We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

NOT: 2 6206 bir avirulent suşu 17, 18 M. tuberculosis mc gibi, bu protokoldeki tüm çalışma Biyogüvenlik Seviye 2 tesis (BSL-2) yapılabilir. M. tuberculosis mc 2 ifade Yeşil Floresan Protein 1. Kültür koşulları 6206 (Mtb GFP) NOT: M. tuberculosis H37Rv auxotroph suşu 6206 mc 2 türetilmiş (Δ panCD, Δ leuCD) plazmid pMN437 ifade GFP bu protokol 16 boyunca kullanılır ile dönüştürdü. Olmayan GFP araştırmacılar için gerginlik daha kolay erişilebilirliği sağlamak için yabani tip gerginlik ifade ile Mtb GFP gerginlik yerine mümkündür. Bununla birlikte, GFP tanımı fagositozu ve Mtb / makrofaj agregaların oluşumunun görsel onay sağlamak için arzu edilir. long süreli depolama, Mtb GFP% 20 gliserol ile desteklenmiş (adım 1.1 tarif edilmiştir) eksiksiz 7H9 ortamında -80 ° C&#39;de dondurulmuştur. <ol><li> % 10 Middlebrook OADC,% 0.02 tiloksapol 24 ug / mL D-pantotenik asit, 50 ug / ml L-lösin ve 50 ug / ml Higromisin B ile 7H9 ortamında ilave tamamlandığını Mtb GFP ortamı (7H9-C) hazırlanması </li><li> Mtb GFP bir şişe çözülme ve 30 ml kare alt PETG şişede 7H9-C 10 mL ekle. </li><li> Döner bir çalkalayıcı (50 rpm) ile 37 ° C&#39;de inkübe edin. Optik yoğunluk ölçümleri (OD 600) birkaç günde OD 600 1.0 (yaklaşık 5-8 gün) ulaşıncaya kadar atın. </li><li> Bir OD 0.2-1.0 arasında 600 korumak için gerektiği gibi sulandırmak ve geçit kültürü. </li><li> Enfeksiyon için, logaritmik büyüme fazı (OD 0.6-1 600) içinde Mtb GFP kurulmuş bir kültür kullanın. Clumpy kültürleri önlemek için, bir su banyosu içinde Mtb kültür şişesi sonikasyon (130 W; 3 x 5 s darbe) ONCE veya haftada iki kez </li></ol> 2. Kültür Koşulları THP-1 hücreleri <ol><li> % 10 ısı ile etkisiz hale getirilen fötal sığır serumu, 2 mM L-glutamin ve 10 mM HEPES ile RPMI 1640 ortamı ilave etmek suretiyle tamamlandı, THP-1 hücre ortamı (RPMI-C) hazırlayın. </li><li> % 5 CO2 bulunan nemli bir atmosferde 37 ° C&#39;de bir T-75 balonuna hücreleri inkübe edin. </li><li> Bir hemasitometre kullanarak hücreleri sayın veya gün aşırı akış sitometrisi ve 0.1 mL başına milyon 0.6 arasında bir yoğunlukta THP-1 hücreleri korumak. </li></ol> 3. Enfeksiyon Protokol Mtb / Makrofaj Agrega Yapıları oluşturmak için <ol><li> THP-1 hücre preparasyonu (96 oyuklu plaka başına) <ol><li> 5 dakika boyunca 250 x g&#39;de santrifüjleyin 7 x 10 6 THP-1 hücreleri. 7 ml RPMI-C süspanse. </li></ol></li><li> Mtb GFP hazırlık <ol><li> Santrifüj 2.8 x 10 8 Mtb GFP sallanan kovası santrifüj 3200 xg 5 dakika süreyle uOD 600, 1.0 = 3 x 10 8 bakteri / ml arasında bir dönüşüm şarkı. </li><li> Adım 3.2.1 olarak RPMI-C ve santrifüj ile bir kez yıkayın. </li><li> 10 s 7 ml RPMI-C süspanse ve vorteks. </li></ol></li><li> enfeksiyon NOT: Şablon için Şekil 1&#39;e bakınız. <ol><li> kültür ortamının buharlaşmasını önlemek için bir su ağız için satırlar A ve H ve sütun 1 ve 12, steril su, 200 uL eklenmesiyle 96 oyuklu bir plaka hazırlanması. </li><li> Arka plan kontrolü (Boş) sütununda 2 (G2 B2) 200 ul RPMI-C ekleyin. </li><li> Bulaştırmak için, THP-1 hücre süspansiyonu (adım 3.1) Mtb GFP süspansiyon (adım 3.2) ekleyin ve pipetleme iyice karıştırın. Nihai THP-1 hücre yoğunluğu mL başına 5 x 10 5 ve enfeksiyonun gelen çokluğu 40. </li><li> 25 ml rezervuar içine THP-1 / Mtb GFP süspansiyon dökün. </li><li> (G11 yoluyla B3), THP-1 kalan 96 oyuklara / MTB GFP süspansiyonu 200 uL usin eklemega çok kanallı pipet. Not: birden çok 96 oyuklu plakalar çalışma durumunda, düzenli daha kanşım her bir göze ilave edilir sağlamak için rezervuar içinde kalan, THP-1 / MTB süspansiyon tekrar süspansiyon. </li><li> 7-10 gün süreyle% 5 CO2 37 ° C&#39;de inkübe edin. </li><li> yavaş yavaş 100 çıkararak her 2 günde bir ortam değiştirme uL 100 uL, çok kanallı bir pipet kullanılarak, RPMI-C önceden ısıtılmış eklenmesi de yavaşça her birinin en ortam geçirdi. Kuyu tekrar süspansiyon ve kuyu dibinde Mtb / makrofaj agrega rahatsız etmeyin. </li><li> Görme Mtb / makrofaj agrega büyüklüğü dikkate alınarak, günlük