نود أن نشكر اليزابيث Guillemet، وكريستوف بويسون Bridoux لودوفيك للمساعدة التقنية الممتازة. نحن مدينون كثيرا لSalamitou سيلفي وFedhila SINDA لإعداد النظام الأولي.

1. تربية الحشرات دورة كاملة من البيض إلى يرقات الطور مشاركة يستمر حوالي 5 أسابيع في C. ° 25 وهناك حاجة إلى واحد أو 2 أسابيع إضافية للحصول على الفراشات الكبار. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع لا يقل عن 100 الشرانق أو دمجها حديثا G. الكبار الفراشات في قفص mellonella شبكات الأسلاك المعدنية 5-لتر. الفراشات الذكور قياس 10 حتي 15 مم. الفراشة الذكور البالغين هو البيج مع الضوء الخافت وعلامات داكنة. أنثى الفراشات تدبير حوالي 20 ملم. الإناث أكثر قتامة من الذكور مع اللون البني / الرمادي. </li><li style=";text-align:right;direction:rtl"> تعليق علبتين من أربع طبقات الورق في قفص لزرع البيضة. بعد 2 أيام، فإن الإناث البالغات وضع البيض على حافة بين الأوراق. </li><li style=";text-align:right;direction:rtl"> مرتين في الأسبوع، ضع البيضة حزم ورقة في صناديق جديدة تربية البلاستيكية التي تحتوي على شبكات للسماح للهواء تعمم، التي تحتوي على حبوب اللقاح وشمع النحل. يفقس البيض في حوالي 3 أيام واليرقات الصغيرة بداية لتطوير طريق تغذية على الشمع وحبوب اللقاح. وبدلا من ذلك، يمكن وضع اليرقات على الغذاء الاصطناعي consistinغرام من مزيج العسل السائل من 500 غرام، 400 غرام الغليسرين، 100 غرام خميرة البيرة المجففة، 250 غرام دقيق القمح، و 200 غرام من مسحوق الحليب الخالي من الدسم و 400 غرام عصيدة من دقيق الذرة. لتوفير نسبة مناسبة من المواد الغذائية في يرقة، فصل بانتظام اليرقات في صناديق جديدة وتجديد الطعام كل يومين. </li><li style=";text-align:right;direction:rtl"> وتربى اليرقات خلال المراحل الست ملعب اليرقة. ويتميز كل ملعب من قبل انزلاق الكبسولة رئيس اليرقات. عندما تصل إلى مرحلة اليرقات قبل التشرنق مشاركة، بحيث يمنعه من التغذية والبدء في بناء شرنقة الحرير الخفيفة. </li><li style=";text-align:right;direction:rtl"> في شرنقتهن واقية، فإن يرقات تتحول إلى الراحة والشرانق. الديدان الشمع تبقى في مرحلة الخادرة لمدة أسبوع أو أسبوعين لنخرج الى العث الكبار. والعث الكبار لا تشرب ولا تأكل. يحدث التزاوج والديدان الشمع دورة &quot;الحياة تبدأ من جديد. </li><li style=";text-align:right;direction:rtl"> لتوحيد فحص الأمراض، واتخاذ اليرقات خلال المرحلة اليرقية الماضي. خلال هذه المرحلة بحيث يمنعه من التغذية، والانتقال إلى غطاء مربع تربية والبدء في إنتاج الحرير. هذا قالمئوية يستمر حوالي 5 أيام. </li></ol> 2. الحشرات اختيار للعدوى <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حدد مشاركة الطور اليرقات، والتي هي 2-3 سم طويلة و180-250 ملغ في الوزن، وعلى مدار 24 ساعة قبل العدوى ووضعها في علبة فارغة لتجويعهم. </li><li style=";text-align:right;direction:rtl"> إزالة شرنقة الحرير الوليدة حول اليرقات. </li></ol> 3. إعداد البكتيرية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد المعلقات البكتيرية العصوية للحصول على تركيزات النهائي تتراوح بين 10 وحدات مستعمرة تشكيل 4 (كفو) / مل إلى 10 8 وت م / مل للحقن داخل وhaemocoelic من 3x10 6 وت م / مل إلى 1x10 8 وت م / مل لابتلاع (كفو للحصول على وLD 50 يعتمد على الأنواع البكتيرية). في حالتنا، B. ويزرع في وسط الشمعية مرق لوريا Bertani-(LB، 10 غرام / لتر تريبتون، 5 خلاصة الخميرة ز / لتر، 10 غرام / لتر كلوريد الصوديوم) عند 37 درجة مئوية تحت الإثارة حتى دخول مرحلة ثابتة، المقابلة لانحناء داخلي للنمو المنحنى. قياس OD 600 نيوتن متر (بين 1 و 2 للB. الشمعية) واستخدامها لتقييم تركيز البكتيريا، وذلك باستخدام منحنى المعايرة التي سبق. خفف الحصاد البكتيريا بواسطة الطرد المركزي عند 5000 x ج لمدة 10 دقيقة في درجة حرارة الغرفة و resuspend في الفوسفات المالحة (PBS: 1M KH 2 PO 4، 1M K 2 HPO 4، كلوريد الصوديوم 5M، ودرجة الحموضة 7.2). سوف تتلقى كل يرقة 10 ميكرولتر من التعليق البكتيرية في التركيز المطلوب. </li><li style=";text-align:right;direction:rtl"> بدلا من ذلك، أن تكون مستعدة لتعليق بوغ العدوى. الحصول على جراثيم من البكتيريا زراعة في تبوغ HCT المتوسطة (5 ز / L تريبتون، 2 ز / L الكازين حلامة، 12.5 مم K 2 HPO 4، 12.5 مم MgSO 4، 0.05 ملم MnSO 4، 1.2 مم ZnSO 4، الحديد 1.2 مم 2 (4 SO) 3، 0.5٪ H 2 SO 4، 25 مم CaCl 2) 20 ° 30 C في مدة 3 أيام. الحصاد الجراثيم بواسطة الطرد المركزي (10،000 XG و 15 دقيقة)، ويغسل مرتين في الماء المقطر المعقم. إعادة تعليق على PEL بوغدعونا في الماء المقطر المعقم والحرارة لمدة 15 دقيقة في 78 ° C لإزالة البكتيريا الخضري المتبقية. أحصى والتعليق من الطلاء التخفيفات المسلسل على لوحات أجار LB. سوف تتلقى كل يرقة 10 ميكرولتر من