מחקר נתמך על ידי ניו המפשייר- INBRE באמצעות פרס פיתוח מוסדי (IDeA), P20GM103506, מהמכון הלאומי למדעי הרפואה הכללית של NIH.

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למחברים אין מה לחשוף.

של מעל 200 מזוהה V. Cholerae serogroups, רק O1 ו O139 יש פוטנציאל מגיפה. ניתן לחלק את הסרוגרופ O1 לשני ביו-טיפוסים: קלאסית ואל טור. עם זאת, זנים היברידיים, הנקראים אל ור גורפים 13 , 17 , התברר כי בעלי רקע ביוטיפ אל Tor, ומאפיינים קלאסיים הנמל 8...

כולרה היא מחלה של המעי הדק הרחוק שנגרם על ידי הצריכה של מזון מזוהם או מים המכילים את החיידק הגראמי שלילי חיידקים Vibrio cholerae. תסמינים של כולרה כוללים הקאות ושילשול מימי בלתי נשלט, מה שמוביל להתייבשות קשה, אשר אם לא מטופלים כראוי, יביא למוות. אשר Cholerae יכול להיות מחולק ליותר מ 200 serogroups מבוסס על המבנה של משטח התא lipopolysaccharide O- אנטיגן. עם זאת, רק 2 serogroups, O1 ו O139, הראו מגיפה או פוטנציאל מגיפה 1 , 2 . יתר על כן, serogroup O139 היה מבודד בעיקר בדרום מזרח אסיה 3 , 4 , בעוד serogroup O1 מופץ ברחבי העולם. יתר על כן, ניתן לחלק את הסרוגרופוס O1 לשני ביו-טיפוסים עיקריים: קלאסית ואל טור. הביוטיפ הקלאסי היה אחראי על מגיפת הכולרה הראשונהS בין 1817 ו 1923. המגיפה השביעית המתמשכת היא תוצאה של ביוטיפ אל טור, אשר גלשו באופן גלובלי את הביוטיפ הקלאסי בסביבה 5 , 6 , 7 . לאחרונה נוצרו זנים המכילים מאפיינים ייחודיים של ביו-טיפוסים קלאסיים ואל-טוריים 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ומאז נקראו אל וריאנטים של אל טור 13 , 17 . כמה גרסאות של אל-טור הראו יכולות ארסיות גבוהות עם התקדמות מחלה מהירה וחמורה יותר ממה שנצפתה קודם לכן, והדגישהצורך בגישה מקיפה יותר לזיהוי סוכן ומניעת מחלות וטיפול 8 , 9 , 18 . בעוד הזיהוי של ביו-טיב אינו מכתיב מיד טיפול, התקדמות נוספת בפיתוח החיסונים ובסוכני הטיפול העתידיים עשויה להפיק תועלת מהבחנה ביו-טייתית. הסדרה הראשונה של הפרוטוקולים המפורטים כאן תאפשר לחוקרים לשמור כראוי V. Cholerae זנים במעבדה הגדרה. עקביות וניתוח שלאחר מכן דורש הכנת מלאי וצמיחה של מבודדים, אשר אינו תלוי ביו-טיב. עם זאת, כדי לגרום באופן אופטימלי ביטוי גנים ארסיות, ביוטיפ עצמאית טכניקות culturing ספציפיים נדרשים 19 . בנוסף, הכנה לבדיקות גנטיות וביוכימיות שונות מתוארים בכתב יד זה. כולרה רעלן (CT) ואת רעלן שיתוף reגלוטן gulated (TCP) הם שני הגורמים העיקריים ארסיות נשלט על ידי הרגולטור הראשי ToxT בשני biotypes של V. Cholerae O1 serogroup 20 . CT הוא רעלן דו-חמצני המורכב מחמש CtxB תת יחידות המקיפות יחידה אחת של CtxA, ואחראית על אובדן אלקטרוליטים מהיר הקשור לכולרה. TCP הוא סוג IV פילוס מקודדים על ידי tcp operon ( tcpABqCRDSTEF ), והוא מעורב בהתקשרות ו קולוניזציה של המעי הדק הרחוק. TcpA הוא הגן הראשון של אופרון tcp אשר מקודד את יחידות יחידת הפרט Pilin חיוני לבנייה של הפילוס 8 . רצף הגן עבור ctxA נשמר לחלוטין בין ביוטיקים קלאסיים אל Tor, בעוד ctxB ו tcpA נבדלים על פני שני biotypes אבל נשמרים בתוך כל ביוטיפ 8 . CtxB נשמר לחלוטין בין biotypes למעט שתי posi בסיסTions (115 ו 203). ב biotype אל Tor, thymine מתגורר בנקודות הבסיס 115 ו 203, בעוד הביוטיפ הקלאסי מכיל ציטוזין בבסיסים אלה. Tcpa נשמר לחלוטין בתוך כל ביוטיפ, אך נבדלים בסיסים מרובים בין biotypes. הבדלים גנטיים אלה משמשים כסימני זיהוי ביו-טיים ראשוניים, ולאחר סידור תגובת תגובת שרשרת הפולימרז (PCR), כולל אלה, ניתן לבודד רצפים מבודדים עם O395 או WT El N1696 הקלאסית מסוג Wild-type כדי לקבוע את הרקע הביוטיפי של CT ו- TCP, בהתאמה, ב V. cholerae נתון לבודד. פרוטוקולים רבים פותחו על מנת לאפיין את ההבחנות הפנוטיפיות בין הביוטיקים הקלאסיים לאל טור , 21 , 22 , 23 . Polymyxin B הוא אנטיביוטיקה פפטיד כי compromises את יושרה של קרום התא החיצוני ב- Gram שלילי bacTeria, ואת התנגדות polyixxin B יכול להיות דמיינו דרך assay התנגדות polymyxin B 21 . ציטרט הוא המצע העיקרי של מחזור של Kreb, ואת היכולת metabolize ציטרט כמקור פחמן יחיד ניתן לקבוע באמצעות assay מטבוליזם ציטראט 22 . HapR מקודד הרגולטור העולמי ו הרגולטור חישה המניין קוורום ב V. cholerae, HapR, אשר נקשר לאזורים שונים האמרגן מסדיר ביטוי הגן האופרה 24 . כמה זנים פתוגניים של V. cholerae יש באופן טבעי מוטציה משמרת מסגרת בגן hapR שגרם זה צפיפות תלויה תקנה של ביטוי גנים ארסיות לאיבוד 24 , 25 . מדידת פעילות פרוטאז HAPR-regulated באמצעות מדיה אגר חלב מאפשר החוקר כדי לזהות אם לבודד מסוים מכיל HAPR תפקודית 23 . הבדיקות המוליזה של המעי הגס על יכולתו של מתח להפריש אנזימים hemolytic כי lyse תאי דם אדומים; את מידת המוליזה יכול להיות דמיינו על צלחות אגר דם 23 . תנועתיות קשורה לעיתים קרובות עם ארסיות V. cholerae וניתן לנתח באמצעות לוחות אגר התנועה 23 . מבחני ה- Voges-Proskauer בודקים את יכולתו של זן להסיס את הגלוקוז כמקור פחמן יחיד ומייצרים את התוצר האסייתי 21 . עם הופעת גרסאות אל Tor, קשה לחזות את התוצאות של assay פנוטיפי נתון ללא בדיקות genotypic נרחב, ולפני deducing את הרקע של ביוטייפ של V. Cholerae מבודד, מומלץ לבצע את הרכבה זו של מבחני 23 ולהשוות את התוצאות זנים התייחסות כמו בטבלה 2 . כאן, יש לנו סדרה מתקדמת של פרוטוקולים, באופן קולקטיבי ניצול foreeeהבחינו genotypic ו מבחני פנוטיפי לגישה מקיפה יותר לאפיון V. ביוטכנולוגי כולרה . יתר על כן, יש לנו תיאר את ההבחנה גנוטיפי פנוטיפי של V ידוע. Cholerae אל Tor וריאנטים (MQ1795 ו BAA-2163), בהשוואה זנים התייחסות ביוטיפ נפוץ (WT קלאסי O395, WT אל Tor C6706, ו ​​WT אל Tor N16961, טבלה 1 ). הופעתה של גרסאות אל Tor הציגה אתגרים האמינות של המועסקים בעבר פרוטוקול יחיד אפיון ביו-טיוב אפיון; עם זאת, זה מספר רב של מערכת זיהוי assay יאפשר אפיון אמין יותר של קליני וסביבתי V. כולסטרס מבודד.