floresan mikroskobu (4-10X objektif) tarafından kuyuları incelemek. Günün 7-10 tarafından, Mtb / makrofaj agrega yeterince büyük olmalıdır ilaç etkinlik testleri (Bölüm 4) için devam etmek (referans için Şekil 2&#39;ye bakınız). </li><li> İstenirse, bir otomatik hücre görüntüleme sistemi kullanılarak 96-kuyuların yakalama görüntüleri monteparlak bir alan ve GFP filtresi ile Mtb / makrofaj agrega oluşumu belgelemek için ayarlar. NOT: Kullanıcıların otomatik görüntüleme sistemine erişimi yoksa, temsilci kuyuların görüntüleri GFP ve parlak bir alan yetenekleri ile herhangi bir uygun mikroskop kullanılarak alınabilir. </li></ol></li></ol> 4. Büyüme İnhibisyon Tahlili Mtb / Makrofaj Agrega türetilen Uyuşturucu Etkinlik Karşı MTB Değerlendirmek için <ol><li> İlaç hazırlama (iki ilaç için üçlü koşullar) NOT: Şablon için Şekil 1A bakınız. <ol><li> Ayrı 96 oyuklu bir plaka içerisinde, G10 üzerine B2&#39;ye 7H9-Cı ortamın 125 uL ekleyin. </li><li> Aşama 4.2.4 seyreltmeden dolayı 7H9-C 1 mL çift en arzu edilen nihai konsantrasyonda iki ilaç hazırlanması. </li><li> üçlü tedaviler için sırasıyla kuyulara B11-C11-D11 ve E11-F11-G11 her ilacın 250 mcL ekleyin. </li><li> Çok kanallı bir pipet kullanılarak, seri Dilute testi ilaçlar B10-G10 içine B11-G11 125 uL hareket ettirerek iki kat. Her adımda 5 kez pipetleme karıştırın. </li><li> plaka üzerinde (sağdan sola) sütun sütun 125 uL taşımak ve sütun 4 sonra durdurmak için devam edin. </li><li> sütunu 4 karıştırıldıktan sonra, bir atık kabı içine 125 uL atın. Sütunlar 2, 3 arka plan (boş) ve pozitif bir büyüme kontrol olarak kullanmak için izin vermek için herhangi bir ilaç içermemelidir. Alternatif test ilaç kütüphaneleri için Şekil 1B şablonu izleyin: Not. Her 96 oyuklu plaka tek bir konsantrasyonda 58 ilaçları verebilir. bu adımda 4.2.4 yarı yarıya seyreltilmiş olacağından çift arzu edilen nihai konsantrasyonu kullanılarak bu ilaç tabak hazırlayın. </li></ol></li><li> İlaç tedavisi: <ol><li> MTB ihtiva eden 96 oyuklu plaka makrofajlar (MTB / makrofaj agregalar) ile enfekte al. </li><li> Dikkatle çok kanallı pipet ile ilgili tüm kuyular (G11 yoluyla B2) süzün.iki adımlı bir şekilde bu gerçekleştirin: ilk plaka eğmeden 150 uL kaldırın ve ardından plakayı devirme ve iyi alt kenarına pipet takarak kalan medya (~ 50 uL) çıkarın. Makrofaj Mtb / agrega, çukurun tabanına yapışık olarak, önemli bir yok olması gerekmektedir. </li><li> Yavaşça Mtb / makrofaj toplulukları ihtiva plakanın ilgili tüm kuyular (G11 ile B2) 7H9-C medya 100 mcL ekleyin. </li><li> Çok kanallı bir pipet kullanılarak, enfeksiyon levhanın tekabül eden gözleri 96 oyuklu plaka (aşama 4.1) ihtiva eden bir ilaç kupasında 100 uL aktarın. </li><li> kapalı torbada yer ve 37 ° C &#39;de 3 gün boyunca inkübe edilir. </li></ol></li></ol> Resazurin microtiter Test kullanma Niceleme İlaç Etkinliğinin 5. <ol><li> 0.8 mg nihai konsantrasyonda stok solüsyonu resazurin Hazırlama / ml H 2 O. sterilizasyon için 0.22 um gözenek boyutlu PVDF zarından filtre.</li><li> 1: 1 oranında bir 2 resazurin stok solüsyonu, H2O ve Tween-80 (H 2 O içinde% 20 çözelti) karıştırılarak çalışma çözeltisi resazurin hazırlayın. Nihai konsantrasyon 0.4 mg / mL resazurin ve% 5 Tween-80 bulunmaktadır. </li><li> Bir plaka okuyucusu, kurulum 530 nm&#39;de uyarma ve 590 nm&#39;de emisyonla 37 ° C&#39;de 24 saat süre ile, her 30 dk floresan okumak için bir programı kullanarak. 37 ° C&#39;ye kadar ön-sıcak plaka okuyucu. </li><li> Çok kanallı bir pipet kullanılarak ilaç tedavi plaka (aşama 4.11) ilgili tüm oyuklara (G11 ile B2) resazurin çalışma çözeltisinin 20 uL ekleyin. </li><li> plaka okuyucu üzerinde plakası yerleştirin ve adım 5.3 kurmak programını başlatın. Not: deney plakası, yüksek hacimli işlenmesini kolaylaştırmak için, 37 ° C kuluçka makinesi içinde bir analiz geliştirmek, tek flüoresan 24 saatte bir okuma yapmak için yeterlidir. Bu aynı zamanda kinetik ve inkübasyon özelliklerine sahip bir plaka okuyucu erişimi olmayan kullanıcılar için geçerlidir. </li></ol>

