使用全基因组测序表征源自Oreochromis spp.农场的致病性大肠杆菌菌株
Summary
使用台式仪器进行全基因组测序（WGS）策略的可行性简化了实验室环境中对每种与公共卫生相关的微生物的基因组询问。描述了细菌WGS工作流程的方法学调整，并介绍了用于分析的生物信息学管道。
Abstract
水产养殖是全世界增长最快的粮食生产部门之一，罗非鱼养殖是养殖的主要淡水鱼品种。由于水产养殖做法容易受到人为来源的微生物污染，因此需要广泛使用抗生素，导致水产养殖系统成为具有临床相关性的抗生素耐药性和致病细菌（如大肠杆菌）的重要来源。在这里，通过全基因组测序（WGS）和计算机分析阐明了从内陆养殖的Oreochromis属中回收的致病性大肠杆菌菌株的抗菌素耐药性，毒力和移动组特征。进行了抗菌药敏试验（AST）和WGS。此外，使用各种可用的网络工具测定系统发育组、血清型、多位点序列分型（MLST）、获得性抗菌素耐药性、毒力、质粒和噬菌体含量。大肠杆菌分离株仅表现出对氨苄青霉素的中等敏感性，并通过基于WGS的分型法表征为ONT：H21-B1-ST40菌株。虽然仅检测到单个抗菌素耐药性相关基因[mdf（A）]，但鉴定出来自非典型肠致病性大肠杆菌（aEPEC）病理型的几个毒力相关基因（VAG）。此外，还检测到来自大型质粒组和18个噬菌体相关区域的质粒复制子的货物。总之，从墨西哥锡那罗亚州一个养鱼场回收的aEPEC分离株的WGS表征可以深入了解其致病潜力以及食用原始水产养殖产品可能造成的人类健康风险。有必要利用下一代测序（NGS）技术来研究环境微生物，并采用一个健康框架来了解健康问题的起源。
Introduction
水产养殖是全球增长最快的食品生产部门之一，其生产实践旨在满足人类消费不断增长的食品需求。全球水产养殖产量从1997年的3400万吨增加到2017年的112吨，增加了两倍1。占产量近75%的主要物种群是海藻、鲤鱼、双壳类、鲶鱼和罗非鱼（Oreochromis spp）。1. 然而，由于集约化养鱼，微生物实体引起的疾病的出现是不可避免的，导致潜在的经济损失2.
众所周知，鱼类养殖实践中的抗生素用于预防和治疗细菌感染，这是生产力的主要限制因素3，4。尽管如此，残留的抗生素在水产养殖沉积物和水中积聚，施加选择压力并改变鱼类相关和居住的细菌群落5，6，7，8。因此，水产养殖环境是抗菌素耐药性基因（ARGs）的储存库，以及抗生素耐药细菌（ARB）在周围环境中的进一步出现和传播9。除了通常观察到的影响鱼类养殖实践的细菌病原体外，还经常遇到肠杆菌科的成员，包括肠杆菌属、大肠杆菌属、克雷伯氏菌属和沙门氏菌属 10 的人类病原体株。大肠杆菌是从鱼类养殖中从鱼粉和水中分离出的最常见微生物11，12，13，14，15。
大肠杆菌是一种多功能革兰氏阴性细菌，栖息在哺乳动物和鸟类的胃肠道中，作为其肠道微生物群的共生成员。然而，大肠杆菌具有高度的适应性，可以在不同的环境生态位中定植和持续存在，包括土壤，沉积物，食物和水16。由于通过水平基因转移（HGT）现象获得和损失，大肠杆菌已迅速进化为适应性强的抗生素耐药病原体，能够在人类和动物中引起广泛的疾病17，18。根据分离的起源，致病变异被定义为肠道致病性大肠杆菌（InPEC）或肠外致病性大肠杆菌（ExPEC ）。此外，InPEC和ExPEC根据疾病表现，遗传背景，表型性状和毒力因子（VF）细分为明确定义的病理型16，17，19。
致病性大肠杆菌菌株的传统培养和分子技术允许快速检测和鉴定不同的病理型。然而，它们可能既费时又费力，并且经常需要高技术培训19。此外，由于其遗传背景的复杂性，没有一种方法可用于可靠地研究大肠杆菌的所有致病变异。目前，随着高通量测序（HTS）技术的出现，这些缺点已被克服。全基因组测序（WGS）方法和生物信息学工具以经济实惠的方式大规模改善了对微生物DNA的探索，有助于在单次运行中对微生物进行深入表征，包括密切相关的致病变异20，21，22。根据生物学问题，可以使用几种生物信息学工具、算法和数据库来执行数据分析。例如，如果主要目标是评估ARG，VF和质粒的存在，则ResFinder，VirulenceFinder和PlasmidFinder等工具及其相关数据库可能是一个很好的起点。Carriço等人22详细概述了应用于微生物WGS分析的不同生物信息学软件和相关数据库，从原始数据预处理到系统发育推断。
一些研究表明，WGS在基因组询问抗菌素耐药性属性、致病潜力以及跟踪来自不同来源的大肠杆菌临床相关变体的出现和进化关系方面具有广泛的实用性23，24，25，26.WGS能够鉴定抗菌剂表型耐药性的分子机制，包括那些罕见或复杂的耐药机制。这是通过检测获得性ARG变异，药物靶基因中的新突变或启动子区域27，28。此外，WGS提供了推断抗菌素耐药性特征的潜力，而无需事先了解细菌菌株的耐药性表型29。或者，WGS允许表征具有抗菌素耐药性和毒力特征的移动遗传元件（MGE），这推动了现有病原体的细菌基因组进化。例如，在2011年德国大肠杆菌疫情调查期间应用WGS揭示了一种明显新颖的大肠杆菌病理型的独特基因组特征;有趣的是，这些暴发菌株起源于肠聚集性大肠杆菌（EAEC）组，该组从肠出血性大肠杆菌（EHEC）病理型30中获得了编码志贺毒素的噬菌体。
