Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
Uploaded: 2016-12-14T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

TB riconosce un assegno di ricerca da Fibrosi Cistica Canada.

Si noti che la ricerca Consiglio di etica (REB) è necessario per raccogliere e conservare campioni di espettorato da soggetti umani. Questi studi sono stati approvati dal Hospital for Sick Children REB # 1.000.019,444 mila. 1. preparazione dei campioni dell&#39;espettorato CF <ol><li> Raccogliere campione di espettorato dai pazienti durante le visite di routine alla clinica fibrosi cistica e tenere in ghiaccio. </li><li> Trasporto del campione di espettorato sul ghiaccio entro la prima ora di raccolta, al laboratorio di ricerca, di sottoporsi a trattamento. </li></ol> 2. dell&#39;espettorato Processing <ol><li> Registrare il volume del campione di espettorato ottenuti. Aggiungere tampone fosfato salino (PBS) per 2 volte il volume del campione (ad esempio, 2 parti di PBS, 1 parte di campione). </li><li> Mescolare il campione bene con una pipetta di trasferimento. Vortex il campione sul valore massimo per 1 minuto per miscelare completamente. </li><li> Aliquota 1 ml della miscela sopra nel numero provette da 1,5 ml microcentrifuga appropriati e spin down a 5.000 xg per20 min a 4 ° C. </li><li> Dopo la centrifugazione, rimuovere il surnatante e scartare il pellet. </li><li> Filtro sterilizzare il surnatante attraverso un filtro di 0,22 micron e raccogliere in una provetta pulita. NOTA: La sterilità del filtrato viene testato da placcatura su LB agar e inoculando mezzi liquidi. </li><li> Conservare il surnatante dell&#39;espettorato a -80 ° C per un uso futuro. NOTA: L&#39;espettorato da più pazienti possono anche essere raggruppati dopo la filtrazione. </li><li> Prima dell&#39;uso, diluire espettorato filtrato 1/10 v / v (100 ml di espettorato, 900 l di media) a supporto desiderato. NOTA: Qui, è stato utilizzato il brodo lisogenia standard (LB) media. </li></ol> 3. Metodo Chambered coprioggetti per la formazione di biofilm <ol><li> Crescere isolato batterico di interesse overnight a supporto desiderato a 37 ° C con agitazione (200 rpm). Nota: sono stati utilizzati una serie di diversi batteri, tra isolati clinici Pseudomonas aeruginosa, Staphylococcus aureus,Complesso Burkholderia cepacia e xylosoxidans Achromobacter. Scelta dei supporti dipende dalle tensioni e le condizioni di interesse, ma i media LB possono essere utilizzati per gli esperimenti iniziali. </li><li> Dalla cultura overnight, inserire 40 ml di coltura in 4 ml di mezzi freschi e crescere per 3-4 ore a 37 ° C con agitazione (200 rpm) per ottenere una coltura con una densità ottica a 600 nm (OD 600) di circa 0,5 -0.6. </li><li> Diluire la cultura dal punto 3.2 a 1/5 nel supporto desiderato con il 10% espettorato filtrato o senza filtrato espettorato (come controllo). Altro concentrazione di espettorato filtrato può essere testato (cioè, 50% o 100%). </li><li> Utilizzare 200 ml di diluizione per seminare pozzi di camere di diapositive. </li><li> Permettere ai batteri di attaccare per 4 ore a 37 ° C senza agitazione. </li><li> Dopo 4 ore, rimuovere il supporto e lavare delicatamente il biofilm con mezzi freschi 1x. Sostituire con 200 ml di mezzi freschi. NOTA: Per studiare gli effetti della sputo sul biofilm, i fresh media dovrebbero contenere espettorato surnatante. </li><li> Consentire biofilm di crescere per quantità desiderata di tempo a 37 ° C senza agitazione, sostituirà supporti ogni 12 ore, senza lavaggio fino al momento per microscopia. NOTA: Per studiare l&#39;effetto dei sovranatanti di espettorato su biofilm sensibilità agli antibiotici, gli antibiotici vengono aggiunti ai media dopo 24 ore di crescita di biofilm e sono mantenute nella media fino a quando la colorazione e l&#39;imaging di biofilm. </li></ol> 4. colorazione biofilm e Microscopia confocale <ol><li> Dopo il tempo di crescita desiderato (24-48 hr funziona meglio), rimuovere i supporti da pozzi da camera e lavare delicatamente ciascuna camera due volte con 300 ml di PBS sterile. </li><li> Preparare la miscela di colorazione per biofilm mescolando 1 ml di ciascun colorante (fornito nel kit fattibilità) per ogni ml di soluzione necessaria. Fai la tintura in acqua o mezzi soluzione. NOTA: L&#39;acqua è consigliato dal produttore. </li><li> Aggiungere 200 ul di miscela colorante in ciascun pozzetto della covergla chamberedss ed incubare a temperatura ambiente, al buio per 45 min. </li><li> Rimuovere miscela colorazione dalle camere e lavare bene ogni con 300 ml di PBS sterile. Rimuovere PBS e sostituirlo con acqua dolce o media. </li><li> Procedere con la visualizzazione di biofilm mediante microscopia confocale. </li></ol> 5. Visualizzazione biofilm con Microscopia confocale <ol><li> Leggi biofilm macchiati in camere immediatamente dopo la colorazione (entro 1 ora). Minimizzare ritardo nella visualizzazione delle diapositive colorando 1 a 2 camere 8 pozzetti alla volta. </li><li> Eseguire l&#39;imaging utilizzando microscopio confocale con i laser per l&#39;eccitazione e di filtro set per l&#39;acquisizione. NOTA: Qui, il disco di sistema confocale di filatura con i laser Borealis spettrali (Verde: 491nm, colore rosso: 561 nm) sono stati utilizzati per l&#39;eccitazione. filtro per le emissioni set di 515/40 e 624/40 sono stati usati per visualizzare le macchie dal kit biofilm viabilità. </li><li> Prendere le immagini utilizzando un obiettivo 25X acqua sul microscopio confocale con la macchina fotografica. </li><li&gt; Usa Z-Stack per modellare il biofilm. Prendere immagini ogni 0,5-1 micron a partire dal primo piano di messa a fuoco per l&#39;ultimo fotogramma di messa a fuoco del biofilm (in genere si estende 30-80 micron per 48 biofilm hr) </li><li> Prendere 3-5 immagini da ogni pozzetto. NOTA: Così per un 8 coprioggetti ben camerata, 24-40 immagini saranno generate. </li><li> Salvare le immagini per l&#39;analisi. NOTA: Le immagini devono essere salvati come file OME-TIFF per essere analizzati utilizzando COMSTAT 18,19. Istruzioni per l&#39;analisi delle immagini biofilm possono essere trovati a http://www.comstat.dk/. Una volta che le immagini vengono importate, parametri come spessore medio, biomassa e la copertura di superficie per ogni canale (rosso e verde) possono essere analizzati. </li></ol>

