Name: quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software
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Los autores desean agradecer a la Fundación de Fibrosis Quística por proporcionar fondos para esta investigación.

1. Recolección de aislados bacterianos
<ol><li>Obtención de aislados de P. aeruginosa de una cohorte de pacientes pediátricos con FQ en tratamiento de erradicación con tobramicina inhalada en SickKids (Toronto). Congele los aislados a -80 °C en citrato de glicerol y subcultivo al menos tres veces antes de su uso.</li>
</ol>2. Formación de biofilm in vitro
NOTA: Utilice un método de cubreobjetos con cámara1 para la formac...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+production+affects+Pseudomonas+aeruginosa+biofilm+development+and+architecture,+but+is+not+essential+for+biofilm+formation.">Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.</a> Journal of Medical Microbiology. 53, 679-690 (2004).</li><li>Reichhardt, C., Parsek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+scanning+microscopy+for+analysis+of+Pseudomonas+aeruginosa+biofilm+architecture+and+matrix+localization.">Confocal laser scanning microscopy for analysis of Pseudomonas aeruginosa biofilm architecture and matrix localization.</a> Frontiers in Microbiology. 10, 677 (2019).</li><li>Yang, X., Beyenal, H., Harkin, G., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+biofilm+structure+using+image+analysis.">Quantifying biofilm structure using image analysis.</a> Journal of Microbiological Methods. 39, 109-119 (2000).</li><li>Yang, X., Beyenal, H., Harkin, G., Lewandowski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biofilm+image+thresholding+methods.">Evaluation of biofilm image thresholding methods.</a> Water Research. 35, 1149-1158 (2001).</li><li>Ross, S. 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No existe un método prescrito para comparar cuantitativamente imágenes tridimensionales de estructuras de biopelículas in vitro , y los procedimientos descritos en este contexto son a menudo difíciles de estandarizar debido a la variabilidad entre operadores20. Por lo tanto, este protocolo ofrece un marco simple y reproducible para las aplicaciones de COMSTAT que buscan cuantificar los cambios en la arquitectura de la biopelícula in vitro bajo diversas condiciones antimicrob...

Los biofilms son agregados de microorganismos orientados en una matriz de sustancias poliméricas extracelulares (EPS) autoproducidas. La matriz de EPS es muy compleja y consta principalmente de células bacterianas, agua, proteínas, polisacáridos, lípidos y ácidos nucleicos1, todos los cuales producen biopelículas claramente diferentes de las células planctónicas de vida libre. Los EP de biofilm se adhieren entre sí y a varias superficies. La matriz de EPS tiene propiedades que median el intercambio de metabolitos de célula a célula, material genético y compuestos utilizados para la señalización y defensa intercelular2. Estas propiedades proporcionan colectivamente a las biopelículas integridad estructural y protección contra los factores estresantes externos, lo que contribuye a la evasión inmunitaria y la resistencia a los antimicrobianos3.
Pseudomonas aeruginosa es un patógeno nosocomial bien reconocido, conocido por adoptar una estrategia evasiva de crecimiento de biopelículas en respuesta a los antimicrobianos. Un buen ejemplo de esto ocurre en pacientes con el trastorno genético recesivo, la fibrosis quística (FQ). Las biopelículas desempeñan un papel fundamental en el desarrollo de P. aeruginosa resistente a los antimicrobianos 4 y permiten el establecimiento de infección pulmonar crónica en pacientes con FQ, lo que provoca un deterioro acelerado de la función pulmonar y una mortalidad prematura5. Por lo tanto, se realizan estudios de biofilm in vitro para probar la eficacia de antibióticos y nuevos agentes antiinfecciosos contra aislados de P. aeruginosa obtenidos de pacientes con FQ 6,7. Después de la formación de la biopelícula, los antimicrobianos se aplican externamente a la estructura y se utiliza la microscopía de escaneo láser confocal (CLSM) para generar reconstrucciones tridimensionales de alta resolución de los segmentos de la biopelícula. Es una práctica común utilizar el software informático, COMSTAT, como complemento de ImageJ, para cuantificar los cambios en la arquitectura de la biopelícula 8,9,10,11.
Aunque COMSTAT es útil para cuantificar la estructura de la biopelícula, la reproducibilidad y la estandarización del análisis de imágenes se abordan con menos frecuencia. Por ejemplo, el procedimiento de procesamiento de imágenes, realizado antes de ejecutar COMSTAT, es objetivo, pero contiene un elemento de subjetividad al establecer umbrales de imagen12,13. De manera similar, el programa COMSTAT permite al operador elegir entre condiciones y parámetros básicos y avanzados para la segmentación de imágenes, así como diez funciones de análisis cuantitativas (por ejemplo, distribución de espesor, área de superficie, biomasa, coeficiente de rugosidad adimensional). La multitud de opciones de usuario, junto con los diferentes niveles de experiencia del operador, puede dar lugar a informes erróneos sobre el comportamiento de la biopelícula.
Por lo tanto, el objetivo de este protocolo es presentar un método relativamente sencillo para la comparación cuantitativa de estructuras de biopelículas in vitro utilizando COMSTAT. En este trabajo, se capturan imágenes tridimensionales de segmentos de biopelícula de un aislado de CF P. aeruginosa a través de CLSM utilizando el modelo14 de cubreobjetos con cámara, una técnica establecida que se utiliza para realizar experimentos de biopelícula in vitro reproducibles. Utilizando COMSTAT como un complemento de ImageJ, este método permite a los investigadores identificar cuantitativamente los cambios en la arquitectura de la biopelícula en presencia de antimicrobianos en condiciones variables. En general, este método tiene como objetivo eliminar las variaciones subjetivas asociadas con la operación manual de COMSTAT, facilitando así la estandarización de los protocolos en todos los centros.

