This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.

A. Biyofilm Oluşumunun 1. Kristal Violet Kantifikasyonu baumannii S1 <ol><li> A. 5 ml&#39;lik bir kültür büyütün lizojeni suyu içinde baumannii S1 (LB) 16 saat süre ile bir çalkalama inkübatöründe (220 rpm) 30 ° C&#39;de (tripton 10 g / L, bira mayası özütü 5 g / L). </li><li> A. kültürünü ayarlayın istenen OD 0.8 600 baumannii S1. Bir 96-yuvalı plaka kullanarak, bakteri kültürünün aşılanması: saflaştırılmıştır GKL enzim (40 mg / ml) 10 ul ihtiva eden taze LB (1 100 seyreltme); Yeni kültürün nihai hacim 100 ul olduğunu. </li><li> Hem de bir kontrol kültürü hazırlayın; Bu herhangi bir enzim içermez. Çoğaltır istenen sayıda elde etmek için benzer koşullar tekrarlayın. </li><li> Bir kapak ile plaka örtün ve sızdırmaz şekilde kapalı bir 10 litrelik plastik bir kap içine koyun. Ortamı yavaşça çıkarmadan önce 3 saat süre ile 30 ° C &#39;de plaka inkübe edin. </li><li> Oyuk taze LB ortamı bir 100 ul ilave edin ve 30 ° C&#39;de 21 saat boyunca inkübe plaka. </li&gt; <li> Inkübasyon ikinci döneminden sonra, yavaşça tüm ortamı çıkarın. 200 ul steril su ile planktonik bakteri hücresini yıkayın. Yıkama sırasında hücrelere sadece minimal rahatsızlık olduğundan emin olun. </li><li> Her çukuruna% 1 kristal viyole çözeltisi 100 ul ilave edin ve oda sıcaklığında 15 dakika inkübe edilir. 200 ul steril su ile kuyusunu yıkayarak kristal viyole çözüm çıkarın. Iki kez yıkama tekrarlayın. </li><li> Her çukuruna% 33 asetik asit, 100 ul eklenir ve yavaşça çalkalanarak 15 dakika boyunca inkübe edilir; Bu boya eriyecektir. </li><li> 600 nm &#39;de kristal viyole absorbansı ölçülerek oluşan biyofilm miktarının belirlenmesi. Kristal viyole miktarı oluşturulmuş, biyofilm miktarı ile orantılıdır. </li></ol> A. 2. konfokal lazer tarama mikroskobu baumannii S1 Biyofilm <ol><li> A. 5 ml&#39;lik bir kültür büyütün 16 saat süre ile bir çalkalama inkübatöründe (220 rpm) 30 ° C&#39;de LB baumannii S1. </li><li> ReklamA. sadece kültür istenen OD 0.8 600 baumannii S1. 35 mm bir cam tabanlı μ-çanağı kullanılarak, bakteri kültürünün aşılanması: saflaştırılmıştır GKL enzim (40 mg / ml) 30 ul ihtiva eden taze LB (1 100 seyreltme); Yeni kültürün nihai hacmi 1 ml olmuştur. </li><li> Kapaklı μ-Dish Kapak ve mühürlü 10 litrelik plastik kaba koyun. Ortamı yavaşça çıkarmadan önce 3 saat süre ile 30 ° C &#39;de μ-Bulaşık inkübe edin. 1 ml &#39;lik bir toplam hacme getiren saflaştırılmıştır GKL enzim ve taze ortam 30 ul ekle. 30 ° C&#39;de bir 21 saat daha inkübe edilir. </li><li> Adımı yineleyin 2.3 ve 30 ° C sıcaklıkta bir 24 saat μ-Dish kuluçkaya yatmaktadır. Sonra, yavaşça ortamı çıkarın. </li><li> Μ-çanağı Hank dengeli tuz çözeltisi (HBSS) içinde eritildi 5 ug / ml Alex Fluo 488-konjuge Buğday tohumu aglutinini (WGA) 500 ul ilave edin ve 30 dakika boyunca 37 ° C&#39;de inkübe edin; Bu oluşan biyofilm leke olacaktır. T boyama çözüm çıkarın ve yıkayınO u-Dish HBSS 2 ml. Yıkama adımı bir kez daha tekrarlayın. </li><li> % 3.7 formaldehit HBSS içinde çözüldü ve 30 dakika boyunca 37 ° C&#39;de inkübe 500 ul ilave et; Bu μ-Dish üzerine biyofilm çözecektir. HBSS 2 ml ile bir kez μ-bulaşık yıkama ve daha sonra tamamen uzaklaştırın. Μ-Bulaşık biyofilm sabit önceden CLSM görüntüleme 4 ° C de karanlıkta tutulmuşlardır edilebilir. </li><li> CLSM görüntüleme ve analiz için yığının başına 0.21 mikron arayla görüntü başına 97 yığınlarının oluşturmak için 63 kat büyütme kullanın. </li></ol>

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The authors declare that they have no competing financial interests.

