Biz çalışma sırasında, yardım ettikleri için Mayo Clinic tüberküloz Laboratuvarı, laboratuvar teknologlar teşekkür etmek istiyorum.

Her laboratuvar, inokulum hazırlanması ve plaka içine inokulum pipetleme için uygun bir biyogüvenlik seviyesini belirlemek için bir risk değerlendirmesi Kurumsal Biyolojik Güvenlik Görevlisi ile işbirliği içinde yerine getirmesi gerekir. Microtiter plakalar inoküle ve plastik bir yapıştırıcı mührü ile mühürlenmiş kadar laboratuar, 3 laboratuvar (BSL3) uygulamaları biyogüvenlik düzeyi kullanabilir. Bu plakalar daha sonra ısı da kapalı bir plastik torba içine yerleştirilir. Kuluçka ve yorumlama okuma mikrotiter plate bir biyogüvenlik düzeyi 2 laboratuvar (BSL2) yapılır. 1. Malzemelerin Etiketleme <ol><li> Etiket kan agar plaka, üç Middlebrook 7H10 plakaları, Middlebrook 7H9 suyu, tuzlu su arasında cam boncuk tüp ve BSL2 uygun tanımlayıcılar (örneğin, hasta ismi, tıbbi kayıt numarası) ile mikrotiter AST plaka. </li><li> Etiket kanlı agar ve &quot;saflık onay&quot; olarak kaplama 7H10. Inokulum Periyodik koloni sayımı, organizmaların uygun bir konsantrasyon testte kullanılan olmasını sağlamak için tavsiye edilir. Yapılan koloni sayımı &quot;1 / 1 koloni sayısı&quot; ve &quot;1 / 50 koloni sayısı&quot; ile etiketlenmiş 7H10 plakaları iki olması gerekir. </li></ol> 2. BSL3 gitmek Teknolog hazırlıkları <ol><li> Uygun kişisel koruyucu ekipmanlar BSL3 çalışması için gerekli olan ve katı ön cüppe, baş örtüsü, eldiven, galoş ve bir maske içerir. Her kurum, kendi kurumu Endüstriyel Hijyen görevlisi ile işbirliği içinde kendi solunum koruma planı oluşturmalıdır. Laboratuvarımızda, motorlu hava temizleme respiratörleri (PAPRs) ve HEPA filtreden donatılmış, N95, yarım yüz gaz maskeleri de dahil olmak üzere çeşitli solunum maskeleri ve var. </li></ol> 3. Biyolojik güvenlik kabini hazırlanması <ol><li> BSL3, yüzey dezenfeksiyon ve yaklaşık 6 inç havalandırma panelinden dezenfektan ile ıslatılmış kağıt havlu yerleştirerek biyolojik güvenlik kabini (BSC) hazırlamak. </li><li> Sol tarafta bir atık kabı ve sağ tarafta küçük nephlometer ve vortexer yerleştirin. </li><li> Aşılamak döngüler, pipetors ve ipuçları kağıt havlu yanında. </li><li> Organizma (et suyu veya katı orta) ve kağıt havlu üzerine McFarland 0.5 standart bir raf yerleştirin. </li></ol> 4. Nephelometeri Kalibrasyonu <ol><li> Nephelometeri tuzlu arasındaki cam boncuk tüp yerleştirin ve tuzlu arasındaki boş kullanarak sıfır araç ayarlamak. </li><li> Nephelometeri McFarland 0.5 standart yerleştirin ve üreticinin talimatlarına göre kalibre. </li></ol> 5. İnokulum Hazırlanması <ol><li> ≥ 0.5 McFarland Standart elde edilinceye kadar uygun etiketli tuzlu arasındaki cam boncuk tüp içerisine steril bir döngü kullanarak katı orta BSC, kazıma koloniler çalışmak. </li><li> Görme örnek incelemek ve McFarland Standart karşılaştırın. </li><li> 30-60 saniye için Vortex örnek. </li><li> 15 dakika boyunca yerleşmek için bir zamanlayıcı ayarlamak inokulum izin verin. </li></ol> 6. Mikrotiter plate Aşılanması <ol><li> McFarland 0.5, 4.1 ve 4.2 adımları kalibre nephelometeri kullanarak tuzlu arasında suyu ile inokulum ayarlayın. Bu 30 dakika (ortalama üretici özellikleri) içinde tamamlanması gerekir. </li><li> 200 mcL pipetter ve tuzlu boncuk arasındaki tüp TOP 1 / 3 üncü inokulum 100 mcL kaldırmak için bir UZUN aerosol dayanıklı pipetlemeyin ucu kullanın. Middlebrook 7H9 Broth inoküle edin. 20 saniye boyunca inoküle 7H9 suyu yavaş yavaş girdap. </li><li> AST mikrotiter plate üreticinin paketinden çıkarın. Folyo paket hasarlı ise kurutucu mavi veya kullanmayın. </li><li> 01:50 seyreltme hazırlanmasında daha sonra kullanmak üzere steril bir tüpe steril su 50 mcL pipetleme 1:50 sulandırma tüpü hazırlayın. </li><li> 7H9 broth inokulum bir çukur içine dikkatlice dökün. AEROSOLS JENERATÖR KAÇININ. </li><li> AST mikrotiter plate etiketi ile karşı karşıya koyun. </li><li> 12-kanal pipetter, her bir kuyunun 100 mcL inokulum eklemek için kullanın. </li><li> Koloni sayımı için dilüsyonları hazırlayın: <ol type="a"><li> 1 / 1 seyreltme, &quot;1 / 1&quot; etiketli 7H10 plaka üzerinde 1 ul döngü şeritli de pozitif kontrol (H12) hazırlanır. </li><li> 1 / 50 dilüsyon de pozitif kontrol (H12) 1 ul döngü yukarıda hazırlanan su borusu 50 ul ile aşılama ve tüp içine dönen tarafından hazırlanmıştır. Bu seyreltme plaka Streak 1 mcL &quot;1 / 50&quot; olarak etiketlenmiş. </li></ol></li><li> Uygun bir şekilde etiketlenmiş kanlı agar ve 7H10 plaka ve izolasyonu için çizgi üzerine 50 mcL pipetleme çukur saflığı plakalarını aşılamak. </li><li> YerAST mikrotiter plate üzerinde yapışkan bir mühür. Beyaz arka mühür kullanılarak yeterli sızdırmazlık sağlamak için her bir kuyunun üzerine sıkıca bastırın. Kırışıklıkların oluşmasını önlemek. </li><li> Mikrotiter plate bir ısı mühür veya bant ile plastik bir torba ve yakın bir tüberkülsit dezenfektan ve yer mikrotiter plate ile ıslatılmış kağıt havlu ile silinerek dezenfekte edin. <ol type="a"><li> Plastik torba birincil plakanın bir sızıntı