Name: Autore in primo piano: Avanzamento della ricerca nell'autoaggregazione microbica mediante citometria a flusso di imaging
Uploaded: 2023-09-29T14:00:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

Questo lavoro è stato sostenuto dalla Israeli Science Foundation (Grant 119/16) e dalla sovvenzione IMoh (3-15656) a IKG. R.S. sostenuto dalla borsa di studio Kreitman.

Il file .ast, con il modello per Lacticaseibacillus rhamnosus GG (LGG) come esempio, è fornito nel file di codifica supplementare 1.
1. Preparazione dei terreni
<ol><li>Preparare il brodo Lactobacilli MRS seguendo le istruzioni del produttore (55 g in 1 L di acqua deionizzata) e le piastre di agar Lactobacilli MRS con agar all'1,5% (p/v) (vedi Tabella dei materiali). Dopo la sterilizzazione in autoclave, il mezzo è pronto per l'us...

Analisi quantitativa dell'autoaggregazione microbica mediante citometria a flusso per immagini

<ol>
	<li>Trunk, T., Khalil, H. S., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+autoaggregation.">Bacterial autoaggregation.</a> AIMS Microbiology. 4 (1), 140-164 (2018).</li><li>Ishikawa, M., Nakatani, H., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtaA,+a+new+member+of+the+trimeric+autotransporter+adhesins+from+Acinetobacter+sp.+Tol+5+mediating+high+adhesiveness+to+various+abiotic+surfaces.">AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.</a> PLoS One. 7, e48830 (2012).</li><li>Inoue, T., 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=Molecular+characterization+of+low-molecular-weight+component+protein,+Flp,+in+Actinobacillus+actinomycetemcomitans+fimbriae.">Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.</a> Medical Microbiology and Immunology. 42 (4), 253-258 (1998).</li><li>Boddey, J. A., Flegg, C. P., Day, C. J., Beacham, I. R., Peak, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+microcolony+formation+by+Burkholderia+pseudomallei+requires+pilA+and+enhances+association+with+cultured+human+cells.">Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and enhances association with cultured human cells.</a> Infection and Immunity. 74 (9), 5374-5381 (2006).</li><li>Suissa, R., 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=Context-dependent+differences+in+the+functional+responses+of+Lactobacillaceae+strains+to+fermentable+sugars.">Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.</a> Frontiers in Microbiology. 13, 949932 (2022).</li><li>Isenring, J., Geirnaert, A., Lacroix, C., Stevens, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistable+auto-aggregation+phenotype+in+Lactiplantibacillus+plantarum+emerges+after+cultivation+in+in+vitro+colonic+microbiota.">Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota.</a> BMC Microbiology. 21, 268 (2021).</li><li>Kos, B., 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=Adhesion+and+aggregation+ability+of+probiotic+strain+Lactobacillus+acidophilus+M92.">Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92.</a> Journal of Applied Microbiology. 94 (6), 981-987 (2003).</li><li>Zommiti, M., 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=In+vitro+assessment+of+the+probiotic+properties+and+bacteriocinogenic+potential+of+Pediococcus+pentosaceus+MZF16+isolated+from+artisanal+tunisian+meat+&amp;#34;Dried+Ossban&amp;#34;.">In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal tunisian meat &amp;#34;Dried Ossban&amp;#34;.</a> Frontiers in Microbiology. 9, 2607 (2018).