这项工作介绍了使用台式测序仪对细菌WGS工作流程的方法学调整。此外，使用基于Web的工具提供生物信息学管道来分析结果序列，并进一步支持生物信息学专业知识有限或没有生物信息学专业知识的研究人员。所描述的方法可以阐明致病性 大肠杆菌 菌株ACM5的抗菌耐药性，毒力和移动组特征，该菌株于2011年从墨西哥锡那罗亚州的内陆养殖 Oreochromis 属中分离出来12。
Protocol
注意： 大肠杆菌 菌株ACM5通过处理和培养鱼样品进行粪便大肠菌群（FC）测定12来回收。在鱼类取样期间，鱼类没有表现出疾病、细菌或真菌感染的临床症状，平均温度为22.3°C。分离后，将 大肠杆菌 分离株进行生化检测，并用DMSO（8%v/v）作为冷冻保护剂冷冻保存在脑心脏输液（BHI）肉汤中。 1. 重新激活冷冻 大肠杆菌 ACM5原液培养物<…
Representative Results
通过椎间盘扩散法测定抗菌药敏性，并通过CLSI断点标准解释跨越六个不同抗菌类别的12种抗生素，即氨基糖苷类、β-内酰胺类、氟喹诺酮类、硝基呋喃类、苯尼科尔和叶酸途径拮抗剂。 大肠杆菌 ACM5表现出对所有抗生素的敏感性，除了一种β-内酰胺类药物。测试了四种β-内酰胺类药物：氨苄西林、羧苄西林、头孢噻嗪和头孢噻肟。其中，测量了氨苄西林的14 mm抑制晕。因此，根据CLSI对氨苄?…
Discussion
本研究介绍了使用台式测序仪和用于致病性 大肠杆菌 变体基因组表征的管道对细菌 WGS 工作流程的适应。根据所使用的测序平台，湿实验室程序（细菌培养、gDNA 提取、文库制备和测序）和序列分析的周转时间 （TAT） 可能会有所不同，尤其是在研究生长缓慢的细菌时。按照上述WGS协议，TAT在4天内，这与目前文献所述（<5天）相当 34。
计算大肠杆?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
墨西哥国家科学技术委员会（CONACyT西班牙语缩写）授予何塞·安东尼奥·马加尼亚-利扎拉加[第481143号]的博士奖学金。
Materials
| Accublock Mini digital dry bath | Labnet | D0100 | Dry bath for incubation of tubes |
| Agencourt AMPure XP | Beckman Coulter | A63881 | Magnetic beads in solution for DNA library purification |
| DeNovix DS-11 | DeNovix Inc. | UV-Vis spectophotometer to check the quality of the gDNA extracted | |
| DNA LoBind Tubes | Eppendorf | 0030108418 | 1.5 mL PCR tubes for DNA library pooling |
| DynaMag-2 Magnet | Invitrogen, Thermo Fisher Scientific | 12321D | Magnetic microtube rack used during magnetic beads-based DNA purification |
| Gram-negative Multibac I.D. | Diagnostic reseach (Mexico) | PT-35 | Commercial standard antibiotic disks for antimicrobial susceptibility testing |
| MiniSeq Mid Output Kit (300-cycles) | Illumina | FC-420-1004 | Reagent cartdrige for paired-end sequencing (2×150) |
| MiniSeq System Instrument | Illumina | SY-420-1001 | Benchtop sequencer used for Next-generation sequencing |
| MiniSpin centrifuge | Eppendorf | 5452000816 | Standard centrifuge for tubes |
