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のバイオフィルム形成の1クリスタルバイオレット定量バウマニ S1 <ol><li> A. 5mlの文化を育て振盪インキュベーター（220 rpm）し、30℃でのLB培地（LB）（トリプトンを10g / L、酵母エキス5グラム/ L）でバウマニ S1 16時間。 </li><li> A.の文化を調整バウマニ S1 0.8の所望のOD 600まで。 96ウェルプレートを使用して、細菌培養物接種：精製GKL酵素（40 mg / mlで）の10μLを含有する新鮮なLBに（1 100希釈）。新しい文化の最終容量は100μlです。 </li><li>同様に対照培養物を準備し;これは、任意の酵素が含まれません。複製の所望の数を得るために同様の条件を繰り返します。 </li><li>蓋付きプレートを覆い、密封された10リットルのプラスチック容器に入れてください。優しくメディアを削除する前に、3時間、30℃でプレートをインキュベートします。 </li><li>新鮮なLB培地の別の100μlをウェルに加え、30℃で21時間静置します。 </li&gt; <li>インキュベーションの第二の期間の後、静かにすべてのメディアを削除します。 200μlの滅菌水でプランクトン細菌細胞を洗浄します。洗浄中の細胞にのみ最小限の乱れがあることを確認します。 </li><li>各ウェルに1％クリスタルバイオレット溶液100μlを加え、室温で15分間インキュベートします。 200μlの滅菌水でよく洗浄することにより、クリスタルバイオレット溶液を除去します。さらに2回の洗浄を繰り返します。 </li><li>各ウェルに33％酢酸を100μl加え、穏やかに振盪しながら15分間インキュベートします。これは、色素を溶解します。 </li><li> 600nmにおけるクリスタルバイオレットの吸光度を測定することによって形成されたバイオフィルムの量を定量します。クリスタルバイオレットの量は、形成されたバイオフィルムの量に比例します。 </li></ol> A. 2.共焦点レーザー走査顕微鏡バウマニ S1バイオフィルム<ol><li> A. 5mlの文化を育て16時間振とう培養（220 rpm）し、30℃でのLBでバウマニ S1。 </li><li>広告A.だけの文化バウマニ S1 0.8の所望のOD 600まで。 35mmのガラス底のμ-皿を使用して、細菌培養の接種：精製されたGKL酵素（40 mg / mlの）の30μLを含む新鮮なLBに（1 100希釈）;新しい文化の最終容量を1 mlです。 </li><li>蓋付きのμ-皿を覆い、密封された10リットルのプラスチック容器に入れてください。優しくメディアを削除する前に、3時間30℃でμ-皿をインキュベートします。 1ミリリットルの全容量にそれをもたらし、精製GKL酵素と新鮮な培地30μlのを追加します。 30℃でさらに21時間インキュベートします。 </li><li>繰り返しステップ2.3および30℃でさらに24時間μ-皿をインキュベートします。そして、そっとメディアを削除します。 </li><li> μ-皿にハンクス平衡塩溶液（HBSS）中に溶解し5μg/ mlのアレックスのFluo 488結合小麦胚芽凝集素（WGA）の500μLを加え、37℃で30分間インキュベートし;これは、形成されたバイオフィルムを染色します。 T染色溶液を除去し、洗浄彼はHBSS 2mlで皿をμ。洗浄ステップをもう一度繰り返します。 </li><li> HBSSに溶解した3.7％ホルムアルデヒド500μlのを追加し、37℃で30分間インキュベートし;これは、μ-皿にバイオフィルムを修正します。 HBSS 2mlで一度μ-皿を洗い、その後、完全に溶液を除去。 μ皿バイオフィルムで固定前CLSM画像に4℃で暗所に保存することができます。 </li><li> CLSMイメージングおよび分析のために、スタックあたり0.21ミクロンの間隔で画像あたり97スタックを生成するために、63倍の倍率を使用しています。 </li></ol>

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The authors declare that they have no competing financial interests.

実験の両方のセットは、A.バウマンニ S1は、高い塩濃度とのNaClなしLB培地で培養したバクテリア15によって形成されたバイオフィルムの量を減少させることができます。このようなアーティファクトの存在は、バイオフィルム形成量、ならびに異なる処理条件全体の定足数消光酵素の影響を過小評価する可能性があります。触媒的に不活性な酵素の使用は、酵素隔離の影響の可?...

