TB Kistik Fibrozis Kanada araştırma bursu kabul eder.

Araştırma Etik Kurulu (REB) toplamak ve insan denekler gelen balgam örnekleri saklamak için gerekli olduğunu unutmayın. Bu çalışmalar Sick Children REB # 1000019444 için Hastanesi tarafından kabul edildi. 1. Hazırlama CF Balgam Örnekleri <ol><li> kistik fibroz kliniğine rutin ziyaretler sırasında hastaların balgam örneği toplayın ve buz üzerinde tutmak. </li><li> işleme geçmesi, araştırma laboratuarına, koleksiyonun ilk saat içinde buz üzerinde balgam örneği taşıyın. </li></ol> 2. Balgam İşleme <ol><li> Elde edilen balgam örneğinin hacmini kaydedin. Örnek (örneğin, 2 parça PBS, 1 kısım örnek) hacminin 2x fosfat tamponlu tuzlu su (PBS) ekleyin. </li><li> Bir transfer pipet yardımıyla iyice örnek karıştırın. Vortex 1 dk tamamen karıştırmak için en yüksek ayarda örnek. </li><li> Kısım uygun sayı, 1.5 ml mikrosantrifüj tüpler içine yukarıdaki karışımın 1 ml ve 5.000 xg aşağı doğru döndürün4 ° C&#39;de 20 dakika. </li><li> santrifüj sonrasında, süpernatant kaldırmak ve pelet atın. </li><li> Filtre 0.22 um&#39;lik bir filtreden süpernatantı steril ve temiz bir mikrosantrfuj tübüne birikir. NOT: süzüntü sterilite LB agar kaplama ve sıvı ortamları aşılayarak test edilir. </li><li> ileride kullanmak üzere, -80 ° C&#39;de, balgam süpernatant saklayın. NOT: Balgam birden hastalar da filtrasyon aşağıdaki havuza edilebilir dan. </li><li> Balgam Süzüntü, 1/10 hac / hac istenen medyada (balgam 100 ul, ortam 900 ul) seyreltilmiş, kullanılmadan önce. NOT: Burada, standart lizojeni suyu (LB) ortamı kullanılmıştır. </li></ol> Biyofilm Oluşumunun 3. odacıklı coverglass Yöntemi <ol><li> (200 rpm) çalkalanarak 37 ° C&#39;de arzu edilen ortam ilgi gece boyunca bakteriyel izolatı büyütün. Not: Farklı bakteri çok sayıda kullanılmış, Pseudomonas aeruginosa, Staphylococcus aureus klinik izolatlarının dahil,Burkholderia cepacia karmaşık ve Achromobacter xylosoxidans. ortam seçimi Ancak LB ortamı ilk uygulamalar için kullanılabilir suşları ve ilgi koşullara bağlıdır. </li><li> Gecelik kültürden taze ortam 4 ml kültür, 40 ul koyun ve yaklaşık olarak 0.5, 600 nm (OD 600) de optik yoğunluğu ile bir kültür elde etmek için (200 rpm) çalkalanarak 37 ° C&#39;de 3-4 saat boyunca büyümeye -0.6. </li><li> % 10 balgam süzüntü ya da (kontrol olarak) balgam filtrattan olmadan istenilen medyada 1/5 adım 3.2 kültürünü sulandırmak. Balgam filtre bulunan konsantrasyonu test edilebilir (örneğin,% 50 ya da% 100). </li><li> slayt odaları kuyu tohum seyreltme 200 ul kullanın. </li><li> Bakteriler çalkalamadan 37 ° C&#39;de 4 saat eklemesini sağlar. </li><li> 4 saat sonra, medyayı çıkarın ve yavaşça 1x taze medya ile biyofilm yıkayın. 200 ul taze medya ile değiştirin. NOT: biyofilm üzerindeki fres balgam etkilerini araştırmakh medya balgam süpernatant içermelidir. </li><li> biyofilm mikroskopi için saatine kadar yıkama olmadan Ortam her 12 saat yerine, sallayarak olmadan 37 ° C&#39;de zaman istenilen miktarda büyümeye olanak sağlar. NOT: biyofilm antibiyotik duyarlılık balgam Süpernatantları etkisini incelemek için, antibiyotikler biyofilm büyüme 24 saat aşağıdaki ortama ilave edilir ve boyama ve biyofilm görüntüleme kadar medyada muhafaza edilir. </li></ol> 4. Boyama Biyofilmler ve Konfokal Mikroskop <ol><li> İstenilen büyüme süresi (24-48 saat en iyi şekilde çalışır) ardından, oda kuyulardan ortamını çıkarın ve yavaşça steril PBS 300 ul ile iki kez her odasına yıkayın. </li><li> gerekli çözeltinin her ml (canlılık kitte sağlanan), her boyanın 1 ul karıştırılarak biyofilm boyama karışım hazırlayın. Su veya ortam çözelti içinde boya sağlayın. NOT: Su üretici tarafından tavsiye edilmektedir. </li><li> bölmeli covergla her bir kuyu için boya karışımının 200 ul eklep, 45 dakika süreyle karanlıkta, oda sıcaklığında inkübe edin ve. </li><li> odalarından boyama karışımı çıkarın ve steril PBS 300 ul her bir yıkama. PBS çıkarın ve tatlı su ya da medya ile değiştirin. </li><li> konfokal mikroskobu ile biyofilm görselleştirme ile devam edin. </li></ol> 5. Konfokal Mikroskop ile biyofilm görselleştirme <ol><li> hemen (1 saat içinde) boyandıktan sonra odalarına lekeli biyofilm okuyun. Bir seferde 2 8-kuyu odalarına 1 boyanarak slaytların görselleştirme gecikmeyi en aza indirmek. </li><li> edinimi için uyarma ve filtre setleri için lazerler ile konfokal mikroskop kullanılarak görüntüleme gerçekleştirin. NOT: Burada, spektral borealis lazerler ile dönen disk konfokal sistemi (Yeşil: 491nm, Kırmızı: 561 nm) uyarma için kullanılmıştır. 515/40 ve 624/40 emisyon filtre setleri biyofilm canlılığı kiti lekeleri görselleştirmek için kullanılmıştır. </li><li> kamera ile konfokal mikroskop bir 25X su objektif kullanarak fotoğraf çekmek. </li><li&gt; Kullan Z-Yığınları biyofilm modeli. Her 0.5-1 mikron biyofilm son olarak odak çerçeveye ilk odak düzlemi başlayarak görüntü almak (genellikle 30-80 mikron 48 saat biyofilm kapsayan) </li><li> her bir kuyunun 3-5 görüntüleri çekmek. NOT: Böylece 8 de odacıklı coverglass için, 24-40 görüntüleri oluşturulur. </li><li> Analiz için görüntüleri kaydedin. NOT: OME-TIFF dosyalarını COMSTAT 18,19 kullanılarak analiz edilecek şekilde Görüntüler kaydedilmesi gerekir. biyofilm görüntü analizi için talimatlar http://www.comstat.dk/ adresinde bulunabilir. görüntüleri ithal edildikten sonra, bu tür her kanala (kırmızı ve yeşil) için ortalama kalınlığı, biyokütle ve yüzey kapsama gibi parametreler analiz edilebilir. </li></ol>