<table>
	<tbody>
		<tr>
			<td>Anti-Psl mAb, Psl0096</td>
			<td>Medimmune</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar (TSA with 5 % Sheep Blood) Medium</td>
			<td>Fisher Scientific</td>
			<td>R01200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eight-well Chambered Coverglass w/ non-removable wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen SYTO 9 Green Fluorescent Nucleic Acid Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S34854</td>
			<td></td>
		</tr>
		<tr>
			<td>LB BROTH (LENNOX), Liquid Autoclave Sterilized</td>
			<td>BioShop Canada</td>
			<td>LBL666</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin, 900 &#181;g/mg</td>
			<td>Alfa Aesar by Thermo Fisher Scientific</td>
			<td>J66040</td>
			<td>It is recommended to perform a minimal inhibitory concentration (MIC) test for every batch made to ensure quality control of antimicrobial potency</td>
		</tr>
		<tr>
			<td>Quorum Volocity 6.3</td>
			<td>Quorum Technologies</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software

Las biopelículas son agregados de microorganismos que dependen de una matriz autoproducida de sustancia polimérica extracelular para su protección e integridad estructural. Se sabe que el patógeno nosocomial, Pseudomonas aeruginosa, adopta un modo de crecimiento de biopelícula, causando infección pulmonar crónica en pacientes con fibrosis quística (FQ). El programa informático, COMSTAT, es una herramienta útil para cuantificar los cambios inducidos por antimicrobianos en la arquitectura de la biopelícula de P. aeruginosa mediante la extracción de datos de imágenes confocales tridimensionales. Sin embargo, el funcionamiento estandarizado del software se aborda con menos frecuencia, lo cual es importante para la generación óptima de informes sobre el comportamiento de la biopelícula y la comparación entre centros. Por lo tanto, el objetivo de este protocolo es proporcionar un marco simple y reproducible para cuantificar estructuras de biopelículas in vitro en diferentes condiciones antimicrobianas a través de COMSTAT. La técnica se modela utilizando un aislado de CF P. aeruginosa , cultivado en forma de réplicas de biopelícula y expuesto a tobramicina y al anticuerpo monoclonal anti-Psl, Psl0096. El enfoque paso a paso tiene como objetivo reducir la ambigüedad del usuario y minimizar la posibilidad de pasar por alto pasos cruciales del procesamiento de imágenes. Específicamente, el protocolo enfatiza la eliminación de las variaciones subjetivas asociadas con la operación manual de COMSTAT, incluida la segmentación de imágenes y la selección de funciones de análisis cuantitativo apropiadas. Aunque este método requiere que los usuarios dediquen tiempo adicional a procesar imágenes confocales antes de ejecutar COMSTAT, ayuda a minimizar la heterogenicidad de la biopelícula tergiversada en las salidas automatizadas.

Se utiliza un aislado de P. aeruginosa cultivado a partir de un paciente infectado con FQ para demostrar las fortalezas de este enfoque en la cuantificación precisa de los cambios inducidos por los antimicrobianos en la arquitectura de la biopelícula in vitro . El flujo de trabajo general de este modelo se representa en la Figura 1. En la Figura 2 se muestra el procedimiento de procesamiento de imágenes y análisis COMSTAT en ImageJ. En la <...

Watch this Scientific Journal Video about Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software at JoVE.com

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

Los cambios inducidos por antimicrobianos en la arquitectura de la biopelícula de Pseudomonas aeruginosa difieren entre los aislados clínicos cultivados de pacientes con fibrosis quística e infección pulmonar crónica. Después de la microscopía confocal, el software COMSTAT se puede utilizar para cuantificar las variaciones en la arquitectura de la biopelícula (por ejemplo, área de superficie, espesor, biomasa) para aislados individuales para evaluar la eficacia de los agentes antiinfecciosos.