التعليق بوغ في التركيز المطلوب. </li></ol> 4. البكاء إعداد السمية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد السم Cry1C من B. سلالة ثورينجينسيس 407 تتحول مع ال 21 pHTF31C بلازميد يحمل الجين الترميز Cry1C. ثقافة السلالة في 100 مل المتوسطة HCT في 30 ° C لمدة 72 ساعة مع المضادات الحيوية (الاريثروميسين 10mg/ml) للسماح تبوغ كامل، إنتاج السم الكريستال والتحرر في طاف الثقافة. </li><li style=";text-align:right;direction:rtl"> تنقية البلورات من طاف على التدرج 72٪ -79٪ سكروز. إضافة أول 17 مل من السكروز 79٪ في أنبوب 40 مل. 17 طبقة بعناية مل من السكروز 72٪ على رأس السكروز 79٪. وأخيرا، طبقة 5-6 مل من ثقافة 10X sporulated المركزة وإغلاق الأنبوب. أجهزة الطرد المركزي لمدة 14 ساعة أنبوب في 20،000 XG، 4 ° C، لفصل البلورات من الجراثيم. جمع بلورات في واجهة التدرج. إعادة تعليق بيليه الكريستال في 25 مل من الماء المعقم البارد لغسل عليه. أجهزة الطرد المركزي في 8000 x ج لمدة 20 دقيقة. ويتكرر ثلاث مرات هذه الخطوة لإزالة كافة السكروز التمسك البلورات. إعادة تعليق بلورات الماء في 5 مل العقيمة ومراقبة تحت المجهر لتقييم النقاء. </li><li style=";text-align:right;direction:rtl"> تقييم السم تركيز البروتين من تلطيخ برادفورد الكلاسيكية على حل الكريستال presolubilized في هيدروكسيد الصوديوم 50 مم. ويمكن تخزين بلورات السم في -20 درجة مئوية حتى الاستخدام. </li></ol> 5. حاقن إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> للسيطرة على حجم الحقن الدقيق، واستخدام مضخة محقنة الآلي (مثل KD العلمية KDS 100) مع سرعة الإعداد إلى 1 ميكرولتر / ثانية (الشكل 1A). </li><li style=";text-align:right;direction:rtl"> ملء حقنة 1 مل تحت الجلد مع 300 ميكرولتر المياه أو الحل البكتيرية (1 حقنة في حالة) للإصابة من 20-25 اليرقات. إزالة الفقاعات التي كتبها تا بعنايةpping الحقنة، ثم نعلق 0،45 X 12 ملم إبرة الحقنة إلى. </li><li style=";text-align:right;direction:rtl"> وضع الحقنة في حاقن. </li><li style=";text-align:right;direction:rtl"> إجراء حقن فارغة من 10 ميكرولتر في أنبوب فارغ للسيطرة على حجم حقن. </li></ol> 6. Intrahaemocoelic حقن الحشرات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع الحشرات يدويا بين الإبهام والسبابة. ثم أدخل إبرة مثبتة في حاقن في الجلد اليرقة (بشرة) (الشكل 1C). </li><li style=";text-align:right;direction:rtl"> الحفاظ على الحشرات في المكان في حين حقن ال 10 ميكرولتر حل البكتيرية (بكتيريا بوغ أو الخضري). </li><li style=";text-align:right;direction:rtl"> إزالة بعناية الحشرة من الإبرة. </li><li style=";text-align:right;direction:rtl"> وضع الحشرات المصابة في طبق بتري صغيرة (5 سم في القطر)، 5 يرقات لكل طبق. </li><li style=";text-align:right;direction:rtl"> تصيب اليرقات لا يقل عن 20 حالة لكل التجريبية. </li><li style=";text-align:right;direction:rtl"> ضع الأطباق في C ° 37 في حاضنة لمدة 48 ساعة. </li></ol> 7. الحشرات التغذية الإجبارية (الابتلاع) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في أنبوب إيبندورف 2 مل، مزيج 0،2 ميكروغرام / م وش؛ لتر من السم Cry1C مع أي تعليق الخلية بوغ أو الخضري للحصول على تركيزات تتراوح بين 3x10 النهائي 4 إلى 7 في 1x10 10 ميكرولتر. </li><li style=";text-align:right;direction:rtl"> ملء حقنة 1 مل مع 30G، 25 مم إبرة تحت الجلد مع 300 ميكرولتر من الحل السم البكتيري-Cry1C لإصابة اليرقات من 20-25. إزالة الفقاعات من الحقنة. </li><li style=";text-align:right;direction:rtl"> وضع الحشرات يدويا بحيث فمه يدخل إبرة مثبتة في حاقن (1D الشكل)، والتغذية القسرية مع 10 ميكرولتر من الحل البكتيرية باستخدام مضخة محقنة الآلي. </li><li style=";text-align:right;direction:rtl"> تصيب اليرقات لا يقل عن 20 حالة لكل التجريبية. </li><li style=";text-align:right;direction:rtl"> وضع الحشرات المصابة في صغير 5 سم طبق بتري (5 يرقات لكل طبق). ضع الأطباق في C ° 37 في حاضنة لمدة 48 ساعة. </li></ol> 8. تسجيل وفيات الحشرات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد تجارب التغذية القسرية أو الحقن، والحفاظ على اليرقات في طبق بتري دون طعام عند درجة حرارة 25 مئوية أو 37 درجة مئوية. تحقق اليرقات موrtality بانتظام على مدى فترة 48 ساعة. اليرقات الميتة هي خاملة وعادة ما تتحول الأسود (1B الشكل). </li><li style=";text-align:right;direction:rtl"> ويمكن تحليل البيانات باستخدام برنامج فيات سجل الاحتمالية-14. هذا البرنامج باختبار الخطي من المنحنيات وفيات الجرعة والجرعة القاتلة يوفر (LD 50). وبدلا من ذلك، يمكن استخدام برامج أخرى، مثل بريزم (الرسم البياني لوحة)، وتحليل البيانات وفيات أو البقاء على قيد الحياة. </li></ol>

