Name: quantifying the effects of antimicrobials on in vitro biofilm architecture using comstat software
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Gli autori desiderano ringraziare la Cystic Fibrosis Foundation per aver fornito finanziamenti per questa ricerca.

1. Raccolta di isolati batterici
<ol><li>Ottenere isolati di P. aeruginosa da una coorte di pazienti pediatrici con FC sottoposti a trattamento di eradicazione con tobramicina per via inalatoria presso SickKids (Toronto). Congelare gli isolati a -80 °C in glicerolo citrato e sottocoltura almeno tre volte prima dell'uso.</li>
</ol>2. Formazione di biofilm in vitro
NOTA: Utilizzare un metodo di coverglass a camera1 per la formazi...

<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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Non esiste un metodo prescritto per confrontare quantitativamente immagini tridimensionali di strutture di biofilm in vitro e le procedure descritte in questo contesto sono spesso difficili da standardizzare a causa della variabilità interoperatore20. Pertanto, questo protocollo offre un quadro semplice e riproducibile per le applicazioni COMSTAT che cercano di quantificare i cambiamenti nell'architettura del biofilm in vitro in condizioni antimicrobiche variabili. I punti di fo...

I biofilm sono aggregati di microrganismi orientati in una matrice di sostanze polimeriche extracellulari (EPS) autoprodotte. La matrice EPS è molto complessa, costituita principalmente da cellule batteriche, acqua, proteine, polisaccaridi, lipidi e acidi nucleici1, che rendono i biofilm nettamente diversi dalle cellule planctoniche a vita libera. I Biofilm EPS sono aderenti l'uno all'altro e alle varie superfici. La matrice EPS ha proprietà che mediano lo scambio cellula-cellula di metaboliti, materiale genetico e composti utilizzati per la segnalazione e la difesa intercellulare2. Queste proprietà forniscono collettivamente integrità strutturale dei biofilm e protezione contro i fattori di stress esterni, contribuendo all'evasione immunitaria e alla resistenza antimicrobica3.
Pseudomonas aeruginosa è un patogeno nosocomiale ben noto, noto per adottare una strategia evasiva di crescita del biofilm in risposta agli antimicrobici. Un primo esempio di ciò si verifica nei pazienti con la malattia genetica recessiva, la fibrosi cistica (FC). I biofilm svolgono un ruolo fondamentale nello sviluppo di P. aeruginosa 4 resistente agli antimicrobici e consentono l'instaurarsi di un'infezione polmonare cronica nei pazienti con FC, causando un declino accelerato della funzione polmonare e una mortalità prematura5. Pertanto, vengono eseguiti studi in vitro sul biofilm per testare l'efficacia di antibiotici e nuovi agenti antinfettivi contro gli isolati di P. aeruginosa ottenuti da pazienti con FC 6,7. Dopo la formazione del biofilm, gli antimicrobici vengono applicati esternamente alla struttura e la microscopia a scansione laser confocale (CLSM) viene utilizzata per generare ricostruzioni tridimensionali ad alta risoluzione di segmenti di biofilm. È pratica comune utilizzare il software per computer, COMSTAT, come plug-in di ImageJ, per quantificare i cambiamenti nell'architettura del biofilm 8,9,10,11.
Sebbene COMSTAT sia utile per quantificare la struttura del biofilm, la riproducibilità e la standardizzazione dell'analisi delle immagini sono meno comunemente affrontate. Ad esempio, la procedura di elaborazione delle immagini, eseguita prima dell'esecuzione di COMSTAT, è oggettiva, ma contiene un elemento di soggettività quando si impostano le soglie delle immagini12,13. Allo stesso modo, il programma COMSTAT consente all'operatore di scegliere tra condizioni e parametri di base e avanzati per la segmentazione delle immagini, nonché dieci funzioni di analisi quantitativa (ad esempio, distribuzione dello spessore, area superficiale, biomassa, coefficiente di rugosità adimensionale). La moltitudine di opzioni per l'utente, combinate con i diversi livelli di esperienza dell'operatore, può portare a una segnalazione errata del comportamento del biofilm.
Pertanto, l'obiettivo di questo protocollo è quello di presentare un metodo relativamente semplice per il confronto quantitativo di strutture di biofilm in vitro utilizzando COMSTAT. In questo articolo, le immagini tridimensionali dei segmenti di biofilm di un isolato di CF P. aeruginosa vengono acquisite tramite CLSM utilizzando il modello14 di vetro di copertura a camera, una tecnica consolidata utilizzata per eseguire esperimenti di biofilm in vitro riproducibili. Utilizzando COMSTAT come plug-in di ImageJ, questo metodo consente ai ricercatori di identificare quantitativamente i cambiamenti nell'architettura del biofilm in presenza di antimicrobici in condizioni variabili. Nel complesso, questo metodo mira a eliminare le variazioni soggettive associate al funzionamento manuale di COMSTAT, facilitando così la standardizzazione dei protocolli tra i centri.