veya dökülme var olmalıdır tüm suyu yer olduğundan emin olmak için ikincil bir konteyner olarak hizmet vermektedir. </li></ol></li></ol> 7. Malzemelerin dezenfeksiyonu ve biyolojik güvenlik kabini <ol><li> Aerosollerin üretimi önlemek için hafifçe atmak konteyner içine koymadan önce başka bir çukur inoküle çukur üstüne yerleştirin. </li><li> Tüberkülsit bir dezenfektan ile silerek nephlometer, vortexer, tabak, tüpler, vb de dahil olmak üzere tüm malzemeler dezenfekte edin ve üreticinin talimatlarına göre BSC çıkarmadan önce dezenfeksiyon sigortalamak için uygun zamanı bekleyin. Örneğin, Prospray, kullandığımız tüberkülsit dezenfektan, mikobakteriler öldürmek için 15 dakika gerektirir. </li><li> Otoklav önce, bir biohazard çanta ve mühür atın kap yerleştirin. </li></ol> 8. Kuluçka <ol><li> Tabaklar medya ile aşağı inkübe edilmelidir ve bakım, herhangi bir sıvı dökmekten veya sıçramasını önlemek için alınmalıdır. </li><li> 35-37 ° C inkübatör AST mikrotiter tabak tabak, 7H10 ve kan agar plaklarına BSL2 laboratuar% 5-10 CO 2 ile% 5-10 CO 2 ile inkübe edin. <ol class="lalpha"><li> Plaka iyi kapalı ve mühürlü bir plastik torba içinde, çünkü bizim laboratuar, bu kabul edilebilir. </li></ol></li><li> 2&#39;den fazla mikrotiter AST plakaları, üreticinin talimatlarına göre kuvöz yığılmış olmalıdır. </li></ol> 9 - Temsilcisi sonuçları Sonuçların yorumlanması: Mühürlü AST microtiter plakalar elle bir ayna kullanarak, bir yardımcı olarak ya da Vizyonumuz (TREK Diagnostics) gibi bir otomatik plaka okuyucu ile okunabilir. Plakalar aşılama sonra en kısa sürede 7 gün olarak incelenebilir. (Yani, iyi altındaki hücreleri mevduat) pozitif olup olmadığını belirlemek için, H11 ve H12 pozisyonları büyümesini kontrol kuyuları inceleyin. Büyümesini kontrol kuyuları olumlu değilse, plaka reincubate ve büyümesini kontrol kuyuları pozitif olana kadar (örneğin, 10 gün, 14 gün) periyodik olarak yeniden gözden. <ol type="a"><li> Ayna okuyun: <ol type="i"><li> Platform üzerine yerleştirin AST mikrotiter plate mühürlü; </li><li> 7 gün inkübasyondan sonra büyümesini kontrol kuyuları okuyun; bu negatif, reincubate AST mikrotiter plate ise; </li><li> Büyümesini kontrol eden kabul edilebilir ise, o zaman ilaç içeren kuyu okuyun. Her ilaç için, büyüme, büyüme kontrolü ile ilaç kuyu karşılaştırarak ilk seyreltme kaydedin. </li><li> Büyüme, bulanıklık gibi ya da bir kuyunun dibinde hücreleri bir depozito olarak görünür. </li></ol></li><li> Enstrüman okuyun: üreticileri yönergeleri izleyin; aşağıdaki talimatları okuma Vizyonumuz platformu ile ne yapılabilir bir örnektir: <ol type="i"><li> Giriş yap </li><li> &quot;MYCOTB&quot; AST plaka yazılımı kullanın. </li><li> Kullanıcı bakan barkod ile mühürlü plaka koyun. Büyüme, bulanıklık gibi ya da bir kuyunun dibinde hücreleri bir depozito olarak görünür. </li><li> Büyümesini kontrol eden bu negatif, reincubate AST mikrotiter plate ise; pozitif büyüme göstermez, her ilacın ilk sıra ile ekrana dokunarak plaka okursanız. </li></ol></li></ol> Saflık plakaları okuyun. Kan agar plaka 48 saat sonra, herhangi bir büyüme göstermesi gerekir ve 7H10 saflık plakası, bir organizma türü konfluent büyüme olmalıdır. Kontamine olursa, AST mikrotiter testi saf kültür tekrarlanması gerekir. Aşağıdaki tabloyu kullanarak pozitif kontrol koloni sayıları hesaplayın: Colony Sayısı Anahtar <table border="1"><tbody><tr><td> # Koloniler &quot;1 / 1&quot; etiketli plaka sayılır </td><td> # Koloniler sayılır &quot;1 / 50&quot; etiketli plaka </td><td> İnokulum Koloni Sayısı (kob / ml) </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 X 10 4 </td></tr><tr><td> 50-100 </td><td> 0-2 </td><td> 5 X 04-1 Ekim X 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤ 10 </td><td> 1 X 10 5 5 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 X 10 5 </td></tr></tbody></table> Mycobacterium tuberculo bazı suşları para-aminosalisilik asit ile sondaki olabilirsis. Bu pelet var tüm kuyu bakıyor ve bitiş noktası olarak bir sonraki seyreltme aynı boyutta pelet ilk iyi kullanarak yorumlanabilir. Direnç, bu ilaç ile nadirdir. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Şekil 1"/> Şekil 1. MYCOTB plaka kullanarak Temsilcisi sonuçlanır. <table border="1"><tbody><tr><td> İlaç kısaltma </td><td> Ilaç </td><td> MIC μ / ml </td></tr><tr><td> Odasındaki </td><td> ofloksasin </td><td> 16 </td></tr><tr><td> MXF </td><td> moksifloksasin </td><td> 8 </td></tr><tr><td> RIF </td><td> rifampin </td><td> &gt; 16 </td></tr><tr><td> AMI </td><td> amikasin </td><td> 0,5 </td></tr><tr><td> STR </td><td> streptomisin </td><td> 16 </td></tr><tr><td> RFB </td><td> rifabutinin </td><td> 8 </td></tr><tr><td> PAS </td><td> p-aminosalisilik asit </td><td> ≤ 0.5 </td></tr><tr><td> ETH </td><td> etionamid </td><td> 5 </td></tr><tr><td> CYC </td><td> sikloserin </td><td> 4 </td></tr><tr><td> INH </td><td> izoniazid </td><td> 2 </td></tr><tr><td> KAN </td><td> kanamisin </td><td> 1,2 </td></tr><tr><td> EMB </td><td> etambutol </td><td> 4,0 </td></tr></tbody></table>