<ol><li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infections+with+biofilm+formation%3A+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy%5BTitle%5D)+AND+%22Pediatr.Infect.Dis.J%22%5BJournal%5D)">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li>
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<li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li>
<li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm-specific+antibiotic+resistance%5BTitle%5D)+AND+%22Future+Microbiol%22%5BJournal%5D)">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li>
<li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li>
<li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario%2C+Canada%5BTitle%5D)+AND+%22Can.+J.+Microbiol%22%5BJournal%5D)">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li>
<li>Vidya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis%5BTitle%5D)+AND+%22Eur.J.Clin.Microbiol.Infect.Dis%22%5BJournal%5D)">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li>
<li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells%5BTitle%5D)+AND+%22Biochem.Biophys.Res.Commun%22%5BJournal%5D)">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li>
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<li>LaFayette, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses%5BTitle%5D)+AND+%22Sci.Adv%22%5BJournal%5D)">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199(2015).</li>
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<li>Kennedy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms%5BTitle%5D)+AND+%22Antimicrob.Agents+Chemother%22%5BJournal%5D)">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li>
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Bu tarifnamede tarif edilen yöntemler eksojen ürünlerin mevcudiyetinde büyütülen bakteri biyofilmlerin görselleştirme için izin verir. bu tip bir sistem kullanılıyorsa Beklendiği gibi, exoproducts üretimi önem taşımaktadır. Örneğin, Ditiyotreitol (DTT), genellikle örnekleri sıvılaştırılması yardımcı insan balgam kullanılır. Bununla birlikte, tek başına DTT etkisi biyofilm gelişimi ve canlılığı azaltılmış olur (veriler gösterilmemiştir). Bu durumda, tüm koşullar için uygun kon...