<table><tbody><tr><td>1 kb DNA Ladder</td><td>New England Biolabs</td><td>N3232S</td><td>https://www.neb.com/products/n3232-1-kb-dna-ladder</td></tr><tr><td>60% Glycerol</td><td>Calbiochem</td><td>356352</td><td>http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352</td></tr><tr><td>Agar</td><td>Becto, Dickinson and Co.</td><td>214030</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch</td></tr><tr><td>Agar with brain-heart infusion</td><td>Becto, Dickinson and Co.</td><td>237500</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch</td></tr><tr><td>Agarose</td><td>Peqlab</td><td>732-2789</td><td>https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&amp;amp;_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&amp;amp;_dynSessConf=1766917479792147141&amp;amp;targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&amp;amp;lastLanguage=en&amp;amp;/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&amp;amp;_D%3AcurrentLanguage=+&amp;amp;currentLanguage=en&amp;amp;_D%3AlastLanguage=+&amp;amp;_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+</td></tr><tr><td>Anhydrous K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P288-500</td><td>https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&amp;amp;searchTerm=P288500&amp;amp;searchType=RAPID</td></tr><tr><td>Blood Agar Plates</td><td>Remel</td><td>R01200</td><td>Store at 4 &amp;deg;C;&amp;nbsp; http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar</td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>A73-500</td><td>https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&amp;amp;searchTerm=A73500&amp;amp;searchType=RAPID</td></tr><tr><td>Bromothymol Blue</td><td>Fisher Scientific</td><td>B388-10</td><td>https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&amp;amp;searchTerm=B38810&amp;amp;searchType=RAPID</td></tr><tr><td>Cirtric acid &amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S72836-3</td><td>https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363</td></tr><tr><td>Deoxynucleotide (dNTP) Solution Kit</td><td>New England Biolabs</td><td>N0446S</td><td>Store at -20 &amp;deg;C;&amp;nbsp; https://www.neb.com/products/n0446-deoxynucleotide-solutionset</td></tr><tr><td>Disodium EDTA</td><td>Fisher Scientific</td><td>S311-500</td><td>https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&amp;amp;searchTerm=S311500&amp;amp;searchType=RAPID</td></tr><tr><td>DNA Clean &amp;amp; Concentrator&amp;trade; -25 Kit</td><td>Zymo Research</td><td>D4007</td><td>http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit</td></tr><tr><td>GelGreen Nucleic Acid Stain</td><td>Biotium</td><td>41005</td><td>https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10,000x-in-water/</td></tr><tr><td>Genesys 10SUV-VIS Spectrophotometer</td><td>Thermo Scientific</td><td>840-208100</td><td>https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100</td></tr><tr><td>Gentra Puregene Yeast/Bact. Kit</td><td>Qiagen</td><td>158567</td><td>https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation</td></tr><tr><td>HCl</td><td>Fisher Scientific</td><td>A144-212</td><td>Corrosive;&amp;nbsp; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&amp;amp;searchTerm=A144212&amp;amp;searchType=RAPID</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217-500</td><td>https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&amp;amp;searchTerm=P217500&amp;amp;searchType=RAPID</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td>https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&amp;amp;searchTerm=P285500&amp;amp;searchType=RAPID</td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td>P250-500</td><td>https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&amp;amp;searchTerm=P250500&amp;amp;searchType=RAPID</td></tr><tr><td>Le Loop</td><td>Decon Labs Inc.</td><td>MP190-25</td><td>http://deconlabs.com/products/leloop/</td></tr><tr><td>Le Stab</td><td>Decon Labs Inc.</td><td>MP186-5</td><td>http://deconlabs.com/products/lestab/</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>M63-500</td><td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&amp;amp;searchTerm=M63500&amp;amp;searchType=RAPID</td></tr><tr><td>Mini-Sub Cell GT Horizontal Electrophoresis System</td><td>Bio Rad Labs</td><td>1704406</td><td>http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&amp;amp;WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&amp;amp;WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373</td></tr><tr><td>MR-VP Broth</td><td>Difco</td><td>216300</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td>https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&amp;amp;searchTerm=S374500&amp;amp;searchType=RAPID</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-10</td><td>https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&amp;amp;searchTerm=S27110&amp;amp;searchType=RAPID</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-500</td><td>https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&amp;amp;searchTerm=S233500&amp;amp;searchType=RAPID</td></tr><tr><td>NaNH&lt;sub&gt;4&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S218-500</td><td>https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&amp;amp;searchTerm=S218500&amp;amp;searchType=RAPID</td></tr><tr><td>NanoDrop Lite Spectrophtometer</td><td>Thermo Scientific</td><td>ND-LITE-PR</td><td>https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR</td></tr><tr><td>Nonfat dry milk</td><td>Nestle Carnation</td><td>N/A</td><td>N/A</td></tr><tr><td>Peptone</td><td>Becto, Dickinson and Co.</td><td>211677</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch</td></tr><tr><td>Petri Dishes (100 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712</td></tr><tr><td>Petri Dishes (150 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875714</td><td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&amp;amp;searchTerm=FB0875714&amp;amp;searchType=RAPID</td></tr><tr><td>Polymyxin B sulfate salt</td><td>Sigma-Aldrich</td><td>P1004-10MU</td><td>Store at 2-4 &amp;deg;C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&amp;amp;region=US</td></tr><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA Polymerase</td><td>New England Biolabs</td><td>M0273S</td><td>Store at -20 &amp;deg;C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td></tr><tr><td>&lt;em&gt;Taq&lt;/em&gt; Reaction Buffer</td><td>New England Biolabs</td><td>M0273S</td><td>Store at -20 &amp;deg;C;&amp;nbsp; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td></tr><tr><td>Thermal Cycler Bio-Rad C1000 Touch&amp;trade;&amp;nbsp;</td><td>Bio Rad Labs</td><td>1840148</td><td>http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&amp;amp;_pageLabel=search_page&amp;amp;sfMode=search&amp;amp;sfStartNumber=1&amp;amp;clearQR=true&amp;amp;js=1&amp;amp;searchString=1840148&amp;amp;database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&amp;amp;tabName=DIVISIONNAME</td></tr><tr><td>Triphenyltetrazolium chloride</td><td>Alfa Aesar</td><td>A10870</td><td>https://www.alfa.com/en/catalog/A10870/</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&amp;amp;searchTerm=BP1521&amp;amp;searchType=RAPID</td></tr><tr><td>Tryptone</td><td>Becto, Dickinson and Co.</td><td>211705</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch</td></tr><tr><td>Yeast Extract</td><td>Becto, Dickinson and Co.</td><td>212750</td><td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&amp;amp;parentCategory=&amp;amp;parentCategoryName=&amp;amp;categoryId=&amp;amp;categoryName=&amp;amp;searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch</td></tr><tr><td>&amp;alpha;-napthol</td><td>MP Biomedicals</td><td>204189</td><td>http://www.mpbio.com/product.php?pid=05204189</td></tr></tbody></table>

laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates

החיידק הגרימי שלילי של גראם הוא Vibrio cholerae הוא הסוכן האטיולוגי של כולרה זיהומית זיהומית. בשל השכיחות והחומרה הגלובלית של המחלה, V. Cholerae נחקרה בהרחבה הן בהגדרות סביבתיות והן במעבדה, המחייבת תחזוקה נאותה וטכניקות טיפוח. קלאסי ואל טור הם שני ביו-טיפוסים עיקריים המרכיבים את ה- V. Cholerae O1 serogroup, שכל אחד מהם מציג מאפיינים גנוטיפיים ופנוטיפיים ייחודיים המספקים מנגנונים אמינים לאפיון ביוטיפ, ודורשים ארסיות מובהקות הגורמות לתרבות. ללא קשר לביוטיפ של זן סיבתי עבור כל זיהום או פרוץ מסוים, הטיפול הסטנדרטי עבור המחלה כולל טיפול rehydration בתוספת משטר של אנטיביוטיקה. עם זאת, סיווג ביוטיפ עשוי להיות נחוץ ללימודי מעבדה ועלולות להיות השפעות רחבות יותר בתחום הביו-רפואי.בתחילת שנות האלפיים, זוהו הבדידות הקליניות, אשר הציגו תכונות גנוטיפיות ופנוטיפיות הן מהביוטיקים הקלאסיים והן מהאל-טור. ההיברידיות שזוהו לאחרונה, המכונה אל וריאציות של אל-טור, גרמו לקליניקה ולסביבה לזהות בידוד ביו-טיפוסי כדי להיות מורכבות יותר מאשר בפרוטוקולי זיהוי מסורתיים בודדים. בנוסף לתיאור V. Cholerae תחזוקה כללית ו culturing טכניקות, כתב היד הזה מתאר סדרה של גנים ספציפיים ( ctxB ו tcpA ) מסכי PCR מבוססי PCR ו מבחנים פנוטיפי (התנגדות polyyxin B, מטבוליזם ציטראט, פעילות proteolytic, פעילות hemolytic, תנועה, ומטבוליזם גלוקוז באמצעות Voges- Assay Proskauer) השתמשו באופן קולקטיבי לאפיין ו / או להבדיל בין ביוטוסים קלאסיים לאל טור. יחד, מבחני אלה מספקים גישה שיטתית יעילה לשמש חלופה, או בנוסף, יקר, ניסויים עתירי עבודה ב characterizaשל V. Cholerae קליני (וסביבתי) מבודד.

עבור תחזוקה נאותה ושימוש בכל זן חיידקי, מומלץ לדעת את זמן הכפלה של זן (ים) של עניין. הנה, את שיעורי הצמיחה המשתנים של V נפוץ. זנים cholerae הודגמו דרך עקומת הצמיחה, ואת זמני הכפלה משוער חושבו באמצעות רגרסיה ליניארית. WT El Tor N16961 ו El Tor וריאנט MQ1795 הוכיחו פע?...

Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

כתב יד זה מתאר ראוי Vibrio cholerae טכניקות תחזוקה בנוסף סדרה של מבחני ביוכימי, מנוצל קולקטיבית עבור הבחנה מהירה ואמינה בין קליני וסביבתי V. ביו-טיפוסים של כולרה במעבדה.

טכניקות מעבדה המשמשות לשמירה על ביו-טיפוסים של<em&gt; ויבריו כולרה</em&gt; בידוד קליני ואיכותי

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

laboratory-techniques-used-to-maintain-differentiate-biotypes-vibrio

Research

JoVE Journal

Immunology and Infection

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

10.8K Views.  Plymouth State University.  כתב יד זה מתאר ראוי Vibrio cholerae טכניקות תחזוקה בנוסף סדרה של מבחני ביוכימי, מנוצל קולקטיבית עבור הבחנה מהירה ואמינה בין קליני וסביבתי V. ביו-טיפוסים של כולרה במעבדה. 

Video: Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

באמצעות assay הפאן ויראלי microarray (Virochip) אל מסך דוגמאות קליניות עבור פתוגנים ויראליים

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - שיטה גנטית לשינוי<em&gt; Vibrio cholerae</em&gt; בהתבסס על שינוי הטבעי וFLP-רקומבינציה

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Culturing ושמירה<em&gt; Difficile Clostridium</em&gt; בסביבת אנאירובית

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

פיתוח בינוני אגר chromogenic רגיש וספציפי יותר לצורך זיהוי של<em&gt; Parahaemolyticus Vibrio</em&gt; ו אחר<em&gt; Vibrio</em&gt; מינים

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

פרוטוקול לאפיין את השינויים המורפולוגיים של<em&gt; Clostridium difficile</em&gt; בתגובה לטיפול אנטיביוטי

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

אינדוקציה של דיפרנציאציה נייד תא הדמיה אחת של<em&gt; Vibrio parahaemolyticus</em&gt; שחיין ותאי נחיל

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

מלח מרה-induced ביופילמים בתוך המעית פתוגנים: טכניקות וכימות

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

לכימות ויבריו cholerae קולוניזציה ושלשולים במודל דג זברה למבוגרים

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

המשך הדגירה של האינטראקציה התחרותית בין vibrio פישרי מבודד

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

ראפיד פרוטוקול להכנת Electrocompetent<em&gt; חיידקי Escherichia</em&gt; ו<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

טכניקות מעבדה המשמשות לשמירה על ביו-טיפוסים של

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באמצעות assay הפאן ויראלי microarray (Virochip) אל מסך דוגמאות קליניות עבור פתוגנים ויראליים

TransFLP - שיטה גנטית לשינוי

Culturing ושמירה

פיתוח בינוני אגר chromogenic רגיש וספציפי יותר לצורך זיהוי של

פרוטוקול לאפיין את השינויים המורפולוגיים של

אינדוקציה של דיפרנציאציה נייד תא הדמיה אחת של

מלח מרה-induced ביופילמים בתוך המעית פתוגנים: טכניקות וכימות

לכימות ויבריו cholerae קולוניזציה ושלשולים במודל דג זברה למבוגרים

המשך הדגירה של האינטראקציה התחרותית בין vibrio פישרי מבודד

ראפיד פרוטוקול להכנת Electrocompetent