JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Immunologia e infezioni

用于维持和区分生物型的实验室技术<em>霍乱弧菌</em>临床和环境隔离

Published: May 30, 2017
doi:
10.3791/55760
, , , ,
1Department of Biological Sciences,Plymouth State University

Summary

该手稿除了进行一系列生物化学测定之外，还描述了适用于霍乱弧菌的维护技术，它们共同用于临床和环境V之间的快速可靠的分化。 霍乱弧菌生物型实验室设置。

Abstract

水生革兰氏阴性菌霍乱弧菌是感染性胃肠道霍乱的病原体。由于这种疾病的全球流行和严重程度， V 。 霍乱弧菌已经在环境和实验室环境中得到广泛的研究，需要适当的维护和培养技术。古典和El Tor是组成V的两个主要生物型。 霍乱弧菌 O1血清群，各自显示独特的基因型和表型特征，为生物型表征提供可靠的机制，并需要不同的毒力诱导培养条件。不管任何给定的感染或爆发的致病菌株的生物型是什么，该疾病的标准治疗涉及补充有抗生素方案的补液治疗。然而，生物型分类对于实验室研究可能是必要的，并且可能在生物医学领域具有更广泛的影响。在2000年初，鉴定出了经典和El Tor生物型的基因型和表型性状的临床分离株。新鉴定的杂种，称为El Tor变体，使得临床和环境分离生物型鉴定变得比以前的传统单试验鉴定方案更复杂。除了描述V。 霍乱弧菌一般维护和培养技术，该手稿描述了一系列基于特定基因（ ctxB和tcpA ）基于PCR的遗传筛选和表型测定（多粘菌素B抗性，柠檬酸盐代谢，蛋白水解活性，溶血活性，运动性和葡萄糖代谢通过Voges- Proskauer测定）共同用于表征和/或区分经典和El Tor生物型。一起，这些测定提供了一种有效的系统方法，作为替代使用，或另外用于代表性的昂贵的劳动密集型实验V。 霍乱临床（和环境）分离株。

Introduction

霍乱是由食用含有水生革兰氏阴性菌霍乱弧菌的污染食物或水引起的远端小肠疾病。 霍乱的症状包括呕吐和不可控制的水样腹泻，导致严重脱水，如果不妥善治疗，将导致死亡。 V。基于细胞表面脂多糖O-抗原的结构， 霍乱弧菌可以分为200多个血清群。然而，只有2个血清群，O1和O139，已显示出流行或大流行的潜力1,2 。此外，血清群O139主要分离到东南亚3,4 ，而血清群O1在全球分布。此外，O1血清群可以分为两种主要生物型：古典和El Tor。经典生物型是造成头6头霍乱流行病1817年和1923年之间。正在进行的第七大流行病是El Tor生物型的结果，全球在环境5,6,7的环境中使古典生物型流行病学变异。最近，出现了具有古典和El Tor生物型8,​​9,10,11,12,13,14,15,16,17的区别特征的菌株，并且之后被称为El Tor变体13,17。一些El Tor变体已经显示出比以前观察到的更迅速和严重的疾病进展的升高的毒力能力，强调需要一个更全面的方法来代理身份识别和疾病预防和治疗8,9,18。虽然生物型鉴定并不立即决定治疗，但疫苗开发和未来治疗剂的进一步进步可能受益于生物型别的区别。

这里列出的第一系列协议将使调查人员能够正确维护V。 霍乱弧菌菌株在实验室设置。一致性和随后的分析需要分离物的储备准备和生长，其不是生物型依赖性的。然而，为了最佳地诱导毒力基因表达，需要独立的生物特异性培养技术。此外，本手稿中概述了各种遗传和生物化学测定的准备工作。

霍乱毒素（CT）和毒素共同作用培养的真菌（TCP）是主要调节剂ToxT在V的两种生物型中控制的两种主要的毒力因子。 霍乱弧菌 O1血清群20 。 CT是由五个CtxB组成的两部分毒素 亚单位围绕单个CtxA亚单位，并且负责与霍乱相关的快速电解质损失。 TCP是由tcp操纵子（ tcpABQCRDSTEF ）编码的IV型pilus ，并且涉及远端小肠的附着和定殖。 tcpA是tcp操纵子的第一个基因，其编码构建真菌8所必需的各个pilin亚单位。 ctxA的基因序列在经典和El Tor生物型之间是完全保守的，而ctxB和tcpA在两种生物型之间不同，但在每种生物型8中都是保守的。除了两个基本位置之外， ctxB在生物型之间是完全保守的（115和203）。在El Tor生物型中，胸腺嘧啶位于基因位置115和203，而经典生物型在这些碱基处含有胞嘧啶。 tcpA在每种生物型中都是完全保守的，但在生物型之间的多个基础上是不同的。这些遗传差异用作初级生物型鉴定标记，并且在测序包括这些位点的聚合酶链反应（PCR）扩增产物之后，可以将分离序列与野生型（WT）经典O395或WT E1 Tor N16961进行比较，以确定生物型背景的CT和TCP分别在给定的霍乱弧菌分离株中。