</li><li>Suissa, R., 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=Metabolic+rewiring+of+the+probiotic+bacterium+rhamnosus&lt;/em&gt;+GG+contributes+to+cell-wall+remodeling+and+antimicrobials+production.">Metabolic rewiring of the probiotic bacterium rhamnosus&lt;/em&gt; GG contributes to cell-wall remodeling and antimicrobials production.</a> bioRxiv. , (2023).</li><li>Connell, J. L., Kim, J., Shear, J. B., Bard, A. J., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+quorum+sensing+in+3D-printed+bacterial+aggregates+using+scanning+electrochemical+microscopy.">Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (51), 18255-18260 (2014).</li><li>Misawa, N., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+autoagglutination+activity+by+Campylobacter+jejuni.">Detection and characterization of autoagglutination activity by Campylobacter jejuni.</a> Infection and Immunity. 68 (11), 6168-6175 (2000).</li><li>Sherlock, O., Schembri, M. A., Reisner, A., Klemm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+roles+for+the+AIDA+adhesin+from+diarrheagenic+Escherichia+coli:+cell+aggregation+and+biofilm+formation.">Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation.</a> Journal of Bacteriology. 186 (23), 8058-8065 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry.">Imaging flow cytometry.</a> Nature Reviews Methods Primers. 2, 87 (2022).</li><li>Wang, Y., 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=Metabolism+characteristics+of+lactic+acid+bacteria+and+the+expanding+applications+in+food+industry.">Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry.</a> Frontiers in Bioengineering and Biotechnology. 9, 612285 (2021).</li><li>Turroni, F., 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=Molecular+dialogue+between+the+human+gut+microbiota+and+the+host:+a+Lactobacillus+and+Bifidobacterium+perspective.">Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective.</a> Cellular and Molecular Life Sciences. 71, 183-203 (2014).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochemica et Cytobiologica. 45, 279-290 (2007).</li><li>Dashkova, V., Malashenkov, D., Poulton, N., Vorobjev, I., Barteneva, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+for+phytoplankton+analysis.">Imaging flow cytometry for phytoplankton analysis.</a> Methods. 112, 188-200 (2017).</li><li>Maan, H., Gilhar, O., Porat, Z., Kolodkin-Gal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+colonization+of+arabidopsis+thaliana+roots+induces+multiple+biosynthetic+clusters+for+antibiotic+production.">Bacillus subtilis colonization of arabidopsis thaliana roots induces multiple biosynthetic clusters for antibiotic production.</a> Frontiers in Cellular and Infection Microbiology. 11, 722778 (2021).</li><li>Maan, H., 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=Imaging+flow+cytometry+reveals+a+dual+role+for+exopolysaccharides+in+biofilms:+To+promote+self-adhesion+while+repelling+non-self-community+members.">Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.</a> Computational and Structural Biotechnology Journal. 20, 15-25 (2022).</li><li>Konieczny, M., Rhein, P., Czaczyk, K., Bialas, W., Juzwa, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+to+study+biofilm-associated+microbial+aggregates.">Imaging flow cytometry to study biofilm-associated microbial aggregates.</a> Molecules. 26 (23), 7096 (2021).</li><li>Niederdorfer, R., Peter, H., Battin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attached+biofilms+and+suspended+aggregates+are+distinct+microbial+lifestyles+emanating+from+differing+hydraulics.">Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.</a> Nature Microbiology. 1, 16178 (2016).</li></ol>

