İş Sağlığı ve Amerikan Akciğer Derneği Ulusal Enstitüsü&#39;nün mali desteği ile gerçekleştirilmiştir.

1. M. yetiştirilmesi biyofilm Bir 250ml vida kapaklı şişe içinde tüberküloz <ol><li> Ortam hazırlanması: su içinde 900 ml gliserol 60mL, ferrik amonyum sitrat ve 0.05g MgSO 4 KH 2 PO 4, 0.5g, L-asparagin 4g, sitrik asit 2g, 0.5g eritilir. NaOH ile pH 7.0 &#39;a ayarlanır. Otoklav, serin ve hemen önce deney başlamadan,% 0.1 w bir son konsantrasyon steril ZnSO 4 / ekleme v Mc 2 7000 pantothenat auxotroph olduğundan bu gerginlik de son konsantrasyon 10μg/mL az pantotenik asit gerektirir. </li></ol> Not: Bu M. tüberküloz için kullanılan Sauton medya standart bir bileşimidir. Gerekirse Bununla birlikte, diğer özel ortam, aynı zamanda, diğer mikobakteriyel türler için de kullanılabilir. <ol start="2"><li> Mikroorganizma hazırlık: M. Grow 0.05% 0.7 ila 1.0 arası bir hafta boyunca Tween-80, veya OD 600 ile 7H9OADC tüberküloz. Culture doğrudan bir 1:100 azından aşı malzemesi olarak kullanılabilir. </li><li> Bir 250ml vida kapaklı polistiren şişe (Corning) için Sauton medyasının 25mL dağıtın. Çok sıkı, orta kap şişe inokulum 250μl ekleyin ve 3 hafta boyunca nemli 37 ° C inkübatör içinde bozulmamış yerleştirin. Hiçbir kirlenme var olmasını sağlamak için her gün bir kez şişe gözlemleyin. </li><li> Üçüncü haftanın sonunda, M. ve daha da büyüme izin vermek için şişe kap gevşetmek arayüzü de tüberküloz. Kap bu aşamada gevşek değilse o kapta yetersiz oksijen konsantrasyonu bakteri daha fazla büyümesini frenleyeceğini. </li></ol> 2. M. Büyüme 12-iyi plakaları tüberküloz biyofilm <ol><li> Kesitler A1 ve A2 açıklandığı gibi mc 2 7000, medya ve inokulum hazırlayın. </li><li> Inokulum 600μl ile medya 60mL karıştırın. 4.5m DağıtmaPlakanın her oyuğuna karışımının L. Kapaklı Kapak plakası. Parafilm ile plakası birkaç kez kaydırmak. 5 hafta boyunca 37 ° C&#39;de nemli inkübatörde rahatsız plaka inkübe edin. </li></ol> 3. M. ilaç tolerans persisters sıklığını belirlenmesi tüberküloz biyofilmler <ol><li> M. Grow B. Bu bölümde tarif edildiği gibi, bir 12-gözlü formata tüberküloz biyofilmler yaklaşık 5-hafta toplam alacaktır. </li><li> Biyofilmler olgunlaştı edildikten sonra (kuluçka 5 hafta sonra), pipetle bir mikrotip kullanarak biyofilm altındaki medya içine istenen konsantrasyonda antibiyotik tercih enjekte. </li></ol> Not: pellicles altındaki medya hacmi yaklaşık 3.0mL azaltır. Bu yüzden araştırmacılar buna göre ilaç miktarı hesaplamak gerekir. <ol start="3"><li> Girdap antibiyotik iyice ortamda yayılır yavaşça böylece plaka. Istatistiksel olarak anlamlı sonuçlar için, antibiyotik enjektedört kuyu otik. Buna paralel olarak, antibiyotik diğer dört kuyu içinde eritildi içinde çözücünün aynı hacimde enjekte ve dokunulmamış plakasının son dört kuyu bırakın. Kapaklı Kapak plakası ve plaka etrafında parafilm taze katmanları koymak. Istenilen süre inkübatöre geri yerleştirin. </li><li> İnkübasyon sonunda, plaka açmak ve kuyu her birinde% 0.1 nihai konsantrasyonu (hacim / hacim) ile Tween-80 ekleyebilir. Girdap deterjan üniform dağıtılması için hafifçe tüm plaka. 15 dakika oda sıcaklığında inkübe plakası. Ki tüm içerik üniform bir 15 mL konik tüpe aktarılır, böylece pipetle birkaç defa, her sıra içeriği karıştırılır. </li><li> Oda sıcaklığında 10 dakika boyunca 4000rpm tüpün içeriği aşağı doğru dönmeye başlayacaktır. Taze yıkama tamponu (% 10 gliserol ve% 0.05 Tween-80 ile birlikte PBS) ile 5ml pelletini. Yıkama üç kez tekrarlayın. 