已经开发了许多方案来表征经典和El Tor生物型21,22,23之间的表型区别。多粘菌素B是一种肽抗生素，其在革兰氏阴性杆菌中损害外细胞膜的完整性通过多粘菌素B抗性测定可以观察到teria和多粘菌素B抗性21 。柠檬酸盐是克雷伯氏循环的主要底物，并且可以使用柠檬酸盐代谢测定法22来确定将柠檬酸盐代谢为唯一碳源的能力。 hapR编码霍乱弧菌 HapR中的全局调节子和主要群体感应调节因子，其结合各种启动子区域并调节基因和操纵子表达24 。 霍乱弧菌的一些致病菌株在hapR基因中具有天然存在的转录突变，导致毒力基因表达的密度依赖性调节丧失24,25 。使用牛奶琼脂培养基测量HapR调节的蛋白酶活性允许研究者鉴定特定分离物是否含有功能性HapR 23 。该溶血分析测试菌株分泌溶血红细胞的溶血酶的能力;溶血程度可以在血琼脂平板23上显现。运动性通常与霍乱弧菌中的毒力相关，可以使用运动性琼脂平板23进行分析。 Voges-Proskauer测定法测试菌株作为唯一碳源发酵葡萄糖并产生副产物乙偶姻21的能力 。随着El Tor变体的出现，很难预测任何给定的表型测定的结果，而没有广泛的基因型筛选，并且在推导V的生物型背景之前。 霍乱弧菌分离物，推荐进行这种测定23的装配，并将结果与表2中的参考菌株进行比较。

在这里，我们提出了一系列方案，集体利用上述方案基因型和表型检测用于更全面的表征V的方法 。 霍乱生物型此外，我们已经描述了已知V的基因型和表型区别。与常用的生物型参考菌株（WT经典O395，WT E1 Tor C6706和WT El Tor N16961; 表1 ）相比， 霍乱弧菌 El Tor变体（MQ1795和BAA-2163）。 El Tor变体的出现对先前使用的单一测定生物型表征方案的可靠性提出了挑战;然而，这种多重测定鉴定系统将允许更可靠地表征临床和环境V。 霍乱弧菌分离株

Protocol

注意：每个测定的时间考虑必须作为单个培养基制备需要不同的时间。例如，应将固体琼脂平板培养基充分冷却并干燥（1-2天）。额外的时间考虑（ 即单菌落和隔夜培养生长）在每个方案下规定， 见表2 。 媒体准备 1x磷酸缓冲盐水（PBS） 称取4.0g NaCl，0.1g KCl，0.72g Na 2 HPO 4和0.12g KH 2 PO 4 。将组分?…

Representative Results

为了妥善维护和使用任何细菌菌株，建议知道感兴趣的菌株的倍增时间。这里，常用的V的变化的生长速率。通过生长曲线证实霍乱弧菌菌株，并使用线性回归计算近似倍数。 WT El Tor N16961和El Tor变体MQ1795比WT经典O395（〜2 h）显示更短的倍增时间（分别为〜1 h和〜1 h）（ 图1 ; 表2 ）。 <p class="jove_content" fo:keep-together.within-pa…

Discussion

在200多个确定的V。 霍乱血清群，只有O1和O139具有流行潜力。 O1血清群可以分为两种生物型：古典和El Tor。然而，已经出现了称为El Tor变体13,17的杂交菌株，具有El Tor生物型背景，并具有古典特征8,9,10,11,12,13,14,15,16,17。本手册中描述的方案旨在提供有兴趣表征和/或区分霍乱弧菌的各种临床和非临床分离株的研究者，具有可靠的多检测鉴定系统。使用可靠的多分析鉴定系统作为?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