Cuantificación de los efectos de los antimicrobianos en la arquitectura de biopelículas in vitro mediante el software COMSTAT

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

The significance of this protocol is that it offers a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions. The main advantage of this technique is that it helps eliminate subjective variations associated with the manual operation of COMSTAT, which ultimately facilitates the standardization and transparency of experiments across research centers. To image the Pseudomonas aeruginosa cultures, place the plate onto the stage of a confocal microscope, equipped with the appropriate laser excitation wavelengths and filter sets.And use a 20 to 25x water immersion lens to capture at least six layered z-stack images from the substratum to the top of each biofilm segment in 0.3 micrometer increments. Then save the images as OME-TIFFs for COMSTAT analysis. When all the images have been acquired, open the COMSTAT2 package in ImageJ and create a source folder on the desktop.Add a single OME-TIFF to the folder and open OME-TIFF from the source folder. Delete any empty layers containing no biomass. Select File, Import, and Image Sequence to import the file into ImageJ.Locate and highlight the source folder without opening it and click Select. A sequence options window will appear, click OK.Select Image, Transform, and Flip Z to flip the orientation of the biofilm so that the substratum is at the top of the stack. To define the image properties, select Image and Properties, a source window will appear.Specify the unit of length as micron, define the voxel depth as 0.3, and click OK.Select Image, Adjust, and Threshold. To adjust the image threshold in the threshold window, position the lower slider to the far right to manually set the maximum threshold value and use the upper slider to set the minimum threshold. Then use the slider in the image window to scroll through each layer to ensure that the background noise is sufficiently removed throughout the stack, and select Set to fix the lower threshold value.In the set threshold levels window, select OK, and set and fix the maximum threshold value similarly. Select Apply, click OK in the convert stack to binary window that appears, and exit the threshold window. Select Plugins, Bio-Formats, and Bio-Formats Exporter to save the file with a new file name in the source folder.A Bio-Formats Exporter multiple files window will appear, select OK.A Bio-Formats Exporter options window will appear, select OK again. To run COMSTAT, select plugins and COMSTAT2, an about window will appear, select OK.In the Observed Directories window, select Add, locate and highlight the source folder, highlight and click Choose. An Images in Directories window will appear that lists the OME-TIFFs to be analyzed.On the COMSTAT2.1 window, deselect Automatic Thresholding Otsu's Method and Connected Volume Filtering to ensure that the software uses preset threshold values for individual OME-TIFFs. In the COMSTAT2.1 window, select Biomass, Thickness Distribution and Surface Area, and click Go to run the program. The output data will be shown in the log window.The measurements will be automatically saved as TXT files in the source folder. In this representative analysis, a Pseudomonas aeruginosa isolate, cultured from an infected cystic fibrosis patient, was used to demonstrate the strengths of this approach, inaccurately quantifying antimicrobial induced changes in in vitro biofilm architecture. Live cell imaging can also visualize changes in Pseudomonas aeruginosa biofilm structure before and after biofilm specific monoclonal and tobramycin treatment.COMSTAT data analysis revealed significant differences between treated biofilm structures compared to control cultures. In tobramycin-treated cultures, a clear reduction in the average thickness and biomass was observed. However, in the presence of antibodies, the P.aeruginosa biofilm was resistant to these tobramycin-induced structural changes.When examining the surface to bio volume ratio, a significant reduction was observed in the presence of antibodies in both tobramycin-exposed and unexposed biofilms, indicating the formation of aggregates in antibody treated cultures. This procedure can be performed to quantitatively assess or compare the effects of different antimicrobials used to disrupt biofilm matrices. Using this technique as a general framework, researchers can explore different ways to refine the protocol to include new mathematical models and algorithmic plugins in ImageJ to better depict biofilm heterogenecy.

Research

JoVE Journal

Immunology and Infection

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

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in an 8-well Chamber Slide

En la formación de biofilm in vitro en una diapositiva Cámara de 8 pocillos

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

Investigación

Inmunología e Infección

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Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Los ensayos cualitativos y cuantitativos para la detección y caracterización de proteínas de antimicrobianos

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

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Un<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro mediante Espectroscopia de Impedancia: Un enfoque en la interacción de células T de la célula endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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

La visualización de los Efectos de esputo en el desarrollo del biofilm mediante un modelo de Septadas cubreobjetos

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

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Cuantificación de la Quimiotaxis de monocitos humanos In Vitro y linfocitos murinos, tráfico en Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualización de la formación de Biofilm de Candida albicans utilizando un dispositivo microfluídico automatizado

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...