5 ml&#39;lik yıkama tamponu içinde pelletini. O için rocker üzerinde tutun4 azından vernight ° C. </li></ol> Not: düşük sıcaklıkta başlangıçta M. smegmatis (dispersiyon sırasında büyümesini en aza indirmek üzere) için geliştirilen ve aynı zamanda M. Tuberculosis için kullanılabilir olmasına rağmen, yavaş büyüyen türler, büyük olasılıkla sonucu üzerinde bir etkisi olmaksızın, oda sıcaklığında sallandı edilebilir. BSL-3 tesisi çalışma durumunda, oda sıcaklığında sallanan gerekli olabilir. <ol start="6"><li> , Uygun boyuta geniş sonunda kesme şırıngaya oturtmadan ve parafilm ile sararak mikrotip (2-200μl) ile donatılmış steril bir şırınga hazırlayın. Ucuna takılmış şırınga yoluyla tüpün tüm içerik geçmesi ve yeni bir 15 mL tüpler birikir. Eğer oldukça homojen bir süspansiyon gözlemlemek kadar bu adımı 5 ila 6 kez tekrarlayın. </li><li> Her iyi canlı koloni sayısını belirlemek için 7H11OADC plaka üzerine süspansiyon ve plaka dilüsyonları seri dilüe. 37 ° C inkübatör üç hafta süreyle inkübe edin. Frekanslar belirleyinÇözücü muamele plakalar üzerinde elde edilenler ile muamele antibiyotik olarak elde koloni sayısı oranı hesaplayarak biyofilm popülasyonda persisters arasında cy. </li></ol> 4. Temsilcisi Sonuçlar M. bir şişe kültüre, büyüme tüberküloz ilk hafta sonuna Şişenin taban görülebilir. Hava-medya arayüzü de büyüme üçüncü haftasında (Şekil 1A) sonunda sürekli görünür olmasına rağmen ikinci haftanın sonunda, hava-medya arayüzü üzerinde bakteri yamalı büyüme görülebilir. Bu zamanda, kabın duvarına bakteri eki de gözlemlenmiştir. Bu noktadan itibaren kültür büyüme esas olarak hava arayüzü ortam üzerinde oluşur. Yüzey büyüme altında sıvı açıktır. Tipik olarak, yapının beşinci hafta (Şekil 1B) sonuna kadar olgunlaşır. İnkübasyon uzamış ise, yapılar kabın dibine çöker başlayacaktır. Şaşırtıcı,Üçüncü haftanın sonuna kadar kapak sıkma sürecinde önemli bir adım olduğunu ve bilinmeyen nedenlerle gevşek kapaklı şişe önemli arayüz 9 büyüme başlatılması geciktirir. 12-gözlü formata olarak, hava-ortam ara yüzeyde sağlam bir biyofilm beş haftalık (Şekil 2A) sonunda her bir kuyu görülür. Plakalar tamamen sarılmış değilse o diferansiyel biyofilm büyüme gözlenmektedir. En kötü durumda, önemli medya buharlaşma bakterilerin büyümesini (Şekil 2B) aksaklık olabilir. Böylece, her iki plakanın sarma buharlaşmasını önlemek için de biyofilm (önceki paragrafta bakınız) için bir ortam sağlamak için gereklidir. Bu teknik ile belirlenen Biyofilmlererde canlı basil sayısı oldukça tekrarlanabilir olması. M. Tepki tüberküloz biyofilmlerin antibiyotiklerin doğa ile değişir. Örneğin, rifampisin gibi bakterisidal antibiyotik için, canlılık kaybı bipha takip<html> sic eğilim 9. Nüfusun küçük bir yüzdesi ne olursa olsun antibiyotik veya maruz kalma süresi konsantrasyon antibiyotiklere tamamen inatçı kalan hangi kalıcı bir fazı izler ilk üç ile dört gün boyunca canlılık içinde hızlı bir düşüş. Şekil 3 rifampisin 50μg/mL için 7 günlük maruziyet (MIC göre 50 kat daha yüksek) sonra olgun Biyofilmlererde canlı basil sayısını gösterir. <img alt="Şekil 1A." src="/files/ftp_upload/3820/3820fig1A.jpg" /> M. Şekil 1A. Erken görünüm inkübasyon 3 hafta sonra bakterilerin hava-medya arayüzü tüberküloz basili. <img alt="Şekil 1B." src="/files/ftp_upload/3820/3820fig1B.jpg" /> Şekil 1B. M. biyofilm Vadesi inkübasyon beş hafta sonra hava-medya arayüzü tüberküloz. 820/3820fig2A.jpg &quot;/&gt; M. Şekil 2A. 5 haftalık biyofilmler Tüberküloz 12-iyi biçimde büyüdü. <img alt="Şekil 2B." src="/files/ftp_upload/3820/3820fig2B.jpg" /> Şekil 2B. Parafilm olmadan 12-iyi plaka M. tuberculosis biyofilm büyümeye bir başarısız girişimi. <img alt="Şekil 3." src="/files/ftp_upload/3820/3820fig3.jpg" /> M. ilaç hoşgörülü persisters sıklığını gösteren 3. Bir temsilci arsa Şekil Tüberküloz 12-iyi biçimde büyüdü ve yedi gün boyunca Rifampisin ile 50μg/mL maruz Biyofilmler.

