. DOI:10.3969/j.issn.1005-944X.2017.02.016. Yu ZF,Xu H,Chen PC,et al. Identification and quarantine towards Rhipicephalus bursa in intercepted timbers imported from Thailand[J]. China Anim Health Insp,2017,34(2):58-60. DOI:10.3969/j.issn.1005-944X.2017.02.016.
[8] 吕洁毅,何振毅,刘谢,等. 佛山口岸首次截获绚丽花蜱[J]. 寄生虫与医学昆虫学报,2016,23(4):237-241. DOI:10.3969/j.issn.1005-0507.2016.04.008. Lyu JY,He ZY,Liu X,et al. An exotic species of Amblyomma intercepted at Foshan frontier port[J]. Acta Parasitol Med Entomol Sin,2016,23(4):237-241. DOI:10.3969/j.issn.1005-0507.2016.04.008.
[9] 徐军,王安东,戴莉,等. 中哈边境阿拉山口口岸输入性亚洲璃眼蜱的种类鉴定与分析[J]. 中国兽医杂志,2016,52(11):17-19. Xu J,Wang AD,Dai L,et al. Species identification of an imported tick sample of Hyalomma asiaticum asiaticum by genetic analysis at Alashankou port,China-Kazakhstan border[J]. Chin J Vet Med,2016,52(11):17-19.
[10] 赵爽,胡文海,闫清丽,等. 南沙口岸首次从入境牛皮中截获盾陷璃眼蜱[J]. 中国媒介生物学及控制杂志,2014,25(5):452-454. DOI:10.11853/j.issn.1003.4692.2014.05.017. Zhao S,Hu WH,Yan QL,et al. Investigation of Hyalomma excavatum Koch first captured at Nansha port,Guangzhou,China[J]. Chin J Vector Biol Control,2014,25(5):452-454. DOI:10.11853/j.issn.1003.4692.2014.05.017.
[11] Chen LH,Xiong ZH,Sun LL,et al. VFDB 2012 update:toward the genetic diversity and molecular evolution of bacterial virulence factors[J]. Nucleic Acids Res,2012,40(D1):D641-D645. DOI:10.1093/nar/gkr989.
[12] Mühlen S,Dersch P. Anti-virulence strategies to target bacterial infections[M]//Stadler M,Dersch P. How to overcome the antibiotic crisis. Cham:Springer,2015:147-183. DOI:10.1007/82_2015_490.
[13] Ruer S,Pinotsis N,Steadman D,et al. Virulence-targeted antibacterials:concept,promise,and susceptibility to resistance mechanisms[J]. Chem Biol Drug Des,2015,86(4):379-399. DOI:10.1111/cbdd.12517.
[14] Harms A,Brodersen DE,Mitarai N,et al. Toxins,targets,and triggers:an overview of toxin-antitoxin biology[J]. Mol Cell,2018,70(5):768-784. DOI:10.1016/j.molcel.2018.01.003.
[15] Yother J. Capsules of Streptococcus pneumoniae and other bacteria:paradigms for polysaccharide biosynthesis and regulation[J]. Annu Rev Microbiol,2011,65:563-581. DOI:10.1146/annurev.micro.62.081307.162944.
[16] Platt TM,Loneragan GH,Scott HM,et al. Antimicrobial susceptibility of enteric bacteria recovered from feedlot cattle administered chlortetracycline in feed[J]. Am J Vet Res,2008,69(8):988-996. DOI:10.2460/ajvr.69.8.988.
[17] Kim Y,Koh I,Rho M. Deciphering the human microbiome using next-generation sequencing data and bioinformatics approaches[J]. Methods,2015,79-80:52-59. DOI:10.1016/j.ymeth.2014. 10.022.
[18] Martello LA,Wadgaonkar R,Gupta R,et al. Characterization of Trypanosoma cruzi infectivity,proliferation,and cytokine patterns in gut and pancreatic epithelial cells maintained in vitro[J]. Parasitol Res,2013,112(12):4177-4183. DOI:10.1007/s00436-013-3609-7.
[19] Manichanh C,Chapple CE,Frangeul L,et al. A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library[J]. Nucleic Acids Res,2008,36(16):5180-5188. DOI:10.1093/nar/gkn496.
[20] Boulanger N,Boyer P,Talagrand-Reboul E,et al. Ticks Tick Borne Dis[J]. Méd Mal Infect,2019,49(2):87-97. DOI:10.1016/j.medmal.2019.01.007.
[21] Xiang LL,Poźniak B,Cheng TY. Bacteriological analysis of saliva from partially or fully engorged female adult Rhipicephalus microplus by next-generation sequencing[J]. Antonie van Leeuwenhoek,2017,110(1):105-113. DOI:10.1007/s10482-016-0780-8.
[22] Chiuya T,Masiga DK,Falzon LC,et al. Tick-borne pathogens,including Crimean-Congo haemorrhagic fever virus,at livestock markets and slaughterhouses in western Kenya[J]. Transbound Emerg Dis,2021,68(4):2429-2445. DOI:10.1111/tbed.13911.
[23] Li YC,Wen XX,Li M,et al. Molecular detection of tick-borne pathogens harbored by ticks collected from livestock in the Xinjiang Uygur Autonomous Region,China[J]. Ticks Tick-Borne Dis,2020,11(5):101478. DOI:10.1016/j.ttbdis.2020.101478.