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O., Ribier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+transduction+in+Bacillus+thuringiensis+var.+berliner+1715,+using+bacteriophage+CP54.">Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54.</a> Ber. J. Gen. Microbiol. 121, 203-212 (1980).</li><li>Sanchis, V., Agaisse, H., Chaufaux, J., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+new+insecticidal+Bacillus+thuringiensis+recombinant+strains+by+using+the+sporulation+non-dependent+expression+system+of+cryIIIA+and+a+site+specific+recombination+vector.">Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.</a> J. Biotechnol. 48, 81-96 (1996).</li><li>Tran, S. L., Guillemet, E., Gohar, M., Lereclus, D., Ramarao, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CwpFM+(EntFM)+is+a+Bacillus+cereus+potential+cell+wall+peptidase+implicated+in+adhesion,+biofilm+formation+and+virulence.">CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation and virulence.</a> J. Bacteriol. 192, 2638-2642 (2010).</li><li>Tran, S. L., 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=Hemolysin+II+is+a+Bacillus+cereus+virulence+factor+that+induces+apoptosis+of+macrophages.">Hemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.</a> Cell Microbiol. 13, 92-108 (2011).</li><li>Fedhila, S., 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=Comparative+analysis+of+the+virulence+of+invertebrate+and+mammalian+pathogenic+bacteria+in+the+oral+insect+infection+model+Galleria+mellonella.">Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella.</a> J. Invertebr. Pathol. 103, 24-29 (2010).</li><li>Daou, N., 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=IlsA,+a+unique+surface+protein+of+Bacillus+cereus+required+for+iron+acquisition+from+heme,+hemoglobin+and+ferritin.">IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin.</a> PLoS Pathog. 5, e1000675 (2009).</li><li>Mason, K. L., 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=From+commensal+to+pathogen:+translocation+of+Enterococcus+faecalis+from+the+midgut+to+the+hemocoel+of+Manduca+sexta.">From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta.</a> MBio. 2, e00065-00011 (2011).</li><li>Goldsmith, M. R., Shimada, T., Abe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+genomics+of+the+silkworm,+Bombyx+mori.">The genetics and genomics of the silkworm, Bombyx mori.</a> Annu. Rev. Entomol. 50, 71-100 (2005).</li><li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+transgenesis:+current+applications+and+future+prospects.">Insect transgenesis: current applications and future prospects.</a> Annu. Rev. Entomol. 57, 267-289 (2012).</li></ol>