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Biz ifşa hiçbir şey yok.

Mycobacterium tuberculosis enfeksiyonu nedeniyle tüberküloz (TB), küresel halk sağlığı için büyük bir tehdit olmaya devam etmektedir. Dünya nüfusunun yaklaşık üçte biri asemptomatik patojen ile enfekte olduğu tahmin edilmektedir, 9 milyon yeni vaka aktif verem ve enfeksiyonun yaklaşık 1,7 milyon kalıp her yıl 11 belirtileri ile her yıl klinikte göstermek. Hastalık büyük bir yük öncelikle bir aşı ve altı ile dokuz ay boyunca uygulanan bir ilaca rejimi içeren bir derece ...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

Mycobacterium tuberculosis, insan tüberküloz etyolojik ajan, antibiyotikler de dahil olmak üzere çevresel streslere karşı hayatta kalabilmek için olağanüstü bir yeteneği vardır. Rağmen M. stres toleransı tüberkülozu, tüm tüberküloz 1 6 aylık uzun kemoterapi olasılıkla katkıda biridir, patojen bu özelliği fenotip altında yatan moleküler mekanizmaları tam olarak bilinmemektedir. Birçok mikrobiyal türlerin biyofilm 2-4 olarak adlandırılan son derece organize, bağlı yüzey ve matris kapsüllü yapılarda kendine montaj tarafından stresli ortamlarda hayatta kalmak için evrildi. Topluluklarda Büyüme mikropların tercih edilen bir hayatta kalma stratejisi gibi görünüyor, ve yüzey eki, hücrelerarası iletişimi ve hücre dışı polimerik maddelerin sentezi (EPS) 5,6 düzenleyen genetik bileşenleri yoluyla elde edilir. Çevresel strese toleransı olasılıkla physiolo belki de EPS kolaylaştırdı ve birbiyofilmlerin 7 karmaşık mimarisi içindeki heterojen mikroçevrelerde bireysel basilinin cerrahi adaptasyonu. Son kağıtları bir dizi biz kurduğu M. tüberküloz ve Mycobacterium smegmatis&#39;in fazla 50 kez anti-tüberküloz ilaçlar izoniazid ve rifampisin 8-10 minimal inhibitör konsantrasyonları tolere edebilir biyofilm olarak adlandırılan organize çok hücreli yapılar büyümeye güçlü bir eğilimi, var. M. ortamı gibi bir atmosfer havası ile 9 arasında, sınırlı değişimi: tüberküloz, bununla birlikte, ilgi çekici tepe boşluğu özellikle 9:01 oranında olgun biyofilmlerde, oluşturmak için spesifik koşulları gerektirmektedir. Özel çevre koşullarının Gereksinimleri muhtemelen o M. gerçeği ile bağlantılı olabilir tüberküloz, zorunlu insan patojeni ve böylece doku ortamlara adapte olmuştur. Bu yayının biz kültür M. yöntemleri göstermek tüberkülozbir şişe ve bakteriyolojik yanı sıra genetik çalışmalar için uygun bir 12 de plaka biçiminde biyofilmler. Biz, M. olarak zayıflatılmış bir suş için protokol tanımlamışlardır patojen 9 in vivo büyüme için kritik olan iki lokus, panCD ve RD1, silinmek ile tüberküloz, mc 2 7000,. Bu şekil değiştirme güvenle ve böylece pahalı bir BSL-3 tesisinin ihtiyacı kaçınarak tuberculosis patojenin temel biyolojisi anlamak için bir BSL-2 çevreleme