وأيد هذا البحث من قبل الولايات المتحدة المنح العامة للخدمات الصحية وAI69233 AI073843 من المعهد الوطني للحساسية والأمراض المعدية. EPS هو زميل ويلكوم بوروز في التسبب في الأمراض المعدية. وقد تم تمويل KPH من قبل الأحياء الدقيقة الجزيئية الخلوية والتدريب منحة البرنامج 5 T32 A107611-10.

1. تنقية الهيموجلوبين من الدم الطازج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصول على الدم البشري الطازج تستكمل مع مضاد للتخثر. إبقاء الدم على الجليد أو عند 4 ° C خلال تنقية. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في الدم لمدة 20 دقيقة في ز X 1،500. وخلايا الدم الحمراء (كرات الدم الحمراء) تكون في الجزء السفلي من الأنبوب. نضح بعناية طاف و resuspend بيليه بلطف في الجليد الباردة محلول كلوريد الصوديوم 0.9٪ (W / V). تكرار الطرد المركزي ويغسل 3 مرات. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق بيليه في 1 حجم الجليد الباردة ملي تريس، حمض الهيدروكلوريك 10 (الرقم الهيدروجيني 8.0). وهذا حمل تحلل كرات الدم الحمراء من المقرر أن الضغط التناضحي. إضافة إلى حجم التولوين ~ النهائي 20٪. </li><li style=";text-align:right;direction:rtl"> احتضان في C ° 4 على المشواة ليلة وضحاها. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في 20000 XG المحللة في ل1 ساعة. جمع الكسر حلالة دموية الأوسط مغادرة يمسها التولوين (العائمة في الأعلى) وبيليه. استخدام ماصة طويلة الرقبة لجمع الكسر الأوسط. </li><li style=";text-align:right;direction:rtl"> تمر عبر فلتر 0،44 ميكرون حقنة. إذا الحل يحتوي على جسيماتلا يمكن أن تنتقل المسألة وعبر المرشح، كرر الخطوة 1.5. </li><li style=";text-align:right;direction:rtl"> تنقية الهيموجلوبين (خضاب الدم) مع اللوني السائل عالي الأداء (HPLC) العمود تبادل أنيون (فاريان، PL-1000 SAX A 8 ميكرومتر، 150 مم × 4.6 ملم). الطور المتحرك (أ) هو 10 ملي تريس، حمض الهيدروكلوريك (درجة الحموضة 8.0) والمتنقلة B المرحلة هو 10 ملي تريس، حمض الهيدروكلوريك (درجة الحموضة 8.0) + 0.5 M كلوريد الصوديوم. يتم تشغيل A٪ -100 0٪ من الانحدار B المذيبات أكثر من 2 دقيقة بمعدل 2،0 تدفق مل / دقيقة. يتم مراقبة شطف يعتمد على امتصاص (λ: 410 نانومتر نانومتر و280). جمع فقط جزء يتميز بلون أحمر فاتح وذروة امتصاص بارزة (الشكل 1). </li><li style=";text-align:right;direction:rtl"> Dialyze وشطف ضد الفوسفات مخزنة المالحة (PBS) بين عشية وضحاها ومن ثم لبضع ساعات مرة أخرى. تعقيم عن طريق تمرير من خلال مرشح ميكرون حقنة 0.22. </li><li style=";text-align:right;direction:rtl"> لقياس الهيموغلوبين التركيز، وإعداد الحلول القياسية هب تركيزات معروفة في برنامج تلفزيوني. تحديد تركيز الهيموغلوبين من في العينة عن طريق خلط الحلول القياسية (انظر الجدول مع الكواشف لناإد) أو محلول العينة مع كاشف لل2X Drabkin (المحضرة من مسحوق) بنسبة 1:1. على سبيل المثال، مزيج 100 ميكرولتر حل هب مع 100 ميكرولتر كاشف 2X Drabkin في 96-وحات جيدة. احتضان لمدة 15 دقيقة وقياس الامتصاصية في 540 نيوتن متر. رسم منحنى القياسية وتحديد تركيز الهيموغلوبين في العينة. خمسة إلى خمسة عشر ملغ / مل غلة هي نموذجية. </li><li style=";text-align:right;direction:rtl"> تشغيل ميكروغرام 15-20 تنقية الهيموجلوبين على صفحة SDS 15٪ من