Deney iki seti olarak, A. baumannii S1 bakteriler 15 tarafından oluşturulan biyofilm miktarını azaltabilir yüksek tuz konsantrasyonu olarak NaCl&#39;siz LB ortamında kültive edilmiştir. Bu tür eserin varlığı oluşturduğu biyofilm miktarını, hem de farklı tedavi koşulları arasında nisabı-söndürme enzimlerin etkisini hafife olabilir. Katalitik inaktif enzim kullanımı enzim sekestrasyonun olası etkilerini ortadan kaldırmak için bir negatif kontrol olarak önemlidir. Inaktif enzimler, çekirdek molekülleri ayırmak bile Şekil 1&#39;de görüldüğü gibi, biyofilm oluşumu kesintiye değildir. A. baumannii S1 96 oyuklu plakanın her bir kuyucuğu içinde hava ve sıvı arayüzü arasında hassas bir halka benzeri biyofilm yapısı oluşturur. Dolayısıyla, bakteri biyofilmlerin tesadüfi kaldırılmasını önlemek için medya çıkarma ve yıkama adımları sırasında aşırı ajitasyon önlemek için çok önemlidir. LB ortamı, her bir inkübasyondan sonra çıkarıldıplanktonik hücreler ortadan kaldırır. Buna ek olarak, 96 kuyucuklu plak hava akımı meydana gelen pertübasyonları en aza indirmek ve biyofilm oluşumunu destekleyen bir mikro anaerobik ortam oluşturmak için bir 10 litrelik plastik kap içine yerleştirilmiştir. Bir 96-yuvalı plaka içinde Biyofilm kantitatif da her oyuğa ilave kristal viyole miktarına bağlıdır. Aşırı kristal viyole kuyuya ilave edilmesi halinde, yıkama adımları sayısı arttırılabilir. Kristal viyole boyama deneme biyofilm bozulmasına nisabı-söndürme verimliliği göreceli bir karşılaştırma sağlar. Kristal viyolet biyofilm oluşumunu ölçmek için niceliksel olmasına rağmen, sadece çekirdek giderici enzimlerin katalitik etkinlik açısından yarı kantitatif. Doğru istatistiksel karşılaştırma için yeterli örneklem büyüklükleri farklı tedavi grupları için önemlidir. Uç da sonuçların hatalı biçimde önlemek için kaldırılmalıdır. Konfokal görüntüleme ve bakteriyel biyofilm analizi usef olannisap-söndürme enzimlerin nitel etkilerinin değerlendirilmesi için ul aracı. Kullanıcı (aktif veya inaktif) uygulanan nisap-söndürme enziminin Çeşidi farkında Ancak, analiz için biyofilmi seçme süreci önyargı için potansiyel bir kanal olabilir. Önyargı olasılığını önlemek için, deney kullanıcıdan enzim bilgileri hariç veya seçim için rasgele bir yaklaşım kullanmak üzere tasarlanmış olabilir. Tarafsız bir seçim stratejisi de CLSM tarafından biyofilm morfolojileri iyi niceliksel karşılaştırılmasına olanak sağlamaktadır. Bununla birlikte, bu bakteriyel biyofilmlerin ilgili çekirdek giderici enzimlerin sonucunu değerlendirmek için bir yöntem tarif ilk çalışmadır. Kristal viyole boyama biyofilm bozulmasına nisabı-söndürme enzimlerin programı gösterdi ve enzimin modi operandorum hücre sayıları ve virulans faktörü ekspresyonunu azaltarak sınırlı olmadığını ortaya çıkarmıştır. Bu arada, CLSM analizi ile belirlenen morfolojik değişiklikler de mümkündür Moleküller içgörüler sağlayabilirBu enzimlerin ar hedefler. Geçerli deney bakteriyel biyofilm üzerindeki enzimler ön etkilerini araştırmak amacıyla tasarlanmış olmasına rağmen, protokol bakteriyel büyüme sonrasında enzimi ekleyerek önceden oluşturulmuş biyofilm üzerinde enzimin etkisini değerlendirmek için veya serum kullanımı yoluyla fizyolojik koşullar altında yetkinliğini incelemek için modifiye edilebilir kültürü için benzeri koşullar. GKL enzimleri incelemek için kullanılan protokol biyofilm bozulması onların ilişkiyi araştırmak için başka nisap giderici enzimler ve patojenlere uzatılabilir.

Bulaşıcı hastalıklar için tedavi seçenekleri, antibiyotik ilaçlar 1 bir geniş bağışıklık çok ilaca dayanıklı bakterilerin hızlı artışın daha karmaşık bir. Dirençli bakteri kaynaklı enfeksiyonlardan yüksek morbidite ve mortalite oranları ile, ilaç geliştirme süreçlerini yükselmek ve / veya tedavi seçenekleri geliştirmek için daha iyi anti-bakteriyel alternatifleri keşfetmek için bir ihtiyaç vardır. Son zamanlarda, anti-virülans yaklaşımı dolayısıyla direnç mekanizmaları 2 risklerini azaltmaya olmayan bakteri yöntemlerle virülansını önlemede potansiyeli verilen faiz kazanıyor. Nisabı algılamalı bakteriyel virülans ve bu sinyal olgusunun bozulması bir &#39;ana şalteri&#39; patogenezinde 3 karşı vadeden bir anti-virülans yöntemdir olduğunu. Kritik bakteriyel nüfus yoğunluğu ulaşıldıktan sonra virülansına başlangıcı dışı ortamda nisabı moleküllerin birikimini gerektirir. As quoROM molekülleri, komşu reseptörlerine bağlanma hastalık oluşturma faktörlerinin aktivasyonu gibi antibiyotik direnci ve biyofilm 4 ile ilişkili genlerin yol açar, hücre içi matris geri yayılır. Genel, nisap algılama bozulması Birincil metabolik etkilemeden nisabı molekülü ve reseptör etkileşimi inhibe içerir. Bu nedenle, hücre büyümesi üzerinde doğrudan bir ima yoktur. Fitness tehlikeye olmadığından, gelişmeye ve bu tür tedaviler 5 karşı direnç kazanmak için bakteriler için en az seçim baskı var. Buna ek olarak, çekirdek-algılama bozulması, anti-bakteriyel maddeler karşı koruma sağlar ve immün yanıtları ana biyofilm, olduğu gibi, doğal bakteri koruyucu mekanizmaların engelleyebilir. Bu Dünya&#39;da mikropların% 99 th içinde yaşayan mikroorganizmalar için çok önemli hayatta kalma avantajı kazandıran, karmaşık biyofilm gibi matrisler var olduğu tahmin edilmektedirese yapılar 6. Daha da önemlisi, bu sesil etki oluşumu en kalıcı ve kronik hastane kökenli enfeksiyonların 7 nedenidir. Acinetobacter baumannii küresel hastane kökenli enfeksiyonlarla ilişkilidir ve virülans büyük ölçüde yeterli çoğunluk algılama atfedilir Belli başlı insan patojenleri biridir aracılı biyofilm oluşumu 8. Çekirdek giderici enzimler, Gram-negatif bakterilerin 9 tarafından üretilen N -asil homoserin laktonlar (AHL&#39;ler) olarak bilinen bir bileşikler grubu hedef, çekirdek-kaynaklı sinyal transdüksiyonunun bozulması başarıyla kullanılmıştır. Çeşitli çalışmalar da biyofilm 10,11 virülans faktörü ekspresyonu ve hücre sayılarının azaltılması yoluyla bakteriyel hastalık oluşumunun engellemek için bu enzimlerin kullanımına bağlı genişletmiştir. Ne yazık ki, bakteriyel patojenler tarafından biyofilm oluşumuna karşı nisabı-söndürme enzimlerin etkin kullanımı palpabl gösteri eksikliği kalır.Yeniden A. bozmaya, nisap inhibitörleri (AHL analogları), yerine nisabı giderici enzimler kullanmayı girişimleri olmuştur baumannii biyofilm oluşumu 12. Küçük moleküller inhibitörleri kullanılarak bu yöntem, geçerli bir yaklaşım olsa da, translasyon kullanımlarda, bio-mevcudiyetini sürdürülmesi zor olabilir. Enzimler terapötik etkileri için biyomedikal cihazların yüzeylerine immobilizasyon doğru daha uygundurlar Aksine, katalitik çekirdek giderici enzimlerin kullanılması biyolojik sorunu aşmak olabilir. Burada, kristal viyole boyama ve konfokal lazer tarama mikroskobu (CLSM) kullanarak, Geobacillus kaustophilus (GKL) bakteriyel biyofilm oluşumu üzerinde 13&#39;ten mühendislik nisap-söndürme lactonases etkilerinin bir değerlendirmesini açıklar. Bu çalışma klinik olarak anlamlı A. biyofilm bozulması ilk başarılı gösteri nisap giderici enzimler kullanılarak baumannii S1 süzün. Yöntem tarifBu çalışmada, patojenik Gram-negatif bakterilere karşı sonraki terapötik geliştirme çabaları diğer çekirdek giderici enzimlerin etkinliğini değerlendirmek için yararlıdır.

<table border="0" cellpadding="0" cellspacing="0" width="780">
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tryptone&#160;</td>
			<td style="width:151px;">BD</td>
			<td style="width:132px;">211705</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast Extract</td>
			<td style="width:151px;">BD</td>
			<td style="width:132px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">96-well plate</td>
			<td style="width:151px;">Costar</td>
			<td style="width:132px;">3596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Crystal Violet</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:132px;">C6158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetic Acid</td>
			<td style="width:151px;">Lab-Scan</td>
			<td style="width:132px;">PLA00654X</td>
			<td>Caution: Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#956;-Dish</td>
			<td style="width:151px;">Ibidi</td>
			<td style="width:132px;">80136</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Alex Fluo 488-conjugated WGA</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:132px;">W11261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:132px;">141475095</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:132px;">F8775</td>
			<td>Caution: Corrosive&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Synergy HT Microplate Reader</td>
			<td style="width:151px;">BioTek</td>
			<td style="width:132px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1X-81 Inverted Fluorescence Microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:132px;"></td>
			<td></td>
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	</tbody>
</table>

anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Doğal tipte GKL ve geliştirilmiş GKL çift mutant (E101G / R230C) kristal viyole niceleme deneyde, iki çekirdek giderici enzimler biyofilm oluşumunu kesintiye uygulanabilirliğini göstermek için kullanılmıştır. Her iki enzim karşı lactonase etkinliğini göstermek için gösterilmiştir 3-hidroksi-dekanoyil-L-homoserin lakton (3-OH-Cı 10 -HSL), A. kullandığı başlıca çekirdek molekülün baumannii S1 14. Biyofilm bozulma geçerli bir değerlendirme için, (daha önce AHL ligandları tecrit olmayan gösterilmiştir) kendi katalitik aktif enzimler aynı zamanda anında kontroller (GKL D266N mutant ve GKL E101G / R230C / D266N mutant) olarak alındı. Yabani tip A kullanılmasının yanı sıra baumannii S1, bir mutan Δ Abai suşu (AHL sentaz eksikliği) biyofilm oluşturma yeteneği de test edilmiştir. Çekirdek giderici enzimler, doğal tipte GKL ve GKL E101G / R230C mutant Hem anlamlı öncesi biyofilm oluşumunu azaltmak mümküntedavi A. baumannii S1 kültürleri (n = 10; 0.0001 ≤ p-değeri) (Şekil 1). Bununla birlikte, her iki enzim için biyofilm bozulma derecesi 3-OH-Cı 10 -HSL karşı etkinlikleri orantılı değildir. GKL E101G / R230C mutant devir hızı (k kedi) 3-OH-C 10 -HSL 14 karşı yabani tip GKL altı kat daha hızlıdır. Ancak, enzimler arasındaki biyofilm bozulması ölçüde farklılık sadece iki yönlüdür. <img alt="figür 1" src="/files/ftp_upload/53243/53243fig1.jpg" /> Şekil 1. A. baumannii biyofilm bozulması deneyi. biyofilm oluşturma kristal mor boyama ile ölçülmüştür. Kırmızı sütunlar vahşi tip A. tarafından oluşturulan biyofilm miktarını temsil çekirdek giderici lactonases ilavesi olmadan baumannii ve Δ Abai mutantı. Mavi sütunlar amo temsilvahşi tip A. tarafından oluşturulan biyofilm unt inaktif GKL D266N mutantın doğal tip GKL inaktif GKL E101G / R230C / D266N mutantı ve GKL E101G / R230C mutant: Dört farklı GKL enzimlerin varlığında baumannii. **** P- ≤ 0.0001 değeri. Antimikrobiyal Ajanlar ve Kemoterapi 58 izni ile çoğaltılmıştır, 1802-1805 (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> A. Konfokal görüntüleme baumannii S1 biyofilm oluşumu bu sesil alanların yapısal morfolojisi, çekirdek-söndürme etkileri nitel ve nicel ölçü sağlamak için kullanıldı. Geliştirilmiş GKL E101G / R230C mutant ve zeolitler katalitik açıdan inaktif enzim karşılaştırma için kullanıldı. Diferansiyel görüntü kontrastı (DIC) görüntüsü (geliştirilmiş GKL E101G / R230C mutant ile tedavi, biyofilm boyutunda bir azalmaya neden olduğunu gösterdi Figüre 2). Floresans görüntüleri analiz da geliştirilmiş GKL E101G / R230C mutant (Tablo 1) ile tedavi edildiğinde yüzey alanı, biyokütle ve biyofilm ortalama kalınlıkta azalma olduğunu göstermiştir. <img alt="Şekil 2," src="/files/ftp_upload/53243/53243fig2.jpg" /> A. Şekil 2. Temsilcisi konfokal lazer tarama mikroskobu görüntüleri baumannii biyofilm. A. baumannii biyofilm etkin GKL E101G / R230C / D266N mutant (A) ve GKL E101G / R230C mutant (B) ile muamele edilmiş ve Alexa ile boyandı Fluor WGA 488-konjüge. Biyofilm (solda) ve biyofilm (sağda) floresan görüntüleri DIC görüntüleri temsilcisi xy (merkez), yz (sağda) ve xz (alt) bölümler için gösterilmiştir. Antimikrobiyal Ajanlar ve Kemoterapi &lt;izniyle yayınlanmıştırstrong&gt; 58, 1802-1805 (2014). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <table fo:keep-together.within-page="1"><tbody><tr><td></td><td colspan="3"> SD a ± Değeri </td></tr><tr><td> Karakteristik </td><td> Hiçbir tedavi </td><td> Inaktif mutant ile tedavi </td><td> E101G / R230C mutant ile tedavi </td></tr><tr><td> Biyokütle (um 3 / mikron 2) </td><td> 2.57 ± 1.65 </td><td> 3.39 ± 1.33 </td><td> 1.37 ** 0.20 ± </td></tr><tr><td> Ort kalınlığı (um) </td><td> 3.68 ± 2.51 </td><td> 3.41 ± 1.31 </td><td> 1.21 ** 0.21 ± </td></tr><tr><td> Yüzey alanı (um) </td><td> 235,920.59 ± 79,456.46 </td><td> 209,872.6 ± 115,094.7 </td><td> 115,354.9 * ± 7,630.3 </td></tr><tr><td colspan="4"> Bir n = 10 resim yığınları. ** P ≤ 0,001; *, P ≤ 0,05 inaktif E101G / R230C / D266N mutant ile muamele ile karşılaştırıldığında. </td></tr></tbody></table> Tablo 1. A. baumannii biyofilm yapısal kantitasyon.

Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Engineered Nisabı giderici Lactonases kullanarak Patojen Biyofilmler Anti-öldürücü bozulması

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K Görüntüleme.  Yong Loo Lin School of Medicine. Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Video: Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Tek hücreli Analizi Bacillus subtilis</emFloresan Mikroskobu ve Akım Sitometri Kullanma> Biyofilmler

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

İmmünoloji ve Enfeksiyon

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 Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Ana Hücreler ile Derneği Patojen Mantarlar miktarının Image for Sitometrinin kullanın

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Hücresel ve Biyofilmler Ekstrasellüler Bileşenleri Eşzamanlı Kantitasyonu

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Yeni Bir Yöntem Filamentli Mantar kolonileri ile ilgili Bakteriyel Biyofilmler Niteliksel Çok ölçekli Analizi için kullanılması Eşodaklı ve Elektron Mikroskobu

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Parafin gömme ve ince mikrobiyal koloni biyofilmler mikroskobik analizlerin yapıldığı için kesit

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

Bir biyogüvenlik seviye 2 ortamda yüksek patojenik Coronaviruses çalışmaya Pseudotyped parçacıklar imalatı

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Konfokal Mikroskopi Ile Krallık Lararası Biyofilmlerde Hücre Dışı pH'ın izlenmesi

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Aydınlatma ve Görüntüleme Candida albicans Biyofilmler

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...