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Bir AST mikrotiter plate pozitif büyüme kontrolleri bir örnek Şekil 1&#39;de gösterildiği ve uç nokta her ilaç için MIC (mcg / ml) daire. Laboratuvarımızda yapılan bir çalışmada AST mikrotiter plate yöntemi Mycobacterium tuberculosis duyarlılık testi için altın standart agar oranı yöntemi eşdeğer olduğunu belirtti. AST mikrotiter plate ve agar oranı yöntemi 14 ve 21 gün için son yorumu ile geleneksel agar oranı yöntemine göre sonuçların daha hızlı raporlama mikrotiter AST mikrotiter plate ile elde edildi, respectively.The AST mikrotiter plate ilk test avantajı vardır ikinci satırda aynı anda ilaçlara dirençli suşlar ile haddini gecikmeler önlemeye yardımcı olabilir. AST mikrotiter plate de APM daha kurmak ve okumak çok daha kolay ve Vizyonumuz gibi manuel ayna ve yarı otomatik bir sistem plakaları okumak için kullanılır. TB ilaç direnç testi için MIC değeri durumu, özellikle &quot;borderline&quot; duyarlılığı olanlar izolatları için, geleneksel kritik konsantrasyon değerine göre hekimlere değerli yeni bilgiler verebilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> AST microtiter plakalar ve yapışkan mühür </td><td> TREK Diagnostics, Cleveland Ohio </td><td> MYCOTB </td></tr><tr><td> McFarland 0.5 standardına </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> E1041 </td></tr><tr><td> Middlebrook 7H10 plakalar </td><td> Balıkçı </td><td></td></tr><tr><td> Kan Agar Tabaklar </td><td> Balıkçı </td><td></td></tr><tr><td> Steril mcL 1 döngüler </td><td> Balıkçı </td><td></td></tr><tr><td> Steril döngüler 10 mcL </td><td> Balıkçı </td><td></td></tr><tr><td> Sensititre Tuzlu-Tween cam boncuk broth </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> 27143 </td></tr><tr><td> 7H9 OADC broth </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> T3440 </td></tr><tr><td> Uzun pipetleyin İpuçları </td><td> Balıkçı </td><td></td></tr><tr><td> Steril Oluklar </td><td> TREK Diagnostics, Cleveland, Ohio </td><td></td></tr><tr><td> M. tuberculosis </td><td> ATCC </td><td> 27294 </td></tr><tr><td> Pipeters: 1000, 200, 20μL </td><td> Balıkçı </td><td></td></tr><tr><td> İpuçları 1000, 20, 20 mcL </td><td> Balıkçı </td><td></td></tr><tr><td> Çok Kanallı Pipetter </td><td> Balıkçı </td><td></td></tr><tr><td> İnkübatör% 5-10 CO 2 </td><td> Balıkçı </td><td></td></tr><tr><td> Plastik torbalar, küçük, CO 2 geçirgen </td><td> Poli Plastik, Delano, PA </td><td></td></tr><tr><td> TB dezenfektan (Böyle ProSpray C-60 gibi) </td><td> Certol, Uluslararası Ticaret City, CO </td><td> PSC60/32 </td></tr><tr><td> Ayna Kutusu Okuyucu </td><td> Balıkçı </td><td></td></tr></tbody></table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