<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

I biofilm sono aggregati di microrganismi che si basano su una matrice autoprodotta di sostanza polimerica extracellulare per la protezione e l'integrità strutturale. Il patogeno nosocomiale, Pseudomonas aeruginosa, è noto per adottare una modalità di crescita del biofilm, causando infezione polmonare cronica nei pazienti con fibrosi cistica (FC). Il programma informatico, COMSTAT, è uno strumento utile per quantificare i cambiamenti indotti da antimicrobici nell'architettura del biofilm di P. aeruginosa estraendo dati da immagini confocali tridimensionali. Tuttavia, il funzionamento standardizzato del software è meno comunemente affrontato, il che è importante per la segnalazione ottimale del comportamento del biofilm e il confronto tra i centri. Pertanto, l'obiettivo di questo protocollo è quello di fornire un quadro semplice e riproducibile per quantificare le strutture del biofilm in vitro in condizioni antimicrobiche variabili tramite COMSTAT. La tecnica è modellata utilizzando un isolato di CF P. aeruginosa , coltivato sotto forma di repliche di biofilm ed esposto alla tobramicina e all'anticorpo monoclonale anti-Psl, Psl0096. L'approccio graduale mira a ridurre l'ambiguità dell'utente e a minimizzare la possibilità di trascurare le fasi cruciali dell'elaborazione delle immagini. In particolare, il protocollo enfatizza l'eliminazione delle variazioni soggettive associate al funzionamento manuale di COMSTAT, tra cui la segmentazione delle immagini e la selezione di appropriate funzioni di analisi quantitativa. Sebbene questo metodo richieda agli utenti di dedicare più tempo all'elaborazione di immagini confocali prima di eseguire COMSTAT, aiuta a ridurre al minimo l'eterogenicità del biofilm travisato negli output automatizzati.

Un isolato di P. aeruginosa coltivato da un paziente infetto con FC viene utilizzato per dimostrare i punti di forza di questo approccio nel quantificare accuratamente i cambiamenti indotti da antimicrobici nell'architettura del biofilm in vitro . Il flusso di lavoro complessivo di questo modello è rappresentato nella Figura 1. La procedura di elaborazione delle immagini e di analisi COMSTAT in ImageJ è illustrata nella Figura 2. Nella <stron...

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

Le alterazioni indotte da antimicrobici dell'architettura del biofilm di Pseudomonas aeruginosa differiscono tra gli isolati clinici coltivati da pazienti con fibrosi cistica e infezione polmonare cronica. Dopo la microscopia confocale, il software COMSTAT può essere utilizzato per quantificare le variazioni nell'architettura del biofilm (ad esempio, area superficiale, spessore, biomassa) per i singoli isolati per valutare l'efficacia degli agenti anti-infettivi.

Quantificazione degli effetti degli antimicrobici sull'architettura del biofilm in vitro utilizzando il 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

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