<ol>
	<li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+with+biofilm+formation:+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy.">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+microbial+life+on+surfaces.">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li><li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giving+structure+to+the+biofilm+matrix:+an+overview+of+individual+strategies+and+emerging+common+themes.">Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.</a> FEMS Microbiol.Rev. 39 (5), 649-669 (2015).</li><li>Katharios-Lanwermeyer, S., Xi, C., Jakubovics, N. S., Rickard, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Microbial+coaggregation:+ubiquity+and+implications+for+biofilm+development.">Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development.</a> Biofouling. 30 (10), 1235-1251 (2014).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation:+a+clinically+relevant+microbiological+process.">Biofilm formation: a clinically relevant microbiological process.</a> Clin.Infect.Dis. 33 (8), 1387-1392 (2001).</li><li>Bjarnsholt, 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=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends Microbiol. 21 (9), 466-474 (2013).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms.">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario,+Canada.">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li><li>Vidya, 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=Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis.">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li><li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells.">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li><li>Beaudoin, 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=The+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa.">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li><li>LaFayette, S. L., 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=Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses.">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199 (2015).</li><li>Staudinger, B. J., 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=Conditions+associated+with+the+cystic+fibrosis+defect+promote+chronic+Pseudomonas+aeruginosa+infection.">Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.</a> Am.J.Respir.Crit.Care Med. 189 (7), 812-824 (2014).</li><li>Kennedy, S., 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=Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms.">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li><li>Tom, S. K., Yau, Y. C., Beaudoin, T., LiPuma, J. J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients.">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li><li>Heydorn, A., 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=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Vorregaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Informatics and Mathematical Modelling. , (2008).</li><li>Jurcisek, J. A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <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+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