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The authors have nothing to disclose.

Burada, ayrıntılı olarak ilaç etkinlik testleri için uygun bir alternatif Mtb enfeksiyonu modeli tarif var. Bu model hesabına erken TB ilaç geliştirme sürecinde daha fazla dikkate alınmalıdır iki önemli faktör dikkate alır: ilaç penetrasyonu ve enfeksiyon sırasında Mtb metabolik değişikliklere fizyolojik ilgili engellerin varlığı. Daha önce bizim enfeksiyon modelinin yararları gösterilmiştir ve verimlilik uyumluluğu 16 enfeksiyon ölçekleme olasılı...

<html> Tüberküloz (TB) 40 yılı aşkın 1 TBC kemoterapi rejimlerinin varlığına rağmen ciddi bir küresel sağlık tehdidi olmaya devam etmektedir. Bu uyumsuzluğu 2 hastaya neden çoklu ilaç kombinasyonları kullanılarak 6 aydan uzun tedavi dönemleri için gereksinimi, kısmen kaynaklanmaktadır. Klinik onaylı ilaçların başarılı gelişimi 3 neredeyse varolmayan olduğu son yıllarda ilaca dirençli TB çıkması ayrıca bir alanda sorunları bileşik etti. Nitekim, ayrıntılı bir anti-TB ilaç geliştirme rağmen, sadece tek bir ilaç FDA son 40 yılda 4 klinik kullanım için onaylanmıştır olmuştur. Bu nedenle, anti-TB ilaçların yeni nesil acilen bu sorunu çözmek için ihtiyaç vardır. TB ilaç keşfinde önemli bir sorun klinik ortamda etkinliğine in vitro aktiviteye sahip bileşikler başarılı transfer olmaması= &quot;xref&quot;&gt; 5, 6, 7. Başlangıçta, hedef bazlı yaklaşımlar tam bakteriyel hücrelere çevirmek için başarısız anti-Mtb Drugs 5, taranması için kullanıldı. Mtb hücreleri kullanılır bile, genellikle doğru vivo 8, 9 ilaç etkinliğini öngörmek olmayan suyu üreyen kültürler, kullanılarak gerçekleştirilir. Bu sorunlar, tanınmış ve MTB veya gizli Mtb içeren makrofajlar karşı ilaç tarama deneyleri başarıyla 8, 10, 11, 12 tespit edilmiştir. Ancak, hatta bu daha gelişmiş analizler ilaçlar non-vaskülarize akciğer lezyonları karşılaştıkları penetrasyon engelleri yeterli önem vermesi ve yok enfeksiyon yerinde nekrotik odaklar. AslındaHatta ilk basamak TB ilaç rifampisin için, sub-optimal doz nedeniyle in vivo doku ve beyin omurilik sıvısı (BOS) &#39;de vasat sorgulanmıştır hücre içi Mtb 8, 9 karşı etkinin azalmasına yanı sıra 13, 14, 15 nüfuz. şüphesiz TB ilaç keşif çabalarını artıracak erken kurşun geliştirme sürecinde dikkate bu parametreleri alacak gibi, yeni modeller ve tahlilleri gibi. Bu ihtiyacı karşılamak için, biz son zamanlarda Mtb ilaç etkinlik testleri 16 için, ucuz, hızlı ve BSL-2 uyumlu alternatif enfeksiyon modeli kurdu. Fizyolojik ilgili hücresel penetrasyon engelleri değinmeyecek ve makrofaj pasajlandı oluşturulan yoğun üretilen büyük makrofaj agrega yapılar içinde Mtb paketlenmiş Bu enfeksiyon modeli, <em&gt; gizli Mtb benzeyen değiştirilmiş bir fizyolojik durum ile Mtb. Bu enfeksiyon modeli türetilmiş Mtb diğer hücre içi enfeksiyon modellerinde tutarlı sonuçlar ilaç etkinliğini değerlendirmek için resazurin mikrotiter tahlilde (REMA) ile birleştirildi ve serum konsantrasyonlarına kıyasla yüksek CSF konsantrasyonlarının elde edilmesi için ortak bir TBC ilaç bildirilen yeteneği ile de korelasyon 16. Burada ayrıntılı olarak rema kullanılarak ilaç duyarlılık testi için makrofaj geçirilmiş Mtb uygun üretmek Mtb / makrofaj agrega yapıların üretilmesini tarif eder. Özel olarak, bu enfeksiyonun sistemi aday anti-TBC ilaçların verimli tarama ile uyumlu bir 96-gözlü formata adapte olabilir şeklini göstermektedir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>BD Biosciences</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma</td>
			<td>T8761</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</td>
		</tr>
		<tr>
			<td>Nalgene Square PETG media bottle</td>
			<td>Thermo Fisher</td>
			<td>2019-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Hyclone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 plate reader</td>
			<td>Biotek</td>
			<td></td>
			<td>Interchangable with any fluorescent plate reader and microscope</td>
		</tr>
		<tr>
			<td>Gen5 Software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of rezasurin coversion</td>
		</tr>
		<tr>
			<td>Rifampicin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
	</tbody>
</table>

a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

96 gözlü plaka formatında bu enfeksiyon modeli uyarlamak sağlamlığını teyit etmek için, burada Şekil verilen şablona göre bir ilaç eden 96 oyuklu türetilen Mtb duyarlılığını enfeksiyonu rifampisin modeli (RIF) ve moksifloksasin (Moxi) adapte incelenmiştir 1 A. Bu deneyde anahtarı Mtb / makrofaj agrega yapıların üretimi güvenilir bir şekilde ve böylece verim uyumluluğu (Şekil 1B) sağlayarak,...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Bir Yüksek verim Uyumlu Deneyi Makrofaj, pasajlamaya karşı ilaç etkinliğini değerlendirmektir<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

Research

JoVE Journal

Immunology and Infection

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

7.7K Görüntüleme.  University of Alabama at Birmingham. New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening. 

Video: A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Büyüme Mycobacterium tuberculosis Biyofilmler

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

İmmünoloji ve Enfeksiyon

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Kuruluş ve Eğitim için bir Yüksek Verimli Kur Optimizasyonu<em&gt; Staphylococcus epidermidis</em&gt; Ve<em&gt; Mycobacterium marinum</em&gt; Enfeksiyon İlaç Keşif için bir model olarak

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Yüksek verim Kantitatif Gerçek zamanlı RT-PCR Testi Türleri I ve III İnterferon Alttipleriyle İfadesi Profilleri belirlenmesi için

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Immünkompetan Kemirgenler bir Sağlam Pnömoni Modeli karşı antibakteriyel etkinliğini değerlendirmektir<i&gt; S. pneumoniae</i&gt;<i&gt; H. influenza</i&gt;<i&gt; K. pneumoniae</i&gt;<em&gt; P. aeruginosa</em&gt; ya da<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

bir<em&gt; In vitro</emTüberküloz Lezyonlarında İlaç Penetrasyonunu Öngören Caseum Bağlanma Testi

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Tüberküloz Mycobacterium marinum enfekte yetişkin zebra balığı içinde modelleme

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...