研究由新罕布什尔州INBRE通过机构发展奖（IDeA）支持，P20GM103506来自美国国家卫生研究院国家综合医学研究所。

Materials

1 kb DNA Ladder New England Biolabs N3232S https://www.neb.com/products/n3232-1-kb-dna-ladder
60% Glycerol Calbiochem 356352 http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade—CAS-56-81-5—Calbiochem,EMD_BIO-356352
Agar Becto, Dickinson and Co. 214030 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch
Agar with brain-heart infusion Becto, Dickinson and Co. 237500 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch
Agarose Peqlab 732-2789 https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&_dynSessConf=1766917479792147141&targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&lastLanguage=en&/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&_D%3AcurrentLanguage=+&currentLanguage=en&_D%3AlastLanguage=+&_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+
Anhydrous K2HPO4 Fisher Scientific P288-500 https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&searchTerm=P288500&searchType=RAPID
Blood Agar Plates Remel R01200 Store at 4 °C;  http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar
Boric Acid Fisher Scientific A73-500 https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&searchTerm=A73500&searchType=RAPID
Bromothymol Blue Fisher Scientific B388-10 https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&searchTerm=B38810&searchType=RAPID
Cirtric acid ·H2O Fisher Scientific S72836-3 https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363
Deoxynucleotide (dNTP) Solution Kit New England Biolabs N0446S Store at -20 °C;  https://www.neb.com/products/n0446-deoxynucleotide-solutionset
Disodium EDTA Fisher Scientific S311-500 https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&searchTerm=S311500&searchType=RAPID
DNA Clean & Concentrator™ -25 Kit Zymo Research D4007 http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit
GelGreen Nucleic Acid Stain Biotium 41005 https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/
Genesys 10SUV-VIS Spectrophotometer Thermo Scientific 840-208100 https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100
Gentra Puregene Yeast/Bact. Kit Qiagen 158567 https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation
HCl Fisher Scientific A144-212 Corrosive;  https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&searchTerm=A144212&searchType=RAPID
KCl Fisher Scientific P217-500 https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&searchTerm=P217500&searchType=RAPID
KH2PO4 Fisher Scientific P285-500 https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&searchTerm=P285500&searchType=RAPID
KOH Fisher Scientific P250-500 https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&searchTerm=P250500&searchType=RAPID
Le Loop Decon Labs Inc. MP190-25 http://deconlabs.com/products/leloop/
Le Stab Decon Labs Inc. MP186-5 http://deconlabs.com/products/lestab/
MgSO4·7H2O Fisher Scientific M63-500 https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&searchTerm=M63500&searchType=RAPID
Mini-Sub Cell GT Horizontal Electrophoresis System Bio Rad Labs 1704406 http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373
MR-VP Broth Difco 216300 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch
Na2HPO4 Fisher Scientific S374-500 https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&searchTerm=S374500&searchType=RAPID
NaCl Fisher Scientific S271-10 https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&searchTerm=S27110&searchType=RAPID
NaHCO3 Fisher Scientific S233-500 https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&searchTerm=S233500&searchType=RAPID
NaNH4HPO4·4H2O Fisher Scientific S218-500 https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&searchTerm=S218500&searchType=RAPID
NanoDrop Lite Spectrophtometer Thermo Scientific ND-LITE-PR https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR
Nonfat dry milk Nestle Carnation N/A N/A
Peptone Becto, Dickinson and Co. 211677 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch
Petri Dishes (100 mm x 15 mm) Fisher Scientific FB0875712 https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712
Petri Dishes (150 mm x 15 mm) Fisher Scientific FB0875714 https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&searchTerm=FB0875714&searchType=RAPID
Polymyxin B sulfate salt Sigma-Aldrich P1004-10MU Store at 2-4 °C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&region=US
Taq DNA Polymerase New England Biolabs M0273S Store at -20 °C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer
Taq Reaction Buffer New England Biolabs M0273S Store at -20 °C;  https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer
Thermal Cycler Bio-Rad C1000 Touch™  Bio Rad Labs 1840148 http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&_pageLabel=search_page&sfMode=search&sfStartNumber=1&clearQR=true&js=1&searchString=1840148&database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&tabName=DIVISIONNAME
Triphenyltetrazolium chloride Alfa Aesar A10870 https://www.alfa.com/en/catalog/A10870/
Tris Base Fisher Scientific BP152-1 https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&searchTerm=BP1521&searchType=RAPID
Tris∙HCl Calbiochem 9310 http://www.emdmillipore.com/US/en/product/OmniPur-TRIS-Hydrochloride—CAS-1185-53-1—Calbiochem,EMD_BIO-9310-OP
Tryptone Becto, Dickinson and Co. 211705 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch
Yeast Extract Becto, Dickinson and Co. 212750 http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&parentCategory=&parentCategoryName=&categoryId=&categoryName=&searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch
α-napthol MP Biomedicals 204189 http://www.mpbio.com/product.php?pid=05204189
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Shimada, T., et al. Extended serotyping scheme for Vibrio cholerae. Curr. Microbiol. 28 (3), 175-178 (1994).
  2. Yamai, S., Tadayuki, O., Toshio, S., Yasuji, K. Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups. Jpn. J. Infect. Dis. 71 (10), 1037-1045 (1997).
  3. Karaolis, D. K., Lan, R., Reeves, P. R. The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae. J. Bacteriol. 177 (11), 3191-3198 (1995).
  4. Albert, M. J., et al. Large epidemic of cholera-like disease in Bangladesh caused by Vibrio cholerae O139 synonym Bengal. Lancet. 342 (8868), 387 (1993).
  5. Barua, D., Barua, D., Greenough III, W. B. History of Cholera. Cholera. , 1-36 (1992).
  6. Morales, R., Delgado, G., Cravioto, A., Faruque, S. M., Nair, G. B. Population Genetics of Vibrio cholerae. Vibrio cholerae-Genomics and Molecular Biology. , 29-47 (2008).
  7. Samadi, A. R., Chowdhury, M. K., Huq, M. K., Khan, M. U. Seasonality of classical and El Tor cholera in Dhaka, Bangladesh: 17-year trends. Trans. R. Soc. Trop. Med. Hyg. 77 (6), 853-856 (1983).
  8. Son, M. S., Megli, C. J., Kovacikova, G., Qadri, F., Taylor, R. K. Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J. Clin. Microbiol. 49 (11), 3739-3749 (2011).
  9. Ghosh-Banerjee, J., et al. Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes. J. Clin. Microbiol. 48 (11), 4283-4286 (2010).
  10. Ansaruzzaman, M., et al. Cholera in Mozambique, variant of Vibrio cholerae. Emerg. Infect. Dis. 10 (11), 2057-2059 (2004).
  11. Ansaruzzaman, M., et al. Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique. Int. J. Med. Microbiol. 297 (6), 443-449 (2007).
  12. Lan, R., Reeves, P. R. Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism. J. Clin. Microbiol. 40 (1), 172-181 (2002).
  13. Nair, G. B., Faruque, S. M., Bhuiyan, N. A., Kamruzzaman, M., Siddique, A. K., Sack, D. A. New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J. Clin. Microbiol. 40 (9), 3296-3299 (2002).
  14. Nair, G. B., et al. Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji. J. Med. Microbiol. 55 (11), 1559-1562 (2006).
  15. Nair, G. B., Mukhopadhyay, A. K., Safa, A., Takeda, Y., Faruque, S. M., Nair, G. B. Emerging hybrid variants of Vibrio cholerae O1. Vibrio cholerae—Genomics and Molecular Biology. , 179-190 (2008).
  16. Safa, A., et al. Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes. J. Med. Microbiol. 55 (11), 1563-1569 (2006).
  17. Nusrin, S., et al. Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic. J. Clin. Microbiol. 42 (12), 5854-5856 (2004).
  18. Carignan, B. M., Brumfield, K. D., Son, M. S. Single nucleotide polymorphisms in regulator-encoding genes have an additive effect on virulence gene expression in a Vibrio cholerae clinical isolate. mSphere. 1 (5), e00253 (2016).
  19. Iwanaga, M., Yamamoto, K., Higa, N., Ichinose, Y., Nakasone, N., Tanabe, M. Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol. Immunol. 30 (11), 1075-1083 (1986).
  20. DiRita, V. J., Claude, P., Georg, J., Mekalanos, J. J. Regulatory cascade controls virulence in Vibrio cholerae. Proc. Natl. Acad. Sci. USA. 88 (12), 5403-5407 (1991).
  21. Kovacikova, G., Lin, W., Skorupski, K. Duel regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive Lys-R type regulator AlsR in Vibrio cholerae. Mol. Microbiol. 57 (2), 420-433 (2005).
  22. Vogel, H. J., Bonner, D. M. Acetylornithinase of Escherechia coli: partial purification and some properties. J. Biol. Chem. 218 (1), 97-106 (1956).
  23. Son, M. S., Taylor, R. K. Genetic screens and biochemical assays to characterize Vibrio cholerae O1 biotypes: classical and El Tor. Curr. Protoc. Microbiol. , 6A.2.1-6A.2.17 (2011).
  24. Kovacikova, G., Skorupski, K. Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol. Microbiol. 46 (4), 1135-1147 (2002).
  25. Wang, Y., et al. The prevalence of functional quorum-sensing systems in recently emerged Vibrio cholerae toxigenic strains. Environ. Microbiol. Rep. 3 (2), 218-222 (2011).
  26. Sanders, E. R. Aseptic laboratory techniques. J. Vis. Exp. (63), e3064 (2012).
  27. Martinez, R. M., Megli, C. J., Taylor, R. K. Growth and laboratory maintenance of Vibrio cholerae. Curr. Protoc. , 6A.1.1-6A.1.6 (2010).

Tags

Vibrio CholeraeClinical IsolatesEnvironmental IsolatesBiotypingGenotypic CharacterizationPhenotypic CharacterizationBiochemical AssaysMetabolic CapabilitiesEnteric BacteriaLaboratory TechniquesCulture MaintenanceCryogenic StorageAgar PlatingLiquid CultureInoculation
check_url/it/55760?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Brumfield, K. D., Carignan, B. M., Ray, J. N., Jumpre, P. E., Son, M. S. Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (123), e55760, doi:10.3791/55760 (2017).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image