Bu araştırma, Ulusal Sağlık Enstitüleri Small Business Innovation Araştırma Programı (NIAID, 1R43AI082698-01) ve Gıda ve Tarım USDA Ulusal Enstitüsü (NIFA, 2009-33610-20028) tarafından desteklenmiştir.

1.. Y. pestis plaka aşılama <ol><li> Streak Y. pestis A1122 Luria-Bertani üzerine stok kültürleri (LB) agar (BeiResources # NR15) (Miller). Steril tekniği kullanın ve tüm Y. gerçekleştirmek sınıf II tipi bir biyogüvenlik kabini. Y. pestis manipülasyonlar pestis A1122 bir Biyogüvenlik Seviyesi (BGS) 2 hariç etken suşun seçin. Bu her ikisi de 75 kb çifti düşük kalsiyum plazmid yanıt (LCR) virülans ve virülans için gerekli olan pgm lokus yoksundur. Y. büyütün 28 pestis ° C, statik sıcaklık, hava inkübatör kontrol 48 saat. Y. &#39;nin büyüme hızı pestis 1.25 saat 9 nesil zaman yavaştır. </li></ol> 2.. Y. pestis sıvı medya aşılama <ol><li> Bir tek Y. transfer LB et suyu, kısır bir metal aşılama döngü kullanarak 2 ml içeren steril bir 17 x 100 mm kültür tüpünün içine pestis koloni . Boyutu tekdüze bir koloni seçin, diğer kolonilerin göstergesidir ve plaka kolonilerin geri kalanından açıkça ayrılır. Vortex kısaca tüp ve 28 kültür büyümeye ° C sallayarak inkübatör (225 rpm) yaklaşık 20 saat. </li></ol> 3.. Y. pestis akıbet, muhabir faj Ayrıca, ve bioluminescent algılama <ol><li> Gecede Y. sulandırınız pestis kültür 50 ml şahin tüp taze LB suyu (örneğin, 9.5 mL orta içine hücre 500 mcL) 1:20 ve 28 ° C (225 rpm) sallayarak ile büyüyecek. 0.2 600 nm (600) bir optik yoğunluk (yaklaşık 5 saat) ulaşılana kadar büyütün. Aktif büyüyen hücreleri bioluminescent yanıt ana bakterilerin canlılığı ve fitness ilişkilidir uyum sağlamak faj yeteneği beri tespiti için tercih edilir. Bununla birlikte, algılama sistemi, büyüme döngüsünün 7 tüm aşamalarında hasat hücreleri ile uyumlu olacak şekilde gösterilmiştir . </li><li> N% 2 dekana çözüm hazırlayarak bioluminescent reaksiyon için gerekli substrat oluşturun. LB et suyu 9.8 mL n-dekana 200 mcL karıştırın. 5 saniye süreyle kuvvetlice Vortex Başbakan, 2 mL% 2 n-dekana çözüm ile mikroplaka luminometre enjektör. Luminometre ve sonra her mikroplak dekana çözüm 67 mcL autoinject hemen 10 sn için örnek okumak için önceden ayarlanmış </li><li> 3 kültür tüp içine 1 mL alikotları LB suyu koyun. Her bir tüp muhabiri faj stok solüsyonu (5 x 10 9 plak oluşturan birim stok [PFU] / ml) 20 mcL ekleyin; faj ve medya tek başına örnekleri negatif kontrol olarak hizmet vermektedir. Y. &#39;nin yokluğunda pestis hücreleri, muhabir faj ek bir bioluminescent cevabı ortaya olmamalıdır. </li><li> Y. 1 mL alikotları Uygulama 6 kültür tüp içine pestis kültür (3.1 adım). Kültürleri (testi kültürler) 3 