La citometria a flusso è un metodo ampiamente utilizzato per quantificare l'intensità della fluorescenza nelle cellule eucariotiche, ma potrebbe non fornire misurazioni accurate per le cellule batteriche a causa delle loro dimensioni maggiori o dei piccoli aggregati. Questi fattori possono influenzare in modo significativo la quantificazione precisa dell'autoaggregazione e il livello basale di formazione degli aggregati in diverse condizioni. Per risolvere questo problema, è stata impiegata la citometria a flusso (IFC...

L'autoaggregazione batterica è considerata un passaggio primario nella formazione del biofilm. In questo processo (a volte chiamato anche autoagglutinazione o flocculazione), i batteri dello stesso tipo formano grumi multicellulari che alla fine si depositano sul fondo delle provette di coltura o si attaccano al tessuto oalla superficie bersaglio.
L'autoaggregazione è un fenomeno ampiamente osservato ed è stato dimostrato finora in patogeni Gram-negativi come il patogeno opportunista Acinetobacter baumannii2, il patogeno dentale Aggregobacter actinomycetemcomitans3 e il patogeno emergente Burkholderia pseudomallei4. L'autoaggregazione è stata descritta anche in diversi ceppi probiotici Gram-positivi 5,6,7,8. Nel caso di Lactobacillus (L.) acidophilus, l'autoaggregazione è stata parzialmente mediata dalle proteine dello strato S e correlata con un'adesione allo xilano7. Allo stesso modo, abbiamo trovato una correlazione tra l'autoaggregazione glucosio-dipendente e l'induzione delle proprietà adesive (giudicate dall'adesione alla mucina) delle specie probiotiche Lacticaseibacillus rhamnosus GG, Lacticaseibacillus casei, L. acidophilus, Lacticaseibacillus paracasei e Lactiplantibacillus plantarum5. L'aumento dell'autoaggregazione molto probabilmente rifletteva i cambiamenti nell'espressione delle adesine cellulari in risposta al glucosio e ai suoi cataboliti9. Mentre i meccanismi molecolari dell'autoaggregazione rimangono da determinare, è stato dimostrato che questo processo altera il fenotipo dei batteri aggregati e garantisce loro una maggiore tolleranza ai fattori di stress ambientale1, nonché una maggiore sensibilità alle molecole quorum sensing10.
Diversi approcci sono stati utilizzati per misurare l'autoaggregazione; Un approccio sperimentale consiste nel lasciare che le colture rimangano staticamente in tubi di coltura stretti per un dato tempo. Le colture di controllo rimangono torbide, mentre le colture di autoaggregazione si depositano sul fondo del tubo. Un approccio più quantitativo misura l'autoaggregazione mediante sedimentazione o saggio di sedimentazione11.
Anche la citometria a flusso è stata sempre più impiegata negli ultimi anni per studiare l'autoaggregazione batterica. Questo metodo è appropriato per l'analisi di particelle di dimensioni comprese tra circa 0,5 e 1000 μm. Il singolo batterio o gli aggregati formati sono sospesi in un fluido, immessi in un flusso e possono essere rilevati uno per uno11. La registrazione della luce diffusa in avanti consente di misurare la dimensione relativa della cella o dell'aggregato. È relativamente veloce e semplice, ma non è in grado di rilevare diversi parametri, come la dimensione dell'aggregato o il numero medio di celle negli aggregati. Pertanto, questo approccio può essere integrato al microscopio, consentendo di controllare più parametri12. Tuttavia, la microscopia tradizionale richiede molto tempo e quindi limita il numero di campioni testati e la potenza statistica dell'analisi. In generale, la citometria a flusso con imaging offre diverse funzionalità rispetto alla citometria a flusso tradizionale, come l'analisi simultanea della morfologia e del fenotipo cellulare, l'esecuzione di analisi basate su immagini, il rilevamento di eventi rari e la convalida dei dati della citometria a flusso13. Questi vantaggi migliorano le capacità della citometria a flusso e facilitano un esame più dettagliato delle popolazioni cellulari.
Questo studio fornisce un prezioso protocollo per l'imaging della citometria a flusso per monitorare l'autoaggregazione nei batteri lattici (LAB). Questi bastoncelli Gram-positivi sono anaerobi facoltativi e appartengono al gruppo LAB. Questi efficienti fermentatori di glucosio generano acido lattico come principale prodotto finale del metabolismo dei carboidrati14. Questi batteri sono membri fondamentali benefici del microbioma e si trovano naturalmente nel tratto gastrointestinale (GIT) dell'uomo e degli animali, nonché nel tratto urogenitale delle femmine15. Pertanto, l'esatta caratterizzazione delle loro proprietà di autoaggregazione è di alto interesse biotecnologico e clinico.
I nostri risultati precedenti hanno indicato che il livello basale di autoaggregazione differisce tra i diversi ceppi probiotici. Questa eterogeneità è influenzata dai diversi carboidrati utilizzati come fonte di carbonio5. Per superare questa proprietà fondamentale dei batteri probiotici, gli effetti dei carboidrati della dieta sono stati monitorati sull'autoaggregazione a livello di singola cellula utilizzando IFC. Questo approccio basato su IFC combina la potenza e la velocità dei citometri a flusso tradizionali con la risoluzione del microscopio. Pertanto, consente misure morfometriche complesse ad alta velocità in un modo fenotipicamente definito16,17. Questo approccio può essere esteso ad altri batteri probiotici e patogeni, combinati con reporter fluorescenti per monitorare l'espressione genica e ceppi marcati in fluorescenza per monitorare la presenza e l'abbondanza di specifiche specie batteriche in aggregati eterogenei.

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	<tbody>
		<tr>
			<td>14 mL culture tubes</td>
			<td>Falcon</td>
			<td>352051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Baeton,Dickinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Typtic Soy Broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Sigma</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Lactobacilli MRS broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>288130</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-LOCK MICROPR. 1.5 mL (Eppendorf)</td>
			<td>FL medical</td>
			<td>23053</td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS Software</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&#38;bd=1</td>
		</tr>
		<tr>
			<td>ImageStream X Mark II</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150121_205948?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>MOPS, 3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher bioreagents</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging flow cytometry to study microbial autoaggregation

I batteri benefici e probiotici svolgono un ruolo essenziale nei loro ospiti, fornendo vari benefici per la salute, tra cui l'immunità alle malattie infettive. La famiglia delle Lactobacillaceae è composta da batteri Gram-positivi con proprietà probiotiche confermate. Questo studio utilizza le specie di Lactobacillaceae come modello per dimostrare l'efficacia dell'analisi ad alto rendimento su singola cellula nello studio dell'aggregazione cellulare. L'attenzione si concentra sull'analisi della risposta di queste specie benefiche ai carboidrati semplici della dieta.
Lo studio mostra come la citometria a flusso (IFC) possa superare le differenze fondamentali nell'assemblaggio dei batteri probiotici in presenza e assenza di carboidrati. L'IFC combina la potenza e la velocità della citometria a flusso convenzionale con la risoluzione spaziale della microscopia, consentendo misurazioni morfometriche complesse ad alta velocità in modo fenotipicamente definito in una libreria di ceppi e condizioni batteriche benefiche. Questo protocollo fornisce informazioni sull'autoaggregazione delle specie di Lactobacillaceae e fa luce sulla loro risposta ai carboidrati alimentari, contribuendo a comprendere i meccanismi alla base degli effetti benefici di questi batteri probiotici.