感染症の治療の選択肢は、抗生物質1の広い範囲に影響されない多剤耐性菌の急速な増加によって複雑にされています。耐性細菌媒介性感染症からの高い罹患率および死亡率と、薬物開発プロセスをエスカレートおよび/または治療選択肢を改善するための優れた抗菌選択肢を探索する必要があります。最近では、抗病原性のアプローチは、したがって耐性機構2のリスクを軽減する、非殺菌方法を介して病原性を防止する上で、その潜在的な特定の関心を集めています。 クオラムセンシングは、このシグナリング現象の細菌の病原性と混乱の「マスタースイッチが「病因3に対する有望な抗病原性の方法です。重要な細菌の人口密度に達した後毒性の発症は細胞外環境における定足数の分子の蓄積が必要です。現状として、ラム酒の分子は、その同族受容体との結合は病原性因子だけでなく、抗生物質耐性およびバイオフィルム形成を4に関連する遺伝子の活性化につながる、細胞内マトリックスに戻って拡散します。一般的には、クオラムセンシング破壊が主要代謝経路に影響を与えることなく、定足数分子と受容体の相互作用を阻害することを含みます。したがって、細胞の成長に直接的な意味合いを持っていません。適応度が損なわれないので、進化し、そのような治療5に対する耐性を得るために、細菌のための最小限の選択圧があります。また、クオラムセンシングの破壊は、抗細菌剤からの保護を提供し、免疫応答をホストしてバイオフィルム形成の場合のように、固有の細菌の防御機構を妨害することができます。 それは、地球上の微生物の99％は目の中に生きた微生物に重要な生存の利点を付与する、複雑なバイオフィルムのようなマトリックス中に存在することが推定されていますESEの構造6。さらに重要なことは、これらの固着ドメインの形成は、最も永続的なおよび慢性院内感染7の原因である。 アシネトバクターバウマニは、グローバルな院内感染に関連付けられており、その毒性が大きくクオラムセンシングに起因する主要なヒト病原体の一つであります媒介バイオフィルム形成8。クォーラム消光酵素は、グラム陰性菌9によって生成されたN -アシルホモセリンラクトン（AHLs）として知られている化合物群をターゲットにして、クォーラムが介在するシグナル伝達を妨害するで正常に使用されています。いくつかの研究はまた、バイオフィルム10,11における毒性因子の発現および細胞数の削減による細菌の病原性を遮断するために、これらの酵素の使用に拡大しています。残念ながら、細菌性病原体によるバイオフィルム形成に対する定足数消光酵素の有効利用の触知可能なデモの欠如が残っています。ザ・再A.を破壊し 、定足数阻害剤（AHL類似体）、代わりの定足数消光酵素を使用する試みてきましたバウマニバイオフィルム形成12。小分子阻害剤を使用して、この方法が有効なアプローチであるが、翻訳の用途におけるその生物学的利用能を維持することが課題であることができます。酵素は治療効果のための生物医学装置の表面上に固定化に対してより敏感に反応するように逆に、触媒定​​足数消光酵素の使用は、生物学的利用能の問題を回避することができます。 ここでは、クリスタルバイオレット染色および共焦点レーザー走査顕微鏡（CLSM）を用いて、細菌のバイオフィルム形成に13（GKL） ゲオバチルスのkaustophilusから操作さクオラムクエンチングラクトナーゼの効果の評価を説明します。この研究は、臨床的に関連するAのバイオフィルム破壊の最初に成功した実証であります定足数消光酵素を用いバウマニ S1株。方法が記載さこの研究において、病原性グラム陰性菌に対する後続の治療の開発努力の他のクオラムクエンチング酵素の有効性を評価するために有用です。

<table border="0" cellpadding="0" cellspacing="0" width="780">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
	</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.

クリスタルバイオレット定量実験では、2つの定足数消光酵素がバイオフィルム形成を破壊で実行可能性を実証するために使用した：野生型GKLおよび改善GKL二重変​​異（E101G / R230C）。両酵素は、3-ヒドロキシデカノイル- L -ホモセリンラクトン（3-OH-C 10 -HSL）、A.で使用される主要な定足数分子に対するラクトナーゼ活性を示すことが示されています...

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.

エンジニアクォーラム焼入れラクトナーゼを使用して、病原性バイオフィルムの抗病原性破壊

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

Research

JoVE Journal

Immunology and Infection

10.5K ビュー.  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.

ビデオ: 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.

の単一細胞解析枯草菌</em蛍光顕微鏡とフローサイトメトリーを用いて>バイオフィルム

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 ...

免疫・感染学

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.

の成長結核バイオフィルム

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.

宿主細胞に関連して病原性真菌の定量化のための画像サイトメトリーの使用

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.

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.

歯科バイオフィルムにおける細胞外のpHのレシオメトリックイメージング

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.

共焦点顕微鏡および電子顕微鏡を用いて糸状菌コロニー上の細菌のバイオフィルムの定性的マルチスケール解析のための新しい方法

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.

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

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.

バイオ セーフティ レベル 2 の設定で高病原性コロナを勉強する新規微粒子の製造

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.

共焦点顕微鏡を用いたクロスキングダムバイオフィルムの細胞外pHのモニタリング

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.

カンディダ・アルビカンスバイオフィルムの明確化とイメージング

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