| Nextera XT DNA Library Preparation Kit | Illumina | FC-131-1024 | Reagents to perform DNA libraries for sequencing. Includes Box 1 and Box 2 reagents for 24 samples |
| Nextera XT Index Kit v2 | Illumina | FC-131-2001, FC-131-2002, FC-131-2003, FC-131-2004 | Index set A, B, C, D |
| PhiX Control v3 | Illumina | FC-110-3001 | DNA library control for sequencing |
| Precision waterbath | LabCare America | 51221081 | Water bath shaker used for bacterial culture |
| Qubit 1X dsDNA HS Assay Kit | Invitrogen, Thermo Fisher Scientific | Q33231 | Reagents for fluorescence-based DNA quantification assay |
| Qubit 2.0 Fluorometer | Invitrogen, Thermo Fisher Scientific | Q32866 | Fluorometer used for fluorescence assay |
| Qubit Assay tubes | Invitrogen, Thermo Fisher Scientific | Q32856 | 0.5 mL PCR tubes for fluorescence-based DNA quantification assay |
| SimpliAmp Thermal Cycler | Applied Biosystems, Thermo Fisher Scientific | A24811 | Thermocycler used for DNA library amplification |
| Spectronic GENESYS 10 Vis | Thermo | 335900 | Spectophotometer used for bacterial suspension in antimicrobial susceptibility testing |
| ZymoBIOMICS DNA Miniprep Kit | Zymo Research Inc. | D4300 | Kit for genomic DNA extraction (50 preps) |
References
- Naylor, R. L., et al. A 20-year retrospective review of global aquaculture. Nature. 591 (7851), 551-563 (2021).
- Quesada, S. P., Paschoal, J. A. R., Reyes, F. G. R. Considerations on the aquaculture development and on the use of veterinary drugs: special issue for fluoroquinolones-a review. Journal of Food Science. 78 (9), 1321-1333 (2013).
- Defoirdt, T., Sorgeloos, P., Bossier, P. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Current Opinion in Microbiology. 14 (3), 251-258 (2011).
- Stentiford, G. D., et al. New paradigms to help solve the global aquaculture disease crisis. PLOS Pathogens. 13 (2), 1006160 (2017).
- Chen, H., et al. Tissue distribution, bioaccumulation characteristics and health risk of antibiotics in cultured fish from a typical aquaculture area. Journal of Hazardous Materials. 343, 140-148 (2018).