Antimikrobiyal direnç hızlı algılama dirençli Mycobacterium tuberculosis (MTB) artışı kontrol etmek için çaba önemlidir. MTB antimikrobiyal duyarlılık testleri (AST), geleneksel oran agar yöntemi veya macrobroth test / BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) veya BacT gibi bir araç tarafından yapılmıştır ALERT (bioMérieux, Hazelwood, MO). Agar oranı yöntemi olarak kabul ederken, AST &quot;altın&quot; standardı, yoğun emek ve uyuşturucu ücretsiz agar göre uyuşturucu içeren agar üzerinde koloni sayımı yaparak direnç hesaplama gerektirir. Uyuşturucu ücretsiz orta karşılaştırıldığında ilaç içeren orta ≥% 1 büyüme varsa, organizma o ilaca dirençli olarak kabul edilir. Macrobroth yöntemleri enstrümantasyon ve ilk satırı ilaçlar için test kırılma noktası (&quot;kritik&quot;) ilaç konsantrasyonları (izoniazid, etambutol, rifampin ve pirazinamid) gerektirir. Burada anlatılan yöntemi 96 mikrotiter plate formatında piyasada bulunan [MYCOTB (TREK Diagnostics)] ve amikasin (her ikisi de ilk (izoniazid, rifampin, etambutol) ve ikinci basamak ilaçlar da dahil olmak üzere, tüberküloz tedavisinde kullanılan 12 antimikrobiyaller artan konsantrasyonlarda içeren, sikloserin, etionamid, kanamisin, moksifloksasin, ofloksasin, para-aminosalisilik asit, rifabutin, ve streptomisin). Pirazinamid, ilk satırı ilaç, asidik test koşulları için ihtiyacı nedeniyle mikrotiter plate dahil değildir. Mikrotiter sistemin avantajları, hem de kurmak kolaylığı ve geleneksel agar oranı (21 gün) ile karşılaştırıldığında daha hızlı dönüş süresi (14 gün) içerir. Buna ek olarak, et suyu ya da katı besiyeri kullanılarak hazırlanan inokulum plaka kurmak olabilir. Mikrotiter plate formatı yeni ve MTB laboratuvar benzersiz güvenlik sorunları beri, bu protokol, güvenli bir şekilde kurulum nasıl anlatacağız bu yana, inkübasyon ve mikrotiter plate okuyun.

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

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

Mycobacterium tuberculosis Antimikrobiyal duyarlılık testi zor ama hasta bakımı için kritik. Bu mikrotiter plate formatı 12 antimikobakteriyel ilaçların test sunar ve uygun tedavi yardımcı olacak minimum inhibitör konsantrasyon (MİK) değerleri sağlar.

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). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

38.0K Görüntüleme.  Mayo Clinic. Mycobacterium tuberculosis Antimikrobiyal duyarlılık testi zor ama hasta bakımı için kritik. Bu mikrotiter plate formatı 12 antimikobakteriyel ilaçların test sunar ve uygun tedavi yardımcı olacak minimum inhibitör konsantrasyon (MİK) değerleri sağlar.

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

İzleme Oluşumu ve Antifungal Duyarlılık Testi için 96 Şey microtiter Levha tabanlı Yöntemi Candida albicans Biyofilmler

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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) ...

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 ...

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 ...

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

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

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

Hızlı Bakteriyel Teşhis ve Antibiyotik Duyarlılık Testi için bir Kan Kültürü Peleti hazırlanması

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

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavansin için Başvuru sıvı mikrodilüsyon yöntemi<em&gt; İn Vitro</emAerobik Olarak Büyüyen Bakterilerin&gt; Duyarlılık Testi

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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 ...

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 ...

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 ...

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 ...