نسختين. وصمة عار واحدة من المواد الهلامية، ونقل البروتينات من الهلام إلى آخر غشاء النيتروسليلوز وطخة مناعية لخضاب الدم (الشكل 2). </li><li style=";text-align:right;direction:rtl"> تجميد وتخزين مليلتر مأخوذة 1 من الهيموغلوبين في النيتروجين السائل. </li></ol> 2. وسائط النمو إعداد الحديد تستنفد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد معهد روزويل بارك التذكاري (RPMI) مرق عن طريق إذابة مسحوق RPMI في الماء، إضافة بيكربونات الصوديوم على النحو الموصى به من قبل الشركة المصنعة و1٪ الأحماض cassamino (CA) (W / V). تعقيم عن طريق تمرير من خلال مرشح ميكرون 0.2 و refrig مخزنمتسارع. </li><li style=";text-align:right;direction:rtl"> إعداد المعادن المنضب RPMI (NRPMI) وذلك بإضافة 7٪ (W / V) Chelex 100 و خلط بين عشية وضحاها على لوحة ضجة. إزالة Chelex 100 من يمر من خلال مرشح 0.2 ميكرومتر وتخزين مبردة. تكملة وسائل الاعلام مع المعادن غير الحديدية الأساسية: 25 ميكرومتر ZnCl 2 و 25 ميكرومتر MnCl 2، 100 ملم CaCl 2 و 1 مم MgCl 2 التي أعدت مسبقا ومحلول معقم X 1،000. استخدام الحاويات البلاستيكية لهذه الخطوة لتجنب تلوث الحديد من إعادة استخدامها الإمدادات. </li></ol> 3. النمو المكورات العنقودية الذهبية باستخدام الهيموجلوبين كمصدر وحيد للحديد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خط S. لعزل المكورات على أجار الصويا تريبسيني (TSA) من الأسهم المجمدة. احتضان في C ° 37 للفترة 20-24 ساعة. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق الإيثيلنديامين-N، N&#39;-مكررا (2-hydroxyphenylacetic حمض) (EDDHA) في الإيثانول اللامائي الى 100 ملم. EDDHA لا يذهب إلى حل ولكن يتم تعقيم بواسطة الايثانول. </li><li style=";text-align:right;direction:rtl"> إضافة إلى EDDHA RPMI إلى تركيز النهائي من 0.5 مم. السماحEDDHA حل ما لا يقل عن 30 دقيقة قبل الانتقال إلى الخطوة التالية. بسبب دفعة إلى دفعة الاختلاف، قد النهائي EDDHA تحتاج إلى تركيز خفضت إلى 0.25 ملم للسماح نمو البكتيريا. </li><li style=";text-align:right;direction:rtl"> تطعيم واحدة من المستعمرات S. المكورات في 5 مل من RPMI تحتوي على أنابيب EDDHA في 15 مل المسمار غطاء مخروطي. احتضان في C ° 37 مع اهتزاز عند 180 التناوب في الدقيقة (RPM) ل16-20 ساعة. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي ليلة وضحاها الثقافات لمدة 5 دقائق في 7500 XG و resuspend بيليه في NRPMI المحتوي على 0.5 ملي EDDHA. تطبيع OD 600 إلى ~ 3. </li><li style=";text-align:right;direction:rtl"> إعداد NRPMI تحتوي على 2.5 ميكروغرام لكل مل الهيموغلوبين و0،1-1،0 ملم EDDHA. بسبب الاختلاف بين دفعات، قد EDDHA تركيز الحديد المطلوب لكلاب الحرة في NRPMI تختلف. </li><li style=";text-align:right;direction:rtl"> ثقافة فرعية 10 ميكرولتر من التعليق البكتيرية من الخطوة 3.5 في 1 مل + هب + NRPMI EDDHA في أنبوب 15 مل المسمار غطاء مخروطي. </li><li style=";text-align:right;direction:rtl"> احتضان الثقافات في C ° 37 لمدة تصل إلى 48 ساعة مع اهتزاز عند 180 دورة في الدقيقة أو على طبلة المتداول. </li><lط&gt; كل ساعة 6-12 اتخاذ OD 600 قراءات عن طريق إزالة 50 ميكرولتر من الثقافة وخلطها مع 150 ميكرولتر من برنامج تلفزيوني في 96 لوحات جيدة. </li></ol>