Bacterial autoaggregation is considered a primary step in biofilm formation. In this process (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes or attach to their target tissue or surface1.
Autoaggregation is a widely observed phenomenon and has been shown so far in Gram-negative pathogens such as the opportunistic pathogen Acinetobacter baumannii2, the dental pathogen Aggregobacter actinomycetemcomitans3, and the emerging pathogen Burkholderia pseudomallei4. Autoaggregation has also been described in several probiotic Gram-positive strains5,6,7,8. In Lactobacillus (L.) acidophilus, autoaggregation was partially mediated by S-layer proteins and correlated with an adhesion to xylan7. Similarly, we found a correlation between glucose-dependent autoaggregation and induction of the adhesive properties (as judged by adhesion to mucin) of the probiotic species Lacticaseibacillus rhamnosus GG, Lacticaseibacillus casei, L. acidophilus, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum5. The increased autoaggregation most likely reflected changes in the expression of cellular adhesins in response to glucose and its catabolites9. While the molecular mechanisms of autoaggregation remain to be determined, it has been shown that this process alters the phenotype of the aggregated bacteria and grants them enhanced tolerance to environmental stressors1, as well as increased sensitivity to quorum sensing molecules10.
Several approaches have been used to measure autoaggregation; one experimental approach is to let cultures stand statically in narrow culture tubes for a given time. Control cultures remain turbid, whereas autoaggregation cultures will settle at the bottom of the tube. A more quantitative approach measures autoaggregation by sedimentation or settling assay11.
Flow cytometry has also been increasingly employed in recent years to investigate bacterial autoaggregation. This method is appropriate for analyzing particles between approximately 0.5 and 1000 &#181;m in size. The single bacterium or formed aggregates are suspended in fluid, fed into a stream, and can be detected one by one11. Recording forward scattered light allows measuring the relative size of the cell or aggregate. It is relatively fast and straightforward but cannot detect several parameters, such as aggregate size or the average number of cells in aggregates. Therefore, this approach can be complemented microscopically, allowing more parameters to be checked12. However, traditional microscopy is time-consuming and thereby limits the number of tested samples and the statistical power of the analysis. In general, imaging flow cytometry provides several features compared to traditional flow cytometry, such as simultaneous analysis of cell morphology and phenotype, conducting image-based analysis, detecting rare events, and validating flow cytometry data13. These advantages enhance the capabilities of flow cytometry and facilitate a more detailed examination of cell populations.
This study provides a valuable protocol for imaging flow cytometry to monitor autoaggregation in lactic acid bacteria (LAB). These Gram-positive rods are facultative anaerobes and belong to the LAB group. These efficient glucose fermenters generate lactic acid as their main end-product of carbohydrate metabolism14. These bacteria are beneficial core members of the microbiome and are naturally found in the gastrointestinal tract (GIT) of humans and animals, as well as in the urogenital tract of females15. Therefore, the exact characterization of their autoaggregation properties is of high biotechnological and clinical interest.
Our previous findings indicated that the basal level of autoaggregation differs between different probiotic strains. This heterogeneity is affected by the different carbohydrates used as a carbon source5. To overcome this fundamental property of probiotic bacteria, the effects of carbohydrates from the diet were monitored on autoaggregation on the single-cell level using IFC. This IFC-based approach combines the power and speed of traditional flow cytometers with the microscope&#39;s resolution. Therefore, it allows high-rate complex morphometric measurements in a phenotypically defined way16,17. This approach can be extended to other probiotic and pathogenic bacteria, combined with fluorescent reporters to monitor gene expression, and fluorescently labeled strains to monitor the presence and abundance of specific bacterial species in heterogeneous aggregates.

Autore in primo piano: Avanzamento della ricerca nell'autoaggregazione microbica mediante citometria a flusso di imaging

Citometria a flusso per lo studio dell'autoaggregazione microbica

This study utilized Lactobacillus species as a model to demonstrate the effectiveness of imaging flow cytometry analysis in studying microbial autoaggregation. The focus is on analyzing the response of autoaggregation of Lactobacillus species to simple carbohydrates from the diet. The most used methods for measuring autoaggregation are spectral photometry, which measures microbial suspensions, turbidity, or optical density, and traditional microscopy techniques, such as bright field, phase contrast, and fluorescent microscopy.When talking about hight-throughput imaging, one major experimental challenge is single event segmentation. In this protocol, it is a single aggregate. Imaging flow cytometry addresses this challenge elegantly and give researchers a powerful tool for studying autoaggregation.

I risultati dimostrano che questo metodo può facilmente misurare le differenze nell'autoaggregazione in risposta agli zuccheri alimentari nei batteri LAB. Separando gli individui dagli aggregati, il metodo consente di calcolare la percentuale della popolazione degli eventi di aggregazione rispetto a tutti gli eventi in risposta agli zuccheri fermentabili o non fermentabili della dieta. Inoltre, è stato possibile misurare se ci sono differenze nella dimensione media della popolazione aggregata tra i trattamenti.
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Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

Questo protocollo descrive un approccio quantitativo per misurare l'autoaggregazione microbica utilizzando la citometria a flusso di imaging.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

