وأيد هذا العمل من قبل العظم Profileringsfonds (PF245).

<ol>
	<li>Wallet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+clinical+study+using+a+multiplex+real-time+PCR+test+for+the+detection+of+bacterial+and+fungal+DNA+directly+in+blood.">Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood.</a> Clin. Microbiol. Infect. 16, 774 (2010).</li><li>Beekmann, S. E., Diekema, D. J., Chapin, K. C., Doern, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+rapid+detection+of+bloodstream+infections+on+length+of+hospitalization+and+hospital+charges.">Effects of rapid detection of bloodstream infections on length of hospitalization and hospital charges.</a> J. Clin. Microbiol. 41, 3119 (2003).</li><li>Raymaekers, M., Bakkus, M., Boone, E., de Rijke, B., Housni, H. E. l., Descheemaeker, P., De Schouwer, P., Franke, S., Hillen, F., Nollet, F., Soetens, O., Vankeerberghen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diagnostics+working+group.+Reflections+and+proposals+to+assure+quality+in+molecular+diagnostics.">Molecular Diagnostics working group. Reflections and proposals to assure quality in molecular diagnostics.</a> Acta. Clin. Belg. 66, 33 (2011).</li><li>Peters, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+the+diagnosis+of+bloodstream+infections.">New developments in the diagnosis of bloodstream infections.</a> Lancet Infect. Dis. 4, 751 (2004).</li><li>Hansen, W. L., Beuving, J., Bruggeman, C. A., Wolffs, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+probes+for+the+diagnosis+of+clinically+relevant+bacterial+infections+in+blood+cultures.">Molecular probes for the diagnosis of clinically relevant bacterial infections in blood cultures.</a> J. Clin. Microbiol. 48, 4432-4432 (2010).</li><li>Beuving, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+susceptibility+testing+of+grown+blood+cultures+by+combining+culture+and+real-time+polymerase+chain+reaction+is+rapid+and+effective.">Antibiotic susceptibility testing of grown blood cultures by combining culture and real-time polymerase chain reaction is rapid and effective.</a> PLoS ONE. 6, (2011).</li><li>Rolain, J. M., Mallet, M. N., Fournier, P. E., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+for+universal+antibiotic+susceptibility+testing.">Real-time PCR for universal antibiotic susceptibility testing.</a> J. Antimicrob. Chemother. 54, 538 (2004).</li><li>Nadkarni, M. A., Martin, F. E., Jacques, N. A., Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+bacterial+load+by+real-time+PCR+using+a+broad-range+(universal)+probe+and+primers+set.">Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.</a> Microbiol. 148, 257 (2002).</li><li>Waites, K. B., Brookings, E. S., Moser, S. A., Zimmer, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+susceptibility+testing+with+positive+BacT/Alert+blood+cultures+by+using+MicroScan+overnight+and+rapid+panels.">Direct susceptibility testing with positive BacT/Alert blood cultures by using MicroScan overnight and rapid panels.</a> J. Clin. Microbiol. 36, 2052 ( ).</li><li>Vliegen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+by+real-time+amplification+and+sequencing+of+the+16S+rRNA+gene.">Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene.</a> J. Microbiol. Meth. 66, 156 (2006).</li><li>Hall, L., Doerr, K. A., Wohlfiel, S. L., Roberts, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+MicroSeq+system+for+identification+of+mycobacteria+by+16S+ribosomal+DNA+sequencing+and+its+integration+into+a+routine+clinical+mycobacteriology+laboratory.">Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory.</a> J. Clin. Microbiol. 41, 1447 (2003).</li></ol>

ليس لدينا ما يكشف.

البروتوكول وصفها هنا يمكن التعرف السريع على مسببات الأمراض، ويقدم لمحة فنية المضادة للجراثيم التي يمكن أن تؤدي إلى الإدارة في وقت مبكر من المضادات الحيوية الكافية وبالتالي تحسين تشخيص المرضى الذين يعانون من التهابات مجرى الدم. تبعا للظروف وطلبت من الاختبار، أي منخفضة التكلفة، وإنتاجية عالية، الحد الأدنى من فترة التسليم، ويمكن تعديل ظروف الاختبار. لا يمكن أن يؤديها الإجراء بأكمله في غضون يوم عمل واحد. وعلاوة على ذلك، يمكن تنفيذ جزأين من البروتوكول في وقت واحد، مما يقلل من وقت دورانها بشكل ملحوظ. كما وردت هنا، لوحة الهوية هي مجموعة من البكتيريا الأكثر صلة سريريا في المستشفى. منذ المبدأ الرئيسي هو استهداف المنطقة الجينات 16S، يمكن تصميم تحقيقات محددة لالكائنات الحية الدقيقة الأخرى، وأضيف إلى فحص. كان من المفترض الفحص الكامل للتحليل السريع للثقافات الدم، ولكن يمكن أن تستخدم أيضا لمعالجة سمواد ذر عينة. هذا هو الحال أيضا بالنسبة للمضادات الحيوية التي تم استخدامها لاختبار الحساسية للمضادات الحيوية: يمكن إضافة المضادات الحيوية أو غيرها، استنادا إلى أنماط المقاومة المحلية والمبادئ التوجيهية.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> كلوريد الصوديوم (كلوريد الصوديوم) </td><td> ميرك للمواد الكيميائية </td><td> 106404 </td><td> 0.9٪ في الماء </td></tr><tr><td> Vacutainer فاصل المصل طائرة أسرع من الصوت الأنبوبة 5 مل </td><td> دينار بحريني أنظمة التشخيص </td><td> 367986 </td><td></td></tr><tr><td> مولر هينتون الثاني مرق </td><td> دينار بحريني تشخيص Dystems </td><td> 212322 </td><td> 44 جم / لتر في المياه </td></tr><tr><td> منبذة Rotixa 50 RS </td><td> أندرياس Hettich GMBH &amp; CO. </td><td> 4910 </td><td></td></tr><tr><td> منبذة 5415 D </td><td> إيبندورف </td><td> توقف </td><td></td></tr><tr><td> الاشعال </td> &lt;TD&gt; سيغما الدريخ </td><td> NA </td><td></td></tr><tr><td> تحقيقات </td><td> Sigma-Aldrich/Applied النظم البيولوجية </td><td> NA </td><td></td></tr><tr><td> TaqManEnvironmental سيد مزيج 2.0 </td><td> النظم البيولوجية التطبيقية </td><td> 4396838 </td><td></td></tr><tr><td> معدل الذكاء SYBRGreen Supermix </td><td> الحيوي راد مختبرات BV </td><td> 170-8880 </td><td></td></tr><tr><td> MicroAmp بصري 96-حسنا بلايت رد الفعل </td><td> النظم البيولوجية التطبيقية </td><td> N8010560 </td><td></td></tr><tr><td> MicroAmp فيلم لاصق بصري </td><td> النظم البيولوجية التطبيقية </td><td> 4311971 </td><td></td></tr><tr><td> معدل الذكاء 96-حسنا لوحات PCR </td><td> الحيوي راد مختبرات BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B لاصق الأختام </td><td> الحيوي راد العملatories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> في الوقت الحقيقي ونظام الكشف PCR </td><td> النظم البيولوجية التطبيقية </td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td> في الوقت الحقيقي ونظام الكشف PCR </td><td> الحيوي راد مختبرات BV </td><td> MyiQ أحادية اللون </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

وترتبط عدوى مجرى الدم مع ارتفاع معدلات الوفيات بسبب ظهور محتمل للتسمم، وتعفن الدم الشديد والصدمة الإنتانية 1. ولذلك، إدارة سريع من العلاج بالمضادات الحيوية الكافي يتسم بأهمية قبل كل شيء في علاج التهابات مجرى الدم. العنصر الحاسم في هذه العملية هو التوقيت، وتعتمد اعتمادا كبيرا على نتائج تحديد البكتيريا للمضادات الحيوية، واختبارات الحساسية. ويتم الحصول بصورة روتينية على حد سواء من هذه المعلمات من قبل ثقافة على أساس الاختبار، والتي تستغرق وقتا طويلا، ويأخذ في 24-48 ساعة في المتوسط ​​2 و 4. وكان الهدف من الدراسة لتطوير المعتمدة على الحمض النووي لتحديد هوية المقايسات السريع للالتهابات مجرى الدم، وكذلك إجراء اختبارات الحساسية للمضادات الميكروبات السريع. الفحص الأول هو 16S eubacterial rDNA القائم على فحص PCR في الوقت الحقيقي مع استكمال الأنواع أو تحقيقات جنس محدد 5. باستخدام هذه التحقيقات، الجراثيم سلبية الغرام بما في ذلك أنواع الزائفة، الزائفة الزنجاريةويمكن تمييزها القولونية، فضلا عن الجراثيم الإيجابية الغرام بما في ذلك النيابة العنقودية الذهبية.، المكورات العنقودية الذهبية، المعوية النيابة.، العقدية النيابة.، والعقدية الرئوية. باستخدام هذا الفحص multiprobe، أعطيت تحديد الأول من هذا الكائن الصغير المسبب للمرض بعد 2 ساعة. ثانيا، قمنا بتطوير فحص شبه الجزيئية لإجراء اختبارات الحساسية للمضادات الحيوية من س. المذهبة، المعوية النيابة. و (اختياري) الميكروب الهوائي سلبية الغرام قضبان 6. واستند هذا الاختبار على دراسة التي استخدمت PCR لقياس نمو البكتيريا 7. يتم تحضين البكتيريا حصادها مباشرة من الثقافات الدم لمدة 6 ساعات مع مجموعة مختارة من المضادات الحيوية، وبعد Sybr الأخضر القائم على فحص PCR في الوقت الحقيقي ويحدد تثبيط نمو. ويمكن الجمع بين هاتين الطريقتين توجيه اختيار العلاج المناسب من المضادات الحيوية في اليوم نفسه (الشكل 1). في الختاموالتحليل الجزيئي لكلا تحديد وقابلية للمضادات الحيوية تقدم أسرع بديلة للكشف عن العوامل المسببة للأمراض، ويمكن تحسين تشخيص العدوى مجرى الدم.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

تصميم خطة واضحة سير العمل ليوم واحد لتشخيص مسببات الأمراض البكتيرية تمكن من التعرف السريع للالتهابات مجرى الدم. إدراج الأهداف الثمانية البكتيرية ذات الصلة سريريا وملفاتهم الشخصية المقاومة للمضادات الحيوية يقدم الطبيب نظرة أولية في نفس اليوم، والتي يمكن أن تؤدي إلى مزيد من العلاج الملائم.

في يوم واحد خطة سير العمل للكشف عن مسببات الأمراض البكتيرية ومضادات الميكروبات اختبار المقاومة من زرع الدم

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

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Immunology and Infection

25.2K Views.  Maastricht University Medical Center. تصميم خطة واضحة سير العمل ليوم واحد لتشخيص مسببات الأمراض البكتيرية تمكن من التعرف السريع للالتهابات مجرى الدم. إدراج الأهداف الثمانية البكتيرية ذات الصلة سريريا وملفاتهم الشخصية المقاومة للمضادات الحيوية يقدم الطبيب نظرة أولية في نفس اليوم، والتي يمكن أن تؤ?...

Video: One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

بروتوكول لقاحات مرض السارس المؤتلف RBD المستندة : إعداد البروتين ، والكشف عن تطعيم الحيوانات تحييد

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

غزو ​​الخلايا البشرية من قبل الممرض الجرثومي

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

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 ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

الإنتاجية العالية لطريقة الكشف عن فيروس الإنفلونزا

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

Nanomechanics من المخدرات التفاعلات المستهدفة وكشف المقاومة للجراثيم

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

إعداد ثقافة بيليه الدم الجرثومي السريع لتحديد واختبار حساسية المضادات الحيوية

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

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 ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.