الإعلان عن أي تضارب في المصالح.

استخدام الحشرات وخاصة مرحلة اليرقات، ونماذج عدة أنواع العدوى، أصبحت متكررة. نموذج المفضل لبعض الجوانب غير ذبابة الفاكهة (ذبابة النموذج) تستخدم كل من البالغين واليرقات 1،2 المرحلة. الحشرة lepidopteran G. كما تم mellonella تستخدم أساسا لمعايرة الفوعة البكتيري...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> الشمع وحبوب اللقاح </td><td> لا كشكش لملابس النساء Roanaise </td><td> 303000 </td><td> أي منتج العسل </td></tr><tr><td> حقنة الآلي مضخة </td><td> KD العلمية </td><td> KDS 100 </td><td></td></tr><tr><td> حقنة 1 مل </td><td> تيرومو </td><td> BS 01T </td><td></td></tr><tr><td> إبرة 0،45 × 12 مم </td><td> تيرومو </td><td> NN 2613R </td><td></td></tr><tr><td> طبق بتري 5 سم </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> 30G إبرة، 25 مم تحت الجلد </td><td> Burkard فيبكو المحدودة </td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

haemocoelic الحقن داخل البكتيريا إلى G. وقد ثبت mellonella مفيدة جدا لتحديد عوامل الفوعة التعامل مع العديد من تلف الأنسجة والمقاومة لعوامل المناعة الفطرية من مسببات الأمراض البشرية عدة. على سبيل المثال، يمثل الرقم 2A فيات الحشرات بعد حقن جرعات مختلفة من B.</em...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

haemocolic العدوى عن طريق الفم وداخل يرقات العث من الشمع أكبر<em&gt; جاليريا mellonella</emيوصف&gt;. ويمكن استخدام هذه الحشرة لدراسة العوامل الممرضة للحشرات من الفوعة وكذلك البكتيريا الانتهازية الثدييات. تربية الحشرات، وطرق العدوى والأمثلة على<em&gt; في الجسم الحي</emموصوفة&gt; التحليل.

والحشرات<em&gt; جاليريا mellonella</em&gt; نموذجا عدوى قوية المعنية بالتحقيق في إمراض البكتيرية

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.6K Views.  INRA, Micalis UMR1319, France. haemocolic العدوى عن طريق الفم وداخل يرقات العث من الشمع أكبر جاليريا mellonella. ويمكن استخدام هذه الحشرة لدراسة العوامل الممرضة للحشرات من الفوعة وكذلك البكتيريا الانتهازية الثدييات. تربية الحشرات، وطرق العدوى والأمثلة على في الجسم الحي الت...

Video: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

تشكيل بيوفيلم في المختبر في غرفة الشرائح 8 جيد

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Luciferase به لالتهابات جرثومية في صورة الفئران

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

اللعاب، الغدد اللعابية، وجمع اللمف الدموي من القراد اللبود الكتفي

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

A الفحص المرئي لمراقبة T6SS بوساطة المسابقة الجرثومي

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

استخدام<em&gt; غاليريا mellonella</em&gt; كما كائن نموذج لدراسة<em&gt; البكتيريا المستروحة</em&gt; عدوى

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

التصوير InlC إفراز المعنية بالتحقيق في العدوى البكتيرية الخلوية من قبل الممرض<em&gt; المستوحدة الليستيريا</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

الزرد نموذجا لتقييم إمكانات المسخية من النطرون

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

أن مدغشقر الهسهسة الصرصور كنموذج بديل غير الثدييات حيوانية للتحقيق في الفوعة، والمرضية، وفعالية المخدرات

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

نموذج السابقين فيفو الابتدائي بورسال خلية ثقافة دجاج لدراسة الأمراض المعدية بورسال الفيروسات المرضية

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...