- Zhou, M., et al. Antibiotics control in aquaculture requires more than antibiotic-free feeds: A Tilapia farming case. Environmental Pollution. 268, 115854 (2021).
- Feng, Y., et al. Ecological effects of antibiotics on aquaculture ecosystems based on microbial community in sediments. Ocean & Coastal Management. 224, 106173 (2022).
- Shen, X., Jin, G., Zhao, Y., Shao, X. Prevalence and distribution analysis of antibiotic resistance genes in a large-scale aquaculture environment. Science of The Total Environment. 711, 134626 (2020).
- Su, H., et al. Contamination of antibiotic resistance genes (ARGs) in a typical marine aquaculture farm: source tracking of ARGs in reared aquatic organisms. Journal of Environmental Science and Health, Part B. 55 (3), 220-229 (2020).
- Oliveira, R. V., Oliveira, M. C., Pelli, A. Disease infection by Enterobacteriaceae family in fishes: a review. Journal of Microbiology & Experimentation. 4 (5), 00128 (2017).
- Barbosa, M. M. C., et al. Sorologia e suscetibilidade antimicrobiana em isolados de Escherichia coli de pesque-pagues. Arquivos do Instituto Biológico. 81 (1), 43-48 (2014).
- Valenzuela-Armenta, J. A., et al. Microbiological analysis of Tilapia and water in aquaculture farms from Sinaloa. Biotecnia. 20 (1), 20-26 (2018).
- Reza, R. H., Shipa, S. A., Naser, M. N., Miah, M. F. Surveillance of Escherichia coli in a fish farm of Sylhet, Bangladesh. Bangladesh Journal of Zoology. 48 (2), 335-346 (2021).
- Liao, C. -. Y., et al. Antimicrobial resistance of Escherichia coli From aquaculture farms and their environment in Zhanjiang, China. Frontiers in Veterinary Science. 8, 806653 (2021).
- Dewi, R. R., et al. Prevalence and antimicrobial resistance of Escherichia coli, Salmonella and Vibrio derived from farm-raised Red Hybrid Tilapia (Oreochromis spp.) and Asian Sea Bass (Lates calcarifer, Bloch 1970) on the west coast of Peninsular Malaysia. Antibiotics. 11 (2), 136 (2022).
- Leimbach, A., Hacker, J., Dobrindt, U. E. coli as an all-rounder: the thin line between commensalism and pathogenicity. Current Topics in Microbiology and Immunology. 358, 3-32 (2013).
- Kaper, J. B., Nataro, J. P., Mobley, H. L. T. Pathogenic Escherichia coli. Nature Reviews Microbiology. 2 (2), 123-140 (2004).
- Croxen, M. A., Finlay, B. B. Molecular mechanisms of Escherichia coli pathogenicity. Nature Reviews Microbiology. 8 (1), 26-38 (2010).
- Croxen, M. A., et al. Recent advances in understanding enteric pathogenic Escherichia coli. Clinical Microbiology Reviews. 26 (4), 822-880 (2013).
- Bertelli, C., Greub, G. Rapid bacterial genome sequencing: methods and applications in clinical microbiology. Clinical Microbiology and Infection. 19 (9), 803-813 (2013).
- Lynch, T., Petkau, A., Knox, N., Graham, M., Van Domselaar, G. A primer on infectious disease bacterial genomics. Clinical Microbiology Reviews. 29 (4), 881-913 (2016).
- Carriço, J. A., Rossi, M., Moran-Gilad, J., Van Domselaar, G., Ramirez, M. A primer on microbial bioinformatics for nonbioinformaticians. Clinical Microbiology and Infection. 24 (4), 342-349 (2018).
- Magaña-Lizárraga, J. A., et al. Draft genome sequence of Escherichia coli M51-3: a multidrug-resistant strain assigned as ST131-H30 recovered from infant diarrheal infection in Mexico. Journal of Global Antimicrobial Resistance. 19, 311-312 (2019).