Bakteri biyofilmleri birbirlerine bağlanmış ve bir öz salgılanan matris kaplı olan mikroorganizmaların gruplarıdır. 1,2 Klasik olarak, fiziksel olarak akış koşulları altında meydana gelen bir abiyotik veya biyotik yüzeye bağlı bakteri temsil etmektedir. Biyofilmler, aynı zamanda bu tür test tüplerinde oluşan ısı havuzları veya ince zarlar hava-sıvı arayüzü itibarıyla yüzeylerin statik koşullar altında (akış olmaması) ve distal, büyümeye gösterilmiştir. Onlar biyolojik kirlenme, paslanma ve tıkanmalara neden su rezervuarları veya borularda oluşabilir gibi bu biyofilm uzun, endüstriyel prosesler için büyük bir kayba ortamında tanınan ve vardır oylandı. 3,4 Onlar kateter ile ilişkili enfeksiyonlar, kistik fibroz hastalarında akciğer enfeksiyonları, yanı sıra çok sayıda diğer enfeksiyonlarda rol gösterilmiştir olarak biyofilm, aynı zamanda sağlık ortamlarında kritik önem taşımaktadır. Biyofilm enfeksiyonlarının işaretlerinden 5,6 biri de olanBakterilerin antibiyotiklere duyarlılığının katlanmış ve doğuştan gelen bağışıklık sistemi tarafından açıklığı engelli. Biyofilm tabanlı enfeksiyonu kapsayan 7-9 en iyi okudu, klinik olarak anlamlı senaryolar kronik Pseudomonas aeruginosa biyofilm bulaşmış kistik fibrozis (KF) olan hastalarda ortaya çıkar. P. aeruginosa çok zor tedavi yapmak, kronik enfeksiyon kurulması sırasında bir dizi değişiklik uğrayabilir. 10,11 Biyofilmler farklı olarak doğuştan gelen bağışıklık etkinleştirmek ve inflamasyonu sürebilirim. Bu enfeksiyonlar KF hastalarında artmış morbidite ve mortalite neden 12-14 gibi, bu bağlamda biyofilm gelişimini etkileyen faktörleri anlamak önemlidir. Yeni yapılan bir çalışmada konak faktörleri P. aeruginosa biyofilm agrega oluşumunda kritik olduğunu göstermektedir. 15 Bu biyofilm antibiyotik ve konak savunma mekanizmaları indirgenmiş duyarlılık katkıda bulunur. preseörneğin nötrofil elastaz gibi CF akciğer mikroorganizmaların salgılanan ürünler olarak ev sahibi türevli faktörlere nce, büyük ölçüde biyofilm oluşumunu ve gelişimini modüle potansiyeline sahiptir. 16 Ayrıca, biyofilm sayısız yolların ifadesini modüle ve inflamasyonu başlatmak için ev sahibi ile etkileşim. Böyle standart kristal viyole deneyi olarak yüksek kapasiteli yöntemleri, biyofilm süreci ile ilgili olarak bazı bilgiler sağlayabilir iken, bu faktörlere yanıt olarak biyofilm görselleştirme daha derinlemesine bilgi sağlar. Bu yazıda in vitro biyofilm gelişimini incelemek için KF&#39;li hastaların balgam faktörlerini kullanmak için bir yöntem açıklanmaktadır. Bu yöntem, bir ticari biyofilm canlılığı kiti kullanılarak balgam içeren konak faktörlerine maruz kalan biyofilm hızlı görselleştirme sağlar. Bu teknik, görsel exogeno mevcudiyetinde biyofilm büyüme sırasında oluşan değişiklikleri tespit etmek için kullanılabilirUS ürünleri ve çeşitli koşullar altında biyofilm gelişme değişiklikleri analiz etmek için geliştirilmiş bir yöntemi temsil eder.