kullanılabilir. Yöntem diğer kültürleştirilebilir mikobakteriyel türlerin biyofilm büyümeye, medya uygun modifikasyon ile uzatılabilir. Genel olarak, kültür mikobakteriyel biyofilm yeknesak bir protokol mikobakteri temel esnek özellikleri eğitim ile ilgilenen araştırmacılara yardımcı olacaktır. Ayrıca, büyüyen mikobakteriyel biyofilmlerin açık ve özlü bir yöntem de, klinik ve ilaç inv yardımcı olacaktırestigators potansiyel bir ilacın etkinliğinin test etmek için.

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

Belli koşullarda kültüre zaman Mycobacterium tuberculosis ilaç hoşgörülü biyofilm oluşturmaktadır. Burada kültür M. yöntemleri tarif tuberculosis biyofilmler ve ilaç tolerans persisters sıklığını belirlenmesi. Bu protokoller M. ilaç hoşgörü mekanizmalarına daha sonraki çalışmalar için faydalı olacaktır tüberküloz.

Büyüme Mycobacterium tuberculosis Biyofilmler

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

growth-of-mycobacterium-tuberculosis-biofilms

Research

JoVE Journal

Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

23.6K Görüntüleme.  University of Pittsburgh. Belli koşullarda kültüre zaman Mycobacterium tuberculosis ilaç hoşgörülü biyofilm oluşturmaktadır. Burada kültür M. yöntemleri tarif tuberculosis biyofilmler ve ilaç tolerans persisters sıklığını belirlenmesi. Bu protokoller M. ilaç hoşgörü mekanizmalarına daha sonraki çalışmalar için faydalı olacaktır tüberküloz.

Video: Growth of Mycobacterium tuberculosis Biofilms

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter Plate Format Broth Seyreltme Birinci ve İkinci Line İlaçları Mycobacterium tuberculosis kompleksi Antimikrobiyal Duyarlılık Testi

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

İmmünoloji ve Enfeksiyon

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Numune Hazırlama<em&gt; Mycobacterium tuberculosis</emNükleer Manyetik Rezonans metabolomik Araştırmaları&gt; özler

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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 ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Kurtarma Bir Roman Mikrodiseksiyon Yaklaşımı<em&gt; Mycobacterium tuberculosis</emAkciğer granülomlara Gömülü Formalinle Sabit Parafin dan&gt; Özel Transkript

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Fonksiyonel Araştırmaları 3D İnsan Akciğer Doku Modeli<em&gt; Mycobacterium tuberculosis</em&gt; Enfeksiyon

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Diş Biyofilmler ekstraselüler pH rasyometrik Görüntüleme

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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 ...

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.

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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...