Questo studio ha utilizzato le specie di Lactobacillus come modello per dimostrare l'efficacia dell'analisi di citometria a flusso di imaging nello studio dell'autoaggregazione microbica. L'attenzione si concentra sull'analisi della risposta dell'autoaggregazione delle specie di Lactobacillus ai carboidrati semplici dalla dieta. I metodi più utilizzati per misurare l'autoaggregazione sono la fotometria spettrale, che misura le sospensioni microbiche, la torbidità o la densità ottica, e le tecniche di microscopia tradizionali, come il campo chiaro, il contrasto di fase e la microscopia fluorescente.Quando si parla di imaging ad alto rendimento, una delle principali sfide sperimentali è la segmentazione di un singolo evento. In questo protocollo, si tratta di un singolo aggregato. La citometria a flusso per immagini affronta questa sfida in modo elegante e offre ai ricercatori un potente strumento per studiare l'autoaggregazione.

imaging-flow-cytometry-to-study-microbial-autoaggregation

Research

JoVE Journal

Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Ricerca

Biologia

Lactobacillaceae Sample Preparation for Flow Cytometric Analysis

Start by inoculating five milliliters of lactobacilli MRS broth with a single colony of the lactobacilli strain, incubated at 37 degrees Celsius under static conditions overnight. Dilute the cell culture in a one-to-100 ratio in three milliliters of 50%supplemented tryptic soy broth, vortex the culture, and incubate it. Then invert the tube to mix the cells in the medium before transferring 200 to 300 microliters of the sample into a 1.5 milliliter micro centrifuge tube.

Lactobacillaceae Preparazione del campione per l'analisi citofluorimetrica

Flow Cytometric Data Acquisition of Microbial Aggregates

To begin the flow cytometric analysis, first set the 785-nanometer laser to a power of five milliwatts for dark field measurements. Use a 60X lens with a numerical aperture of 0.9. Then select the high-sensitivity setting and set the speed to low.To ensure calibration bead stability, press the Play button in the fluidics box and examine the bead images, which should appear sharp and clear. Generate a new scatterplot in the workspace box by selecting New Scatterplot. Then select the area in the Brightfield channel that corresponds to the SSC channel intensity and exclude the calibration beads that are running concurrently within the sample.Next, press the Load button and position a tube containing the Lactobacilli sample in the holder. In the Acquisition Settings box, enter the sample name and specify the location for data storage. Set the number of events to be collected, typically within the range of 10, 000 to 20, 000 events.Now, begin the acquisition process by clicking the Record button located in the Acquisition box. The process will halt after reaching the specified volume of events. Press the Return button to unload the tube and remove it from the holder as prompted in the popup window.After repeating the process for all samples, save the template to maintain data acquisition uniformity. For data analysis, to load the raw file into the analysis software, click on File and open the rif file. In the opened window, select Use Acquisition Analysis and then click OK.Next, to create a histogram of the gradient root mean square, click the New Histogram button and choose the population from the acquisition to analyze.Under the X Axis feature, select Gradient RMS of the Brightfield channel. Next, place a gate to exclude non-focused events by clicking the Create Line Region button in the analysis area. Create a scatterplot of the area versus the aspect ratio by clicking the New Scatterplot button in the analysis area.Select the focused population in the New Scatterplot window. Then select the area of the Brightfield channel for the X-Axis feature. Choose the aspect ratio for the Y-Axis feature.Draw a gate on the scatterplot using the Create Rectangle button based on the area value for the aggregate events population. Similarly, draw another gate for the singles and small aggregates population. Save your data analysis as a template by clicking the File tab, choosing Save As Template ast, and then open the next sample file under the same template to create the data analysis file.To measure the aggregate size distribution, start by plotting a histogram of the area of the aggregation events population. Save the data analysis by clicking on the file and selecting the Save As Template option. After saving, open the next sample file under the same template for the data analysis file.Single cells, small aggregates, large aggregates, and chains were observed in LGG and L.paracasei but it was not possible to distinguish between chains and larger aggregates. Significant variations in the aggregation characteristics of different LAB strains were observed in response to fermentable or non-fermentable sugars.