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

Biyofilmler kendinden salgılanan matris kaplı bakteri gruplarından oluşur. Bu endüstriyel kirlenme hem de bir çok sağlık ile ilgili enfeksiyonların gelişmekte ve önemli bir rol oynamaktadır. insan hastalığında en iyi tarif ve çalışılan biyofilm biri kistik fibroz hastalarının kronik akciğer enfeksiyonu meydana gelir. konak bağlamında biyofilm öğrenim görürken, birçok faktör biyofilm oluşumunu ve gelişimini etkileyebilir. Ev sahibi faktörleri biyofilm oluşumunu ve gelişimini etkileyen nasıl tespit etmek amacıyla, balgam yüzer şeklinde bir ev sahibi türevli faktör varlığında, biyofilm büyümeye statik odacıklı coverglass yöntemi kullanılmıştır. Bakteri odalarına tohumlanmış ve balgam süzüntüler maruz kalmaktadır. büyüme 48 saat sonra, biyofilm önce konfokal mikroskopi ve analiz için ticari bir biyofilm canlılığı kiti ile boyandı. Görüntü alınmasının ardından, biyofilm özellikleri farklı yazılım platformları kullanarak değerlendirilebilir.Bu yöntem antibiyotikler de dahil olmak üzere, farklı maddelerin varlığında biyofilm büyüme önemli özelliklerini görselleştirmek sağlar.

Deney genel tasarımı, Şekil 1 &#39;de gösterilmiştir. Bu protokolün kullanılması farklı zaman aralıklarında (örneğin, 24, 48 veya 72 saat) için yetiştirilen biyofilm değişiklikleri görselleştirmek için uygun bir yöntem sağlar. Önemli bir şekilde, örneğin, balgam süzüntüler gibi dış sinyaller, biyofilm gelişme değişiklikleri görselleştirmek için ilave edilebilir. Şekil 2&#39;de de görüldüğü gibi,% 10, ba...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Research

JoVE Journal

Immunology and Infection

8.3K Görüntüleme.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Video: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-kültür Modelleri Pseudomonas aeruginosa</emCanlı İnsan Havayolu Hücreler Yetiştirilen> Biyofilmler

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

İmmünoloji ve Enfeksiyon

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8-iyi Odası Slayt in vitro Biyofilm Oluşumu

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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 ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Karşı Antibiyotik Etkinlik Test Yapay Balgam Orta Kullanımı<em&gt; Pseudomonas aeruginosa</em&gt; Kistik Fibrozis Akciğer Daha İlgili Koşullar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

WinCF Modeli - Bir Mukus bir Ucuz ve Çekilebilir Microcosm Akciğer İnfeksiyonları Mikrobiyoloji Eğitim için bronşiole Takıldı

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Antimikrobiyallerin İn vitro Biyofilm Mimarisi Üzerindeki Etkilerinin COMSTAT Yazılımı Kullanılarak Ölçülmesi

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Kistik Fibrozis Akciğerinde Pseudomonas aeruginosa ve Staphylococcus aureus Biofilms Ex vivo Modelinde Antibiyotik Etkinliği Testi

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

<meta charset="utf8"></head><body>Antimikrobiyal Test ve Mikrobiyal Dinamik Analizi için Uyarlanabilir CF Modeli

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

Özet

Daha Fazla Video Keşfet

Co-kültür Modelleri Pseudomonas aeruginosa Biyofilmler

8-iyi Odası Slayt in vitro Biyofilm Oluşumu

Büyüme Mycobacterium tuberculosis Biyofilmler

Karşı Antibiyotik Etkinlik Test Yapay Balgam Orta Kullanımı

Araştırma yöntemleri

WinCF Modeli - Bir Mukus bir Ucuz ve Çekilebilir Microcosm Akciğer İnfeksiyonları Mikrobiyoloji Eğitim için bronşiole Takıldı

Antimikrobiyallerin İn vitro Biyofilm Mimarisi Üzerindeki Etkilerinin COMSTAT Yazılımı Kullanılarak Ölçülmesi

Kistik Fibrozis Akciğerinde Pseudomonas aeruginosa ve Staphylococcus aureus Biofilms Ex vivo Modelinde Antibiyotik Etkinliği Testi

Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi

Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi