We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. Biyofilm Başlatma <ol><li> bir besleyici zengin ortam içinde biyofilm üreten bir organizmanın bir kültürü büyütün. Bir gliserol stokundan Mueller-Hinton ortamı, 10 ml Staphylococcus aureus suşu Newman inoküle. S. taşıma ile ilgili tüm çalışmaları gerçekleştirmek eldiven ve bir biyogüvenlik kabini içinde aureus. </li><li> bir döner sallayıcı (100-200 rpm) ile 37 ° C&#39;de 16 saat süreyle inkübe edilir. </li><li> Bir spektrofotometre ve standart bir küvet (: 1cm yol uzunluğu) ile kültür OD 600 belirleyin. </li><li> Formül kullanılarak chelexed Roswell Park Memorial Institute ortam (cRPMI) 9, 0.1 OD 600 (inokulum) ile inokulum 20 mi hazırlamak için gerekli kültür hacmi (V) hesaplayın: [V = 20 mi x 0.1 / OD 600 (kültür )] Not: Referans 9 cRPMI hazırlanması ayrıntıları sağlar. <ol><li> 10.000 x g&#39;de 1 dakika için iplik, bir santrifüj tüpüne ve topak hücrelere (yukarıdan) hesaplanan kültür hacim. hazırlık aşamasında içinbiyofilm inokulum yeniden 20 ml cRPMI harcanan büyüme ortamı ve tekrar süspansiyon hücre pelletini aspire. </li></ol></li><li> 25 ml&#39;lik bir hazne içine inokulum dökün. </li><li> Dikkatle yukarı doğru çekerek steril 96-yuvalı peg plaka kapağı çıkarın. Not: Bakım aşağı kapağın sarkan mandal kirletmesine dikkat edilmelidir. kapak bir biyogüvenlik kabini içinde temiz bir yüzeyin üstünde ters yerleştirilebilir. </li><li> 200 ul çok kanallı pipet kullanarak, taban plakasının G sıraları her oyuğuna 125 ul inokulum ekleyin. </li><li> sterilite kontrolü olarak H sırası, her oyuk 125 ul steril cRPMI orta doldurun. </li><li> peg-kapağı atın ve aşağı bakacak şekilde mandal ile plaka alt yukarıda tutun. Dikkatle kendi kuyuları ile bireysel mandal aynı hizaya getirin. plaka ve mandal arasındaki fiziksel temastan kaçının. hizalanmış sonra, dikkatlice aşağı kapağı indirin. mandal sonra gerçekleştirilen yeniden düzenlemeler alt plaka devam edebilir dokundu olarak misalignments kaçınınSatır H. aminat kısırlık kontrolleri </li><li> sıkı sızdırmazlık, bir yapışmalı plastik bir torba içinde buharlaşmasını ve yer önlemek için parafin film ile plaka kapağı kapatılmalıdır. buharlaşmasını en aza indirmek için mümkün olduğunca düşük çantada hava hacmini tutun. </li><li> % 5 CO2 inkübatöründe 48 saat süreyle 37 ° C&#39;de çalkalanmadan inkübe edin. Not: büyüme 48 saat S. için optimal olmuştur aureus, ancak değişir kullanılan organizmaya göre. Not: Statik inkübasyon iyi bir başlangıç ​​noktası olmasına rağmen hafifçe bir mikro-çalkalayıcı üzerinde 150 rpm&#39;de sallayarak, tahlil optimize etmek için bazı S. biyofilm oluşumunu artırabilir olası bir varyasyon aureus izole eder. </li></ol> Biyofilm Mücadelesi Plate 2. Hazırlık <ol><li> tutarsız sonuçlar önlemek için biyofilm meydan okumanın yapıldığı gün taze 96-yuvalı plaka almak ve H. B sıranın her çukuruna, oda sıcaklığına dengelendi cRPMI 100 ul, ilave 200 ul tutabilecek bir 96 oyuklu plaka kullanılmasıortam mandal içinde, ve biyofilm başlangıç ​​plakasının özdeş boyutlara sahip kuyu olduğu takdirde,. </li><li> Aşama 2.7 nihai bir seyreltme için hesaba katmak için, arzu edilen nihai konsantrasyonu 2x cRPMI 1.0 ml ayrı 4 test bileşiklerine kadar seyreltilir. Not: İyi bir başlangıç ​​noktası planktonik hücreler inhibitör konsantrasyonun üzerinde 8 kat daha fazladır. </li><li> A3, kuyu A12 A10 A9 kuyu A7 A6 kuyu A4 bileşik 2, bileşik 3 ve 4 bileşiği için kuyu A1 bileşik 1 200 ul ekle. </li><li> Seri, çok kanallı pipet kullanarak test bileşiklerinin sulandırmak ve B satır ve karıştırmak için satır A 100 ul aktarın. </li><li> Bu şekilde, iyi iyi 100 ul transfer devam etmektedir. Her aktarımdan sonra karıştırın ve satır F durdurmak </li><li> karıştırıldıktan sonra, bir atık kabı içine satır F 100 ul sınırdışı. satırlar G ve H. herhangi bir sıvı transferi yapmayın </li><li> Bir çok kanallı pipet kullanılarak plakanın her oyuğuna cRPMI 100 ul ekle200 ul hacim getirmek. Nihai plaka düzeni için Şekil 1&#39;e bakınız. </li></ol> 3. Biyofilm Mücadelesi <ol><li> biyofilm başlangıç ​​plakasının özdeş boyutlara sahip kuyu özellikleri taze, steril 96 gözlü plaka, fosfat tamponlu salin (PBS) içinde 200 ul ekle. </li><li> kuluçkaya biyofilm başlatma plakadan plastik torba ve parafin filmi çıkarın. </li><li> yukarı doğru kapağını kaldırın. biyofilm zarar verebilir, bu kadar mandal ve plaka alt kısmı arasındaki temastan kaçının. (3.8 bakınız) sonraki OD 600 analiz için alt kısmını tutun. </li><li> (Adım 3.1) PBS içeren plaka üzerine tahta kapağı koyarak planktonik hücreler durulayın. PBS üzerinden kaldırın, 5 sn mandal Batmak ve iyi tarafı karşı mandal vurmak için dikkatli olmak, bir kez daha daldırın. </li><li> meydan plakası içine durulanır topaç kapağı yerleştirin. biyofilm giden zarar verecek bu kadar mandal ve alt plaka arasında gereksiz temastan kaçınıntutarsız sonuçlar. </li><li> Adım 1.10 açıklandığı gibi mühür parafin film ile plaka ve yapışmalı plastik torbaya koyun. </li><li> % 5 CO2 inkübatöründe 37 ° C&#39;de 24 saat boyunca çalkalamadan plaka inkübe edin. </li><li> 3.3 ile biyofilm başlangıç ​​plakanın alt alın. Iyi, çok kanallı pipet kullanarak her bakteri tekrar süspansiyon ve satır H. tüm kuyulara ama sterilite kontrolü meydana gelen büyüme onaylamak için uygun bir mikroplaka okuyucu ile OD 600 ölçmek </li></ol> 4. Biyofilm Belirlenmesi <ol><li> dönüşüm resazurin tespit etmek için bir kuluçka odası ve kinetik floresan okumaları alma yeteneğine sahip ile donatılmış bir plaka okuyucu kullanın. 530 nm uyarma ve 590 nm emisyon kullanarak 24 saat kinetik floresan ölçümü ayarlayın. </li><li> 37 ° C oda sıcaklığını ayarlamak ve plaka her 20 dakikada okudum. üreticinin Okt. göre plaka tabanından ölçmek için plaka okuyucusu ayarlamaleri. </li><li> Bir steril 96 oyuklu bir plaka çok kanallı bir pipet ile 200 ul PBS ilave edilerek Plakayı hazırlayın. </li><li> 0.8 mg, 400 ul ekleyerek biyofilm geri orta hazırlayın / 16 ug / ml resazurin nihai bir konsantrasyona kadar cRPMI ortamı 20 ml stok çözeltisi resazurin ml. Not: Resazurin bilinen bir cilt tahriş edicidir. işlenirken bir laboratuvar önlüğü, gözlük sıçrama ve eldiven kullanın. biyofilm kurtarma ortamı olarak cRPMI en düşük arka plan sinyali sağlar, çünkü kullanılan, ancak uygun eğer Mueller Hinton medya tarafından değiştirilebilir. </li><li> taze 96-yuvalı plakanın her oyuğuna çok kanallı bir pipet ile biyofilm geri kazanım aracına 150 ul ekle. </li><li> Bir biyogüvenlik kabini içine inkübatör transfer meydan plakası ve dikkatle plastik torba ve mühürleme filmi çıkarın. </li><li> meydan plaka tahta kapağını çıkarın ve 3.4 açıklandığı gibi PBS içinde planktonik hücreler durulayın. Sonraki OD 600 ana için zorluk plakanın alt tutunerimesi (4.10 bakınız). </li><li> duruladıktan sonra, biyofilm kurtarma ortamı içeren plaka dibine tahta kapağı yerleştirin. </li><li> çevre kuyuların dibine engel için dikkatli olmak, parafin film ile plakanın yan sarın. Hemen plaka tamamlamasından sonra, plaka okuyucusu üzerine yerleştirin ve adım 4.1 daha önce kinetik protokol resazurin yapılan başlatarak 24 saatlik bir süre boyunca, bir kinetik okuma başlar. </li><li> Ya da zorlukla sırasında meydana olmayabilir planktonik hücrelerin büyümesini ölçmek için, bir mikro-plaka okuyucusu kullanılarak, tüm zorluk plakası alt OD 600 oku. Adım 3.8&#39;de verilen talimatları izleyin. Not: Dikkatle iyi biyofilm kapalı dökülme hücreler kuyunun dibinde yığınlarda büyürler her içeriğini tekrar süspansiyon. Kuyunun içinde herhangi bir baloncuklar kuvvetle OD 600 okuma engeller ve kaçınılması veya okuma öncesinde çıkarılmalıdır. </li></ol>

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The authors have nothing to disclose.

Burada, S. üzerinde test inhibitörlerinin aktivitesinin belirlenmesi için bir tadil edilmiş biyofilm tahlili tarif etmişlerdir biyofilm ilişkili hücrelerin metabolik durumuna odaklanan aureus biyofilm. açıklanan biyofilm başlatma ve meydan prosedürleri çoğunlukla taklit üretici tavsiyelerine rağmen, 24 saat inhibitörü meydan hayatta biyofilm ilişkili hücreleri tespit ve ölçmek için boya resazurin kullanımı dramatik hücre (su banyosu sonification aracılığıyla) kurtarma ve so...

Patojen mikropların olmayan akut kronik enfeksiyonlara 1 giden in vivo biyofilm oluşturabilir. Biyofilm ilişkili enfeksiyon yabancı cisimler (örneğin, suni kemik değişimleri, meme implantları) veya trakea tüpler veya üriner kateterlerin 2 kurulumu implantasyonu içeren tıbbi prosedürler ciddi bir risk faktörüdür. Biyofilm tabanlı enfeksiyonlar nadiren kendi temizlenir olarak bu bağlamlarda, anti-enfektif tedavi bile immün sistemi sağlıklı bireylerde, hemen hemen her zaman gereklidir. Staphylococcus aureus kullanımı sırasında ortaya çıkan biyofilm ilişkili komplikasyon karıştığı En sık görülen patojenlerden biridir invazif tıbbi cihazlar 3. Ne yazık ki, koruyucu bir bariyer olarak biyofilm doğası planktonik hücreler 1,4 den tedaviye onları daha dirençli hale getirir ve tahmin klinik etkinliği değerlendirilmesi hem ilk kritik bir parçasıdırilaç geliştirme yanı sıra ilaç direnç gözetim. Planktonik kültürler üzerinde duruldu laboratuvar koşulları, sadakatle gerçek dünya hastalığı 5 temsil olmayabilir artan bildirim yoktur. Sorunu daha bileşik, biyofilm fenotipleri çoğaltma zordur ve mevcut biyofilm modelleri sıkıcı ve yüksek içi ve tahlil değişkenliği 6 muzdarip. Böylece, birçok araştırmacı, zorunluluk yoluyla, böylece potansiyel bakteriyel virulans ve hastalığın önemli bir yönünü ihmal, uyuşturucu duyarlılık planktonik hücre deneyleri için varsayılan. Burada özellikle, bakteri tahlil için S. protokolü açıklamak aureus, 96 oyuklu bir göre biyofilm sistemi 7,8 kullanarak önceden büyümüş Biyofilmlererde. biyofilm oluşumu ve meydan okuma için protokol esas üreticinin tavsiye takip ederken, biz biof canlılığı ölçülmesi için alternatif bir bilgi açısından zengin bir metodoloji sunmakmücadeleden sonra ilm. Kısaca, bakteriler, biyofilm levhanın kapak takılı çıkıntı yapan mandal üzerinde oluşan mandal plakası, kültürlenir. biyofilm sonra mandal yavaşça planktonik hücrelerini uzaklaştırmak için PBS ile dolu yeni bir levhanın gözleri içine daldırılır. PEG kapaklı ekli biyofilm ile deneye tabi antibiyotik çeşitli konsantrasyonlarını içeren, yeni bir meydan okuma plakasına aktarılır. ikinci bir inkübasyondan sonra, kapaklar daha çıkarılır, yıkanır ve son bir inkübasyon maruz boya, resazurin ihtiva eden bir geri kazanma plakasına aktarılır. Resazurin dönüşüm kinetik kaydedilmiş veya tanımlanmış bir iyileşme döneminden sonra bir bitiş noktası okuma olarak alınabilir. Biyofilm canlılığını nicel Bu boya temelli yöntem sıkıcı CFU (koloni oluşturan birim) önemli ölçüde orijinal protokolde 7&#39;de tarif saymak tabanlı bir metodoloji farklıdır. İlaç meydan plaka ve dönüşüm kinetiği resazurin OD 600 ölçümler canlılığı okundu olarak hizmetbiyofilm hayatta kalmak için hızlı, güvenilir, bilgi açısından zengin ve teknik basit tahlil sunan sırasıyla planktonik ve biyofilm hücrelerinin çıkışları.

<table><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></td></tr></tbody></table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

resazurin deney güvenilir mücadeleden sonra, ilaç barındırmayan ortam içinde replike edebilen çok az canlı hücreleri saptamak için yeterince hassastır. Bu metodoloji, biz hiçbir resazurin dönüşüm 24 saat içinde görülür ki en düşük konsantrasyon olarak en az biyofilm ortadan kaldırılması konsantrasyonunu tanımlar. resazurin tahlil pembe maviden boya rengi dönüştürmek metabolik olarak aktif hücrelerde bulunan oksidatif moleküller dayanır. Boya dönüşüm?...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Biyofilm İlişkili Hedefleme<em&gt; Staphylococcus aureus</emKullanma&gt; Resazurin Tabanlı İlaç-duyarlılık Deneyi

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad de Cartagena UDEC

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JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K Görüntüleme.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

Video: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Antibiyotik Dirençli Stafilokok bakteri tespiti için biyosensör

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Biyomühendislik

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 ...

İmmünoloji ve Enfeksiyon

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Çip üzerinde Stres Kaynaklı Antibiyotik Duyarlılık Testi

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Doğal Bileşik Kütüphaneleri Exploration uygundur Anti-biyofilm Tahliller Bir Platform

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

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 ...

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 ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

İnsan Nötrofiller ve Staphylococcus aureus Biyofilmleri Arasındaki Etkileşimleri Görselleştirmek ve Ölçmek için Standartlaştırılmış In vitro Testler

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Antikorların Candida tropicalis Biyofilmleri Üzerindeki Etkisini Değerlendirmek için Çözünür Tetrazolyum Bazlı İndirgeme Testi

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Antimikrobiyal Bileşiklerin Staphylococcus aureus Internalization ve Hücre İçi Etkinliğini İncelemek için Geliştirilmiş Enzim Koruma Tahlilleri

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biyoloji

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Source: Schaaf, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

Mycobacterium tuberculosis Enfeksiyon Modeline Karşı İlaç Etkinliğini Değerlendirmek İçin Bir Test

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

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