muhabir faj stokunun 20 mcL ekleyin. Kalan 3 kültür, hücreleri tek başına &#39;negatif kontrol (arka plan autobioluminescence) olarak hizmet vermektedir. Kısaca vorteks kültürlerin karıştırın ve her bir örnek hasat 200 mcL (0 zaman okumak için) ve beyaz 96-iyi bir mikrotiter plate içine dağıtmak. Kalan kültür 28 ° C&#39;de sallayarak (225 rpm). </li><li> Mikroplak luminometre kullanarak biyoparlaklık (bağıl ışık birimleri, RLU) için 0 örnekleri ölçün. </li><li> Hasat 200 10 saniye sonra her bir örnek mcL, 20, 30, 40, 50, ve 60 dakika sonrası muhabiri faj Ayrıca, biyoparlaklık için örnek ölçmek. Eğer Y. pestis hücreleri mevcuttur, muhabir faj, özellikle hücre bağlamak DNA&#39;sı enjekte ve luxAB muhabiri genleri (Şekil 1) yazıya çevirmek için ev sahipliği yapan, transkripsiyonel ve translasyonel makine kullanım olacak . </li><li> Sinyal gücü ve sinyal tepki süresi, hücre sayısı ile mevcut orantılıdır. 20 dakika içinde hücreler (10 5 -10 8 CFU / mL), kontrollere göre önemli bir artış RLU yüksek konsantrasyonlarda belirgin olmalıdır. Alt hücreye konsantrasyonlarda (10 2 -10 4 CFU / ml), RLU önemli bir artış, 40-60 dakika içinde belirgin olmalıdır . </li><li> Alternatif olarak, Y. için gerek kalmadan algılama sürecini hızlandırmak için pestis akıbet taze yetişen bir tabak, bir koloni doğrudan muhabiri faj barındıran LB suyu 200 mcL ile 1.5 ml Eppendorf tüp (vorteks) karışık olabilir. Mikrotiter plate bir kuyunun içine hücre / faj karışımı koyun. Mikrotiter plate 28 ° C&#39;de inkübe edin ve 60 dk sonra (sadece tek bir zaman noktasında) biyoparlaklık için örnek okuyun. </li></ol> 4. Temsilcisi sonuçları: Bir temsilci, muhabir faj ders deney Y. algılama aracılı pestis, Şekil 2&#39;de gösterilmiştir . 1 negatif kontroller) faj tek başına (hücreleri), ya da yalnız 2) hücreleri (yok faj), 60 dakika inc boyunca yaklaşık 20 RLU biyoparlaklık başlangıç ​​düzeyleriubation (taban seviyelere luminometre özgüdür). Buna karşın, test örnekleri (muhabiri faj ve hücreleri) biyoparlaklık bir artış faj eklenmesinden sonra 15 dakika açıktır. Sinyal gücü, 60 dakika boyunca sürekli artmalıdır. Inkübatörü, uzun süre süreler (80 dk), ana hücreler aracılı lizis faj nedeniyle zirve sinyali azalacak bir sinyal neden olur. ° C olsa Y. için optimum sıcaklık 37 inkübasyon sıcaklıkları benzer sonuçlar elde pestis büyüme 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Şekil 2"/> Şekil 2. Y. Faj aracılı bioluminescent algılama pestis. süresi 0, 28 ° C&#39;de inkübe muhabiri faj ve hücreler, karışık, ve bioluminesans (RLU) zaman içinde takip edildi. RLU önemli bir artış (* Öğrenciler t-testi, p &lt;0.05), 15 dakika içinde açıktır.