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ليس لدينا شيء في الكشف عنها.

الحديد من المغذيات الأساسية اللازمة للكائنات من جميع ممالك الحياة 15. في الفقاريات، وعزلها الحديد لتجنب السمية الناجمة عن هذا العنصر. هذا تنحية يخفي أيضا من غزو الميكروبات الحديد في عملية تعرف باسم الحصانة الغذائية 16. وردا على ذلك تطورت مسببات الأمراض الا...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف / المواد </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> HPLC عمود أنيون تبادل </td><td> فاريان </td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkin كاشف </td><td> سيجما </td><td> D5941-6VL </td><td></td></tr><tr><td> الهيموغلوبين القياسية </td><td> بوانت العلمية </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex شكل الصوديوم 100 </td><td> سيجما </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC معايير محدودة </td><td> ANC 001 </td><td></td></tr><tr><td> الهيموجلوبين الأجسام المضادة </td><td> سانتا كروز التكنولوجيا الحيوية، وشركة </td><td> SC-21005 </td><td> &lt;/ TD&gt; </tr><tr><td> تريبسيني الصويا أجار </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. المذهبة هي نوع من البكتيريا المسببة للأمراض التي تتطلب الحديد لتنفيذ وظائف الأيض الحيوية وتسبب المرض. الخزان الأكثر وفرة من الحديد داخل الإنسان هو المضيف الهيم، والذي هو العامل المساعد من الهيموغلوبين. للحصول على الحديد من الهيموجلوبين، S. المكورات يستخدم نظام محكم المعروفة باسم المحدد سطح الحديد التنظيم (ISD) نظام 1. مكونات أول نظام ISD ربط الهيموغلوبين المضيف، ثم استخراج واستيراد الهيم، وأخيرا تحرير الحديد من الهيم في السيتوبلازم البكتيري 2،3. وقد تم تشريح هذا المسار من خلال العديد من الدراسات في المختبر 4-9. علاوة على ذلك، كانت مساهمة نظام ISD للإصابة أظهرت مرارا وتكرارا في نماذج الماوس 8،10-14. إنشاء مساهمة منظومة ISD إلى الهيموغلوبين المستمدة من اقتناء الحديد والنمو وقد ثبت أن تكون أكثر تحديا. معقدة المقايسات النمو باستخدام الهيموجلوبين كمصدر وحيد الحديد بY عدم استقرار الهيموجلوبين المتاحة تجاريا، وتلوث الحديد حرة في المتوسط ​​النمو، وسمية المرتبطة chelators الحديد. هنا نقدم طريقة التي يتغلب هذه القيود. مستعدة عالية الجودة من الهيموغلوبين في الدم الطازجة ويتم تخزينها في النيتروجين السائل. وتستكمل الهيموغلوبين في تنقية الحديد تستنفد المتوسطة محاكاة البيئة الحديد للفقراء التي تواجهها مسببات الأمراض داخل المضيف الفقاريات. عن طريق تجويع S. المكورات من الحديد حرة والمكمل مع نموذج التلاعب الحد الأدنى من الهيموغلوبين نحن لحث على النمو بطريقة تعتمد اعتمادا كليا على القدرة على ربط الهيموغلوبين، واستخراج الهيم، من خلال تمرير الهيم المغلف الخلية البكتيرية وتتحلل الهيم في السيتوبلازم. وسوف يكون هذا الاختبار مفيدا للباحثين تسعى إلى إلقاء الضوء على آليات اكتساب الحديد في hemoglobin-/heme-derived S. المكورات وربما غيرها من مسببات الأمراض البكتيرية.

لدينا تنقية الهيموجلوبين الإنسان من حلالة دموية مع HPLC (البروتوكول خطوة 1.7). ويظهر الشكل 1 سجلت الامتصاصية من شطافة في موجات نانومتر 280 و 410. كان جزء 5 جمعها والتخلص منها وكسور أخرى. عادة يتم الحصول على غلة 5-15 ملليغرام من الهيموغلوبين في الملليمتر الواحد من شطافة...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

نحن هنا وصف لفحص النمو<em&gt; المكورات العنقودية الذهبية</em&gt; باستخدام الهيموجلوبين كمصدر وحيد للحديد المغذيات المتاحة. هذا الاختبار يحدد دور العوامل البكتيرية المشاركة في الهيموغلوبين المستمدة من اكتساب الحديد.

المكورات العنقودية الذهبية النمو باستخدام الهيموجلوبين الإنسان باعتبارها المصدر الحديد

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K Views.  Vanderbilt University Medical School. نحن هنا وصف لفحص النمو المكورات العنقودية الذهبية باستخدام الهيموجلوبين كمصدر وحيد للحديد المغذيات المتاحة. هذا الاختبار يحدد دور العوامل البكتيرية المشاركة في الهيموغلوبين المستمدة من اكتساب الحديد.

Video: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

استخدام الري بالتنقيط وتدفق مفاعلات الأقراص الدوارة ل المكورات العنقودية الذهبية التحليل بيوفيلم

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

تحت الجلد من العدوى المكورات العنقودية المذهبة المقاومة للميثيسيلين (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

طريقة فعالة من حيث التكلفة لتعقب مصدر الميكروبات عن طريق محددة الفيروسات البشرية والحيوانية

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

نموذج تجريبي من التهاب الشغاف مقاومة مثسلين<em&gt; المكورات العنقودية الذهبية</em&gt; (MRSA) في الجرذ

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

دراسة تفاعلات<em&gt; المكورات العنقودية الذهبية</em&gt; مع العدلات بواسطة التدفق الخلوي والوقت الفاصل بين المجهري

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

استهداف بيوفيلم أسوشيتد<em&gt; المكورات العنقودية الذهبية</em&gt; استخدام ريسازورين استنادا الفحص المخدرات-القابلية

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

التنميط الجيني ل<em&gt; المكورات العنقودية الذهبية</em&gt; من جانب خاص بالريبوسوم فاصل PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

منهجية لدراسة نقل أفقي جين في<em&gt; المكورات العنقودية الذهبية</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

نموذج الماوس لتقييم الفطرية الاستجابة المناعية للعدوى المكوّرات العنقودية الذهبية

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

تحديد كميه السمية الخلوية للسبحيه الذهبية ضد الكريات البيضاء البشرية الاحاديه النواة

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...