نشكر السيد Channey، E. تشاو، و ك توماس لما قدموه من مساعدة في هذا المشروع. وقد تم تمويل وازم المشروع جزئيا من قبل جامعة ولاية كاليفورنيا بوليتكنيك.

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Vibrio+spp.+in+raw+shrimps+(Parapenaeus+longirostris)+and+performance+of+a+chromogenic+medium+for+the+isolation+of+Vibrio+strains.">Prevalence of Vibrio spp. in raw shrimps (Parapenaeus longirostris) and performance of a chromogenic medium for the isolation of Vibrio strains.</a> Lett Appl Microbiol. 61 (3), 224-230 (2015).</li><li>Food Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+guidance+on+reporting+results+from+studies+evaluating+diagnostic+tests.">Statistical guidance on reporting results from studies evaluating diagnostic tests.</a> http://www.fda.gov/RegulatoryInformation/Guidances/ucm071148.htm. , (2007).</li><li>Burd, E. 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Some media were generously provided by Hardy Diagnostics, Santa Maria, CA. Thorsen conducted this study while a student at California Polytechnic State University. He is currently an employee of Hardy Diagnostics.

وتركز هذه الدراسة على تطوير وسائل الإعلام والثقافة، والتقييم. تقليديا، TCBS هو انتقائية والتفضيلية تستخدم سيلة لعزل وكشف V. نظيرة الحالة للدم، ضمة الكوليرا والخامس. vulnificus 12. ومع ذلك، فقد تم الإبلاغ عن القيود لهذه الوسيلة، مثل عدم القدرة على التفريق V. ?...

كعضو من جنس الضمة، V. نظيرة الحالة للدم هو، المنحني، على شكل قضيب البكتيريا سالبة الجرام، وتشكيل غير بوغ. فإنه يسلك حركية عالية في كل من البيئات السائلة وشبه الصلبة. معظم V. سلالات نظيرة الحالة للدم هي غير الممرضة للإنسان، ومع ذلك فقد الأنواع الفرعية الممرضة تسبب الأوبئة والجوائح، وبالتالي يعتبر هذا النوع أن يكون الممرض تنتقل عن طريق الأغذية في العديد من البلدان 1،2. وقد أظهرت حدوث العدوى الضمة في الولايات المتحدة اتجاها تصاعديا منذ عام 2000 3. ومن بين الضمة النيابة.، V. نظيرة الحالة للدم هو النوع الأكثر ذكرت في كثير من الأحيان تسبب الأمراض في الولايات المتحدة 4،5. وتشمل الأنواع الأخرى ذات الصلة سريريا خامسا alginolyticus، V. vulnificus، V. الكوليرا، وما يحدث هناك نسبة صغيرة من الأمراض عن طريق أنواع متعددة في وقت واحد. خامسا نظيرة الحالة للدم هو ط الطبيعيnhabitant من المياه البحرية، وبالتالي توزيعها على نطاق واسع في المياه البحرية في جميع أنحاء العالم بما في ذلك مصبات الأنهار. تم اكتشاف الأنواع في عام 1950 بعد تفشي التسمم الغذائي في اليابان. في الولايات المتحدة، والأنواع عزله للمرة الأولى في مياه البحر والرواسب، والمحار في المنطقة بوجيه ساوند 6،7. مرشح مغذيات في الموائل البحرية مثل المحار الصدفتين، يمكن أن تأوي V. نظيرة الحالة للدم كجزء من النباتات الطبيعية 8. على هذا النحو، V. وغالبا ما ترتبط العدوى نظيرة الحالة للدم في الانسان الى استهلاك الأغذية البحرية الملوثة، وخاصة المحار النيئ أو. يحدث الطريق أقل شيوعا من دخول عندما يتعرض الجرح مفتوحا لمياه البحر، مما يؤدي إلى إصابة الجلد. معظم V. سلالات نظيرة الحالة للدم لا تسبب الأمراض التي تصيب البشر، ولكن بعض الأنواع الفرعية إيواء عوامل الفوعة مثل دموية بالحرارة مباشرة (TDH) والمسببة للأمراض. الأعراض الأكثر شيوعا من الأمراض المنقولة بالغذاء V. عدوى نظيرة الحالة للدم همالاسهال وآلام في البطن، تليها الغثيان والقيء، والحمى. كما تم الإبلاغ عن الصداع والقشعريرة. فترة الحضانة متوسط هو 15 ساعة، ولكن يمكن أن تصل إلى 96 ساعة بعد استهلاك كمية كافية من السلالات المسببة للأمراض 9. يستمر المرض من سنتين إلى ثلاث أيام. أعراض التهاب المعدة والأمعاء الناجمة عن V. نظيرة الحالة للدم وإلى حد كبير محدودة ذاتيا والعلاج وبالتالي الخاصة ليست ضرورية. الحالات الخفيفة من التهاب المعدة والأمعاء ويمكن علاجها على نحو فعال من خلال معالجة الجفاف عن طريق الفم. ويمكن علاج أمراض خطيرة أكثر من المضادات الحيوية مثل التتراسيكلين أو سيبروفلوكساسين 10. معدل وفيات حوالي 2٪ للحالات التهاب المعدة والأمعاء، ولكن قد يصل إلى 29٪ لأولئك الذين يطورون عدوى مجرى الدم أو تسمم الدم. أي الشخص الذي يستهلك المأكولات البحرية أو لديه جرح مفتوح تتعرض لمياه البحر في خطر من V. عدوى نظيرة الحالة للدم. شكل أكثر شدة من الأمراض، وتسمم الدم تهدد الحياة، هو أكثر شيوعا في جزء من السكان مع زملاء الطبي الأساسيnditions 11، والتي تشمل إدمان الكحول، وأمراض الكبد، ومرض السكري، وأمراض الكلى والأورام الخبيثة، وغيرها من الشروط التي تؤدي إلى استجابة مناعية ضعيفة. والجدير بالذكر أن هذه المجموعة من الأفراد هي أيضا في خطر أعلى للتعاقد الأمراض الحادة الناجمة عن V. vulnificus، التي يمكن العثور عليها في الموائل الطبيعية مشابهة لV. نظيرة الحالة للدم. يتم عزل خامسا نظيرة الحالة للدم بشكل روتيني باستخدام وحمض-سترات الصفراء أملاح-السكروز (TCBS) أجار كوسيلة انتقائية والتفاضلية. تخصيب اليورانيوم في البيبتون القلوية قد تسبق العزلة على TCBS أجار. وثم مزيد من الاختبارات المستعمرات الظني على TCBS في سلسلة من الاختبارات البيوكيميائية و / أو المقايسات الجزيئية التي تستهدف وجود جينات الأنواع المحددة. وغالبا ما تستخدم أساليب PCR القائم لتأكيد هوية V. نظيرة الحالة للدم عن طريق تضخيم الجين دموية بالحرارة، TLH 12. بغض النظر عن الفصلOICE أساليب تأكيد، فمن المهم أن يكون وسيلة فعالة لعزل وتمييز V. نظيرة الحالة للدم من ضمات البحرية الأخرى في المقام الأول. وقد استخدمت بشكل روتيني TCBS للتمييز الأنواع داخل جنس الضمة وفقا لقدراتهم للتخمر السكروز 12. ويرافق رد فعل إيجابي التخمير عن تغيير لون مؤشر الرقم الهيدروجيني زرقة البروموثيمول. ف. المستعمرات نظيرة الحالة للدم هي مميزة إلى حد ما على TCBS، واظهار اللون الأزرق إلى اللون الأخضر. ومع ذلك، هذه الوسيلة لا يمكن التفريق بسهولة V. alginolyticus والخامس. الكوليرا. الأنواع بروتيوس-تخمر السكروز قد ينتج مستعمرات صفراء تشبه V. الكوليرا أو V. alginolyticus 13. على العزلة الأولية على TCBS، V. ويمكن أيضا أن أخطأ في التعرف نظيرة الحالة للدم كما الإيروموناس هايدروفيلا، القوربية الشيغيلانية، وأنواع الزائفة 14. سلالات مع سعر الصرف الثابت السكروز تأخرقد يتم الخلط بين entation مع السكروز الآخرين nonfermenting الضمة 13، والتي تشمل V. نظيرة الحالة للدم. تم العثور على TCBS أن تكون ليست حساسة ضد كولاي، الجرية الزائفة، من بين أمور أخرى. تسفر عن العديد من الأنواع الأخرى خضراء إلى رمادية المستعمرات التي يحتمل الخلط بينه وبين V. نظيرة الحالة للدم أو V. vulnificus 15. ونتيجة لذلك، فمن المستحسن لتطوير وسائل الإعلام ثقافة بديلة مع أفضل حساسية وخصوصية نحو كشف وعزل خامسا نظيرة الحالة للدم وغيرها من الأنواع ذات صلة وثيقة. وقد وضعت مؤخرا عدة بدائل وسائل الإعلام. بالإضافة إلى إدراج وكلاء انتقائية، فإن معظم إدماج ركائز اللونية للتمييز الأنواع على أساس الأنشطة الإنزيمية التفاضلية الخاصة بهم. على سبيل المثال، استخدمت الإندوكسيل-β-جلوكوسيدي والإندوكسيل-β-غالاكتوزيد باعتبارها ركائز اللونية للتمييز V. الفقرةالمستعمرات لدم (والتي تظهر لون أخضر مزرق) من تلك التي V. الكوليرا (الأرجواني) بسبب قدراته التفاضلي على إنتاج بيتا جلوكوزيد وβ غالاكتوزيداز 16. تم تقييم تركيبات مختلفة من أجار مولدات اللون وضعت من قبل العديد من الجماعات ووذكرت لأداء نسبيا أو أفضل من TCBS 17،18،19. ميزة استخدام وسيلة مولد اللون هو أن التلوين من الوسط المحيط هو الحد الأدنى مما يسهل عزل المستعمرات معينة. في هذه الدراسة، قمنا بتقييم قدرة متوسطة مولد اللون وضعت حديثا لكشف وعزل خامسا الكوليرا، V. نظيرة الحالة للدم، والخامس vulnificus. مع التركيز بشكل خاص على قدرته على التفريق V. نظيرة الحالة للدم من الأنواع الأخرى.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent/Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-15-4</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >BHI powder</td>
			<td>Fisher Scientific</td>
			<td>DF0418-17-7</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Blender</td>
			<td>Any</td>
			<td></td>
			<td>to blend oyster meat</td>
		</tr>
		<tr >
			<td >CampyGen gas generator</td>
			<td>Hardy Diagnostics</td>
			<td>CN035A</td>
			<td>to provide a microaerophilic atmosphere; may use other brands</td>
		</tr>
		<tr >
			<td >Chocolate agar plates</td>
			<td>Hardy Diagnostics</td>
			<td>E14</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Common PCR reagents (dNTPs, MgCl2, Taq Polymerase)</td>
			<td>Any</td>
			<td></td>
			<td>or use PCR beads (Fisher Sci 46-001-014)</td>
		</tr>
		<tr >
			<td >Culture tubes</td>
			<td>Fisher Scientific</td>
			<td>S50712</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>S348903</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Gel doc</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >HardyChrom Vibrio agar plates</td>
			<td>Hardy Diagnostics</td>
			<td>G319</td>
			<td>This study evaluates this medium</td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculating loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-606</td>
			<td>10 microliter-size was used in this study</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Oysters</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Fisher Scientific</td>
			<td>R23701</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >Pipette and tips</td>
			<td>Any</td>
			<td></td>
			<td>Sterilized tips</td>
		</tr>
		<tr >
			<td >Primers for tlh</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Scale</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Spreader</td>
			<td>Fisher Scientific</td>
			<td>08-100-11</td>
			<td>Beads may be used instead</td>
		</tr>
		<tr >
			<td >Stomacher blender</td>
			<td>Stomacher</td>
			<td >400</td>
			<td>Samples were homogenized at 200 rpm for 30 sec.&#160; Other homogenizer can be used.</td>
		</tr>
		<tr >
			<td >Sterile filter bags for blenders</td>
			<td>Fisher Scientific</td>
			<td>01-812-5</td>
			<td></td>
		</tr>
		<tr >
			<td >TCBS powder</td>
			<td>Hardy Diagnostics</td>
			<td >265020</td>
			<td>This study evaluates this medium</td>
		</tr>
		<tr >
			<td >Thermocycler</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TSB powder</td>
			<td>Fisher Scientific</td>
			<td>DF0370-07-5</td>
			<td>may use other brands</td>
		</tr>
		<tr >
			<td >UV viewing cabinet</td>
			<td>Any</td>
			<td></td>
			<td>Emit long-wave UV light</td>
		</tr>
		<tr >
			<td >Water bath</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Name</td>
			<td>Sources</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr >
			<td >Bacterial species and strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Aeromonas hydrophila</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Candida albicans</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Campylobacter jejuni</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Escherichia coli</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Proteus mirabilis</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pseudomonas aeruginosa</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Staphylococcus aureus</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Salmonella Choleraesuis</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella boydii</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella flexneri</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Shigella sonnei</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vibrio alginolyticus</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. cholerae (serotypes include O139, O1, non O1, El Tor biovars)</td>
			<td >FDA, ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. damsela</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. fisherii</td>
			<td >Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. fluvialis</td>
			<td >CDC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. furnissii</td>
			<td >CDC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. hollisae</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. metschnikovii</td>
			<td >ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. mimicus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. parahaemolyticus(serotypes include O3:K6, O1:K56, O4:K8, O5:K15, O8, etc)</td>
			<td >ATCC, FDA, CDC, Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. proteolyticus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >V. vulnificus</td>
			<td >FDA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a more sensitive and specific chromogenic agar medium for the detection of vibrio parahaemolyticus and other vibrio species

وقد أظهرت العدوى تنتقل عن طريق الأغذية في الولايات المتحدة التي تسببها أنواع الضمة اتجاها تصاعديا. في جنس الضمة، V. نظيرة الحالة للدم هو المسؤول عن غالبية الإصابات -associated الضمة. وهكذا، والتفريق الدقيق بين الضمة النيابة. وكشف V. نظيرة الحالة للدم هو في غاية الأهمية لضمان سلامة الإمدادات الغذائية لدينا. على الرغم من أن التقنيات الجزيئية شائعة على نحو متزايد، لا تزال تفعل أساليب اعتمادا الثقافة بشكل روتيني وأنها تعتبر الأساليب القياسية في ظروف معينة. وبالتالي، تم اختبار رواية المتوسطة أجار مولد اللون مع الهدف المتمثل في توفير أفضل طريقة لعزل والتفريق بين النيابة الضمة ذات الصلة سريريا. مقارنة بروتوكول الحد الحساسية والنوعية والكشف للكشف عن V. نظيرة الحالة للدم بين المتوسطة مولد اللون الجديد والمتوسطة التقليدية. خامسا مختلف سلالات نظيرة الحالة للدم (ن = 22) إعادةعرض الأنماط المصلية ومصدر من أصول مختلفة استخدمت. كانوا تحديدها من قبل إدارة الغذاء والدواء (FDA) ومراكز السيطرة على الأمراض والوقاية منها (CDC)، وكذلك التحقق في مختبرنا من قبل TLH -PCR. في أربعة على الأقل محاكمات منفصلة، ​​تم تلقيح هذه السلالات على اللونية أجار ووحمض-سترات الصفراء أملاح-السكروز (TCBS) أجار، التي هي وسيلة الموصى بها لزراعة هذا النوع، تليها الحضانة في 35-37 درجة مئوية لمدة 24 -96 ساعة. ثلاثة V. لم سلالات نظيرة الحالة للدم (13.6٪) لا ينمو بالشكل الأمثل على TCBS، ومع ذلك أظهرت مستعمرات خضراء إذا كان هناك نمو. لم سلالتين (9.1٪) لم تسفر المستعمرات السماوي المتوقعة على أجار مولد اللون. خامسا عدم تم اختبار سلالات نظيرة الحالة للدم (ن = 32) أيضا لتحديد خصوصية أجار مولد اللون. ومن بين هذه السلالات، لم لا تنمو 31 أو عرضت الأشكال التضاريسية مستعمرة أخرى. يعني استرداد V. نظيرة الحالة للدم على chromogeكانت شركة الاستثمارات الوطنية أجار ~ 96.4٪ بالمقارنة مع أجار الصويا زيتية تستكمل مع 2٪ كلوريد الصوديوم. في الختام، أجار مولد اللون الجديد هو وسيلة فعالة للكشف عن V. نظيرة الحالة للدم ولتمييزه عن ضمات أخرى.

في هذه الدراسة، تم تجميع 54 سلالات ميكروبية، والتي شملت 22 سلالات داخل V. الأنواع نظيرة الحالة للدم، 19 نوعا الضمة أخرى، و 13 أنواع الضمة غير (الجدول 1). معظم V. إما تلقى سلالات نظيرة الحالة للدم من ادارة الاغذية والعق?...

Watch this Scientific Journal Video about Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species at JoVE.com

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.

تطوير أكثر حساسية وخصوصية اللونية آجار المتوسطة للكشف عن<em&gt; الضمة نظيرة الحالة للدم</em&gt; وغيره<em&gt; الضمة</em&gt; الأنواع

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

development-more-sensitive-specific-chromogenic-agar-medium-for

Research

JoVE Journal

Immunology and Infection

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

14.4K Views.  California Polytechnic State University. 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. 

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

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

استعمار Euprymna scolopes الحبار بواسطة الضمة fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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 - طريقة لتعديل وراثيا<em&gt; ضمة الكوليرا</em&gt; بناء على التحول الطبيعي وإعادة التركيب، FLP

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

استخدام متوسط ​​البلغم الاصطناعي لاختبار فعالية المضادات الحيوية لمكافحة<em&gt; الزائفة الزنجارية</em&gt; في شروط أكثر ملاءمة لالرئة التليف الكيسي

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

متساوي التضخيم (LAMP) فحوصات بوساطة حلقة للكشف عن أنواع محددة من<em&gt; الأيمرية</em&gt; التي تصيب الدجاج

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

تطوير بوساطة حلقة-المجففة بالتبريد متساوي التضخيم الكواشف للكشف عن<em&gt; الكوكسيلا البورنيتية</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

التصور من الفحم آجار ريسازورين الفحص لشبه الكمي، متوسطة الإنتاجية تعداد مايكوباكتيريا

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

تطوير المستمدة الخلية الجذعية خلايا T التنظيمية مستضد محددة ضد المناعة الذاتية

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

استخدام تقنية تراكب لينة أجار للكشف عن المركبات المثبطة إنتاج البكتريا

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

كشف وإثراء نادر محددة مستضد الخلايا B لتحليل النمط الظاهري وظيفة

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

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

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.