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	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

Bu yöntem, hızla Y. tespit muhabiri faj yeteneği gösterir muhabir faj beri pestis kültürlü Y. bioluminescent sinyal yanıt transduce pestis faj eklenmesinden sonra 20 dakika içinde hücreler. Muhabir faj da doğrudan Y. algılama yeteneğine sahiptir izolasyon ve sonraki ekimi 7 önkoşul olmadan klinik matrisler pestis. Genellikle 48 saat tamamlanması için gereken standart faj lizis deneyleri ile karşılaştırıldığında, önemli ölçüde b...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Difco LB agar, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB suyu, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm kültür tüpleri </td><td> ABD Bilimsel </td><td> 1485-0810 </td></tr><tr><td> n-dekana </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Veritas mikroplaka luminometre </td><td> Tuna Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite mikrotiter 96-plaka </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis ve Bacillus anthracis sırasıyla 1, veba ve şarbon etken bakteriyel patojenler Kategori. Her iki hastalık &#39;doğal olarak ortaya çıkan, nispeten nadir olmasına rağmen, bioweapon olarak bu patojenlerin kullanarak terörist grupların olasılığı gerçek. Hastalığın doğasında Bulaşıcılık, hızlı klinik seyri ve yüksek ölüm oranı nedeniyle bir salgın hızlı bir şekilde tespit edilmesi kritik öneme sahiptir. Bu nedenle hızlı algılama ve teşhis sağlayacak metodolojileri, halk sağlığı önlemlerinin ve kriz yönetimi aktivasyonu derhal uygulanmasını sağlamak için gerekli. Rekombinant muhabiri faj Y. &#39;nin tespiti için hızlı ve spesifik bir yaklaşım sağlayabilir pestis ve B. anthracis Hastalık Kontrol ve Önleme Merkezleri, şu anda bu bakteriyel patojenler 2-4 teyit tanımlanması için klasik faj lizis testleri kullanabilirsiniz. Bu deneyleri, doğal olarak bakteriyel bilgisayarlar için özel ve litik faj meydana gelen yararlanmak. Özel faj varlığında ekili bakterinin gecede büyüme sonra, plaklar oluşumunu (bakteriyel lizis) bakteri hedef olumlu bir tanımlama sağlar. Bu testlerin güçlü olmasına rağmen, üç eksiklikleri zarar: 1) laboratuvar bazlı, 2) şüpheli numune bakteri izolasyonu ve kültivasyonu gerektirir, ve 3) tamamlamak için 24-36 saat alır. Bu sorunları ele almak için, rekombinant &quot;ışık etiketli&quot; muhabiri faj genetik Vibrio harveyi luxAB genler Y. genom içine entegre ederek, mühendislik pestis ve B. anthracis özel faj 5-8. LuxAB muhabiri faj hızla kendi özel hedef tespit edebiliyoruz (dakika içinde) ve hassas alıcı hücrelere bioluminescent fenotip görüşmekte. Önemlisi, algılama ekili alıcı hücreleri ile veya sahte-enfekte klinik örneklerden 7 ya da elde edildi. Gösteri amaçlı, burada bilinen bir Y. faj aracılı tespiti için bir yöntem tarif pestis CDC veba teşhis faj ΦA1122 6,7 (Şekil 1) inşa luxAB muhabiri faj kullanılarak izole. Küçük değişiklikler (örneğin büyüme sıcaklık ve ortam değişikliği), B. tespiti için benzer bir yöntem kullanılıyor olabilir anthracis B. kullanarak izole anthracis muhabiri faj Wβ: luxAB 8. Yöntemi ekili Y. biolumescent fenotip faj aracılı transdüksiyon açıklar sonradan mikroplak luminometre kullanarak ölçülür pestis hücreleri. Bu yöntemin geleneksel faj lizis deneyleri üzerinde önemli avantajları, kolay kullanımı, hızlı sonuçlar ve 96-iyi mikrotiter plate formatı aynı anda birden fazla örneklerini test etmek için yeteneği. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Şekil 1"/> Şekil 1. Algılama şematik faj örneği ile karıştırılır, faj hücre, luxAB ifade edilir ve hücre bioluminesces bozar. Örnek işlem gerekli değildir; faj ve hücreler karıştırılır ve daha sonra ışık ölçülür.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Öncelikli bakteriyel patojenlerin tespiti için basit bir yöntem, genetiği muhabiri faj kullanmaktır. Kendi özel konak türe özgü Bu muhabir faj, konak hücreleri için bioluminescent sinyal yanıt hızla uyum sağlama yeteneğine sahiptir. Burada, biz tespiti için muhabir faj kullanımını tanımlamak<em&gt; Yersinia pestis</em&gt;.

Kategori Algılama Bir Bakteriyel Patojenler &#39;Bioluminescent&#39; Muhabir Faj

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Research

JoVE Journal

Immunology and Infection

13.4K Görüntüleme.  Guild Associates, Inc.. Öncelikli bakteriyel patojenlerin tespiti için basit bir yöntem, genetiği muhabiri faj kullanmaktır. Kendi özel konak türe özgü Bu muhabir faj, konak hücreleri için bioluminescent sinyal yanıt hızla uyum sağlama yeteneğine sahiptir. Burada, biz tespiti için muhabir faj kullanımını tanımlamak Yersinia pestis.

Video: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

İmmünoloji ve Enfeksiyon

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Hücre içi bakteriyel patojenler ile Zebra balığı Embryolarının Enfeksiyon

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNA gen Sıralama ve MALDI-TOF MS tarafından Nadir Bakteriyel patojenlerin tanımlanması

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nano PCR ile Hayvan Örnekler Solunum Patojenler yüksek verimlilik Algılama

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...