- Pérez-Vázquez, M., et al. Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS. Journal of Antimicrobial Chemotherapy. 74 (12), 3489-3496 (2019).
- Massella, E., et al. Snapshot study of whole genome sequences of Escherichia coli from healthy companion animals, livestock, wildlife, humans and food in Italy. Antibiotics. 9 (11), 782 (2020).
- Magaña-Lizárraga, J. A., et al. Genomic profiling of antibiotic-resistant Escherichia coli isolates from surface water of agricultural drainage in north-western Mexico: detection of the international high-risk lineages ST410 and ST617. Microorganisms. 10 (3), 662 (2022).
- Saracino, I. M., et al. Next Generation sequencing for the prediction of the antibiotic resistance in Helicobacter pylori: a literature review. Antibiotics. 10 (4), 437 (2021).
- Ghosh, A., Saha, S. Survey of drug resistance associated gene mutations in Mycobacterium tuberculosis, ESKAPE and other bacterial species. Scientific Reports. 10 (1), 8957 (2020).
- Su, M., Satola, S. W., Read, T. D. Genome-based prediction of bacterial antibiotic resistance. Journal of Clinical Microbiology. 57 (3), 01405-01418 (2019).
- Brzuszkiewicz, E., et al. Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC). Archives of Microbiology. 193 (12), 883-891 (2011).
- . CLSI Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed. CLSI standard M02. Wayne, PA: Clinical and Laboratory Standards Institute Available from: https://clsi.org/standards/products/microbiology/documents/m02/ (2018)
- CLSI Performance Standards for Antimicrobial Susceptibility Testing. 31st ed. CLSI supplement M100. Clinical and Laboratory Standards Institute Available from: https://clsi.org/standards/products/microbiology/documents/m100/ (2021)
- Ewing, B., Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research. 8 (3), 186-194 (1998).
- Quainoo, S., et al. Whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis. Clinical Microbiology Reviews. 30 (4), 1015-1063 (2017).
- Desai, A., et al. Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data. PLoS ONE. 8 (4), 60204 (2013).
- Nishino, K., Yamada, J., Hirakawa, H., Hirata, T., Yamaguchi, A. Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to β-lactams. Antimicrobial Agents and Chemotherapy. 47 (9), 3030-3033 (2003).
- Li, M., et al. The resistance mechanism of Escherichia coli induced by ampicillin in laboratory. Infection and Drug Resistance. 12, 2853-2863 (2019).
- Ménard, L. -. P., Dubreuil, J. D. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1): a new toxin with an old twist. Critical Reviews in Microbiology. 28 (1), 43-60 (2002).
- Dubreuil, J. D. EAST1 toxin: An enigmatic molecule associated with sporadic episodes of diarrhea in humans and animals. Journal of Microbiology. 57 (7), 541-549 (2019).
- Goldstein, S., Beka, L., Graf, J., Klassen, J. L. Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Genomics. 20 (1), 23 (2019).
- Guerrero, A., Gomez-Gil, B., Lizarraga-Partida, M. L. Genomic stability among O3:K6 V. parahaemolyticus pandemic strains isolated between 1996 to 2012 in American countries. BMC Genomic Data. 22 (1), 38 (2021).
- FAO Applications of Whole Genome Sequencing (WGS) in food safety management. Food and Agriculture Organization of the United Nations Available from: https://www.fao.org/documents/card/es/c/61e44b34-b328-4239-b59c-a9e926e327b4/ (2016)
- Rantsiou, K., et al. Next generation microbiological risk assessment: opportunities of whole genome sequencing (WGS) for foodborne pathogen surveillance, source tracking and risk assessment. International Journal of Food Microbiology. 287, 3-9 (2018).
Cite This Article
Magaña-Lizárraga, J. A., Gómez-Gil, B., Enciso-Ibarra, J., Báez-Flores, M. E. Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing. J. Vis. Exp. (190), e64404, doi:10.3791/64404 (2022).