[24] Jiang J,Choi YJ,Kim J,et al. Distribution of Rickettsia spp. in ticks from northwestern and southwestern provinces,Republic of Korea[J]. Korean J Parasitol,2019,57(2):161-166. DOI:10.3347/kjp.2019.57.2.161.
[25] Merhej V,Angelakis E,Socolovschi C,et al. Genotyping,evolution and epidemiological findings of Rickettsia species[J]. Infect Genet Evol,2014,25:122-137. DOI:10.1016/j.meegid. 2014.03.014.
[26] 魏琼,刘翔,张燕飞,等. 宁夏地区食源性与人源沙门菌耐药性与血清型对比研究[J]. 中国抗生素杂志,2016,41(9):707-709,717. DOI:10.3969/j.issn.1001-8689.2016.09.012. Wei Q,Liu X,Zhang YF,et al. Serotype distribution and drug resistance of food-born and human orign Salmonella in Ningxia[J]. Chin J Antibiot,2016,41(9):707-709,717. DOI:10.3969/j.issn.1001-8689.2016.09.012.
[27] Bottone EJ. Bacillus cereus,a volatile human pathogen[J]. Clin Microbiol Rev,2010,23(2):382-398. DOI:10.1128/CMR. 00073-09.
[28] Torkar KG,Bedenić B. Antimicrobial susceptibility and characterization of metallo-β-lactamases,extended-spectrum β-lactamases,and carbapenemases of Bacillus cereus isolates[J]. Microb Pathog,2018,118:140-145. DOI:10.1016/j.micpath. 2018.03.026.
[29] Wu HJ,Wang AHJ,Jennings MP. Discovery of virulence factors of pathogenic bacteria[J]. Curr Opin Chem Biol,2008,12(1):93-101. DOI:10.1016/j.cbpa.2008.01.023.
[30] 尹磊,祁克宗,宋祥军,等. 大肠杆菌Ⅲ型分泌系统2毒力岛研究进展[J]. 微生物学通报,2017,44(12):3031-3037. DOI:10.13344/j.microbiol.china.170468. Yin L,Qi KZ,Song XJ,et al. Type Ⅲ secretion system 2 pathogenicity islands of Escherichia coli[J]. Microbiol China,2017,44(12):3031-3037. DOI:10.13344/j.microbiol.china. 170468.
[31] Francetic O,Belin D,Badaut C,et al. Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion[J]. EMBO J,2000,19(24):6697-6703. DOI:10.1093/emboj/19.24.6697.
[32] 余乐正,柳凤娟,鄢南南,等. 革兰阴性菌分泌系统及其分泌产物研究进展[J]. 动物医学进展,2017,38(8):80-84. DOI:10.16437/j.cnki.1007-5038.2017.08.017. Yu LZ,Liu FJ,Yan NN,et al. Progress on secretion systems and secretory products of gram-negative bacteria[J]. Prog Vet Med,2017,38(8):80-84. DOI:10.16437/j.cnki.1007-5038.2017. 08.017.
[33] Shon AS,Bajwa RPS,Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae:a new and dangerous breed[J]. Virulence,2013,4(2):107-118. DOI:10.4161/viru.22718.
[34] Osorio CR,Juiz-Río S,Lemos ML. A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island[J]. Microbiology (Reading),2006,152(Pt 11):3327-3341. DOI:10.1099/mic.0.29190-0.
[35] Blair JMA,Webber MA,Baylay AJ,et al. Molecular mechanisms of antibiotic resistance[J]. Nat Rev Microbiol,2015,13(1):42-51. DOI:10.1038/nrmicro3380.
[36] Fournier PE,Richet H,Weinstein RA. The epidemiology and control of Acinetobacter baumannii in health care facilities[J]. Clin Infect Dis,2006,42(5):692-699. DOI:10.1086/500202.
[37] Kempf M,Rolain JM. Emergence of resistance to carbapenems in Acinetobacter baumannii in Europe:clinical impact and therapeutic options[J]. Int J Antimicrob Agents,2012,39(2):105-114. DOI:10.1016/j.ijantimicag.2011.10.004.
[38] Vila J,Martí S,Sánchez-Céspedes J. Porins,efflux pumps and multidrug resistance in Acinetobacter baumannii[J]. J Antimicrob Chemother,2007,59(6):1210-1215. DOI:10.1093/jac/dkl509.
[39] Haley BJ,Kim SW,Cao H,et al. Genomic and metagenomic analysis of antibiotic resistance in dairy animals[J]. J Anim Sci,2017,95 Suppl 4:S131. DOI:10.2527/asasann.2017.265.
[40] Kastner S,Perreten V,Bleuler H,et al. Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food[J]. Syst Appl Microbiol,2006,29(2):145-155. DOI:10.1016/j.syapm.2005.07.009.
[41] Garofalo C,Vignaroli C,Zandri G,et al. Direct detection of antibiotic resistance genes in specimens of chicken and pork meat[J]. Int J Food Microbiol,2007,113(1):75-83. DOI:10.1016/j.ijfoodmicro.2006.07.015.
[42] 袁立霞,罗晓,张文丽,等. 制药废水厂微生物群落和多种抗性基因相关性分析[J]. 河北科技大学学报,2019,40(2):175-181. DOI:10.7535/hbkd.2019yx02012. Yuan LX,Luo X,Zhang WL,et al. Correlation between antibiotic resistance genes and microbial communities in pharmaceutical wastewater[J]. J Hebei Univ Sci Technol,2019,40(2):175-181. DOI:10.7535/hbkd.2019yx02012.

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