I metodi qui descritti consentono la visualizzazione dei biofilm batterici cresciute in presenza di prodotti esogeni. Non sorprendentemente, la produzione dei exoproducts è di importanza quando si utilizza questo tipo di sistema. Per esempio, ditiotreitolo (DTT), è spesso usato su campioni di espettorato umani per aiutare a liquefare i campioni. Tuttavia, l&#39;effetto di DTT solo può diminuire sviluppo biofilm e vitalità (dati non mostrati). Quindi, controlli appropriati per tutte le condizioni sono necessarie. Ino...

biofilm batterici sono gruppi di microrganismi che sono attaccati l&#39;uno all&#39;altro e racchiuso in una matrice di auto-secreta. 1,2 Classicamente, rappresentano i batteri fisicamente collegati a una superficie abiotico o biotico formata in condizioni di flusso. I biofilm hanno anche dimostrato di crescere in condizioni statiche (assenza di flusso) e distale da superfici, come ad esempio all&#39;interfaccia aria-liquido di piscine termali o pellicole formate in provetta. Questi biofilm sono da tempo riconosciuti nell&#39;ambiente e sono un grande danno per processi industriali, in quanto possono formare in serbatoi di acqua o in tubi, con conseguente biofouling, corrosione e blocchi. 3,4 I biofilm sono anche critici in ambienti sanitari, in quanto è stato dimostrato essere coinvolti nelle infezioni correlate al catetere, infezioni polmonari nei pazienti affetti da fibrosi cistica, così come in numerose altre infezioni. 5,6 Una delle caratteristiche di infezioni biofilm è la desgualcita sensibilità dei batteri agli antibiotici e compromessa rinvio del sistema immunitario innato. 7-9 Il più studiati, scenari clinicamente rilevanti che coinvolgono infezione biofilm-based si verifica in pazienti affetti da fibrosi cistica (FC), che sono cronicamente infettati con Pseudomonas aeruginosa biofilm. P. aeruginosa può subire una serie di modifiche durante la creazione di infezione cronica che lo rendono molto difficile da trattare. 10,11 I biofilm possono differenziale attivare l&#39;immunità innata e guidare l&#39;infiammazione. 12-14 Poiché queste infezioni portano ad un aumento della morbilità e mortalità nei pazienti con FC, è fondamentale per capire i fattori che possono influenzare lo sviluppo del biofilm in questo contesto. Un recente studio suggerisce che ospitano-fattori critici nella formazione di P. aeruginosa aggregati biofilm. 15 Questi biofilm contribuiscono alla ridotta sensibilità agli antibiotici e meccanismi di difesa dell&#39;ospite. il PreseSNO di fattori dell&#39;ospite-derivati, come elastasi neutrofila, così come i prodotti secreti da microrganismi presenti nel polmone CF, hanno il potenziale di modulare notevolmente la formazione di biofilm e sviluppo. 16 Inoltre, biofilm interagiscono con l&#39;host di modulare l&#39;espressione di numerosi percorsi e avviare l&#39;infiammazione. Mentre i metodi high throughput, quali il test cristalvioletto standard possono fornire alcune informazioni per quanto riguarda il processo di biofilm, visualizzazione del biofilm in risposta a questi fattori fornire informazioni più approfondite. In questo manoscritto si descrive un metodo per l&#39;utilizzo di fattori dalla nell&#39;espettorato di pazienti con FC per studiare lo sviluppo di biofilm in vitro. Questo metodo consente la visualizzazione rapida dei biofilm esposti a espettorato contenente fattori host utilizzando un kit di biofilm redditività commerciale. Questa tecnica può essere utilizzata per identificare visivamente i cambiamenti che si verificano durante la crescita di biofilm in presenza di exogenonoi prodotti, e rappresenta un metodo migliore per analizzare i cambiamenti nello sviluppo del biofilm in varie condizioni.

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

I biofilm sono costituiti da gruppi di batteri racchiusi in una matrice di auto-secreto. Essi svolgono un ruolo importante nella contaminazione industriale e nello sviluppo e la persistenza di molte infezioni salute correlati. Uno dei biofilm più ben descritti e studiati nelle malattie umane avviene in infezione cronica polmonare dei pazienti affetti da fibrosi cistica. Studiando biofilm nel contesto dell&#39;ospite, molti fattori possono influenzare la formazione e lo sviluppo del biofilm. Al fine di individuare come fattori dell&#39;ospite possono influenzare la formazione di biofilm e sviluppo, abbiamo usato un metodo coprioggetti Chambered statica a crescere biofilm in presenza di fattori dell&#39;ospite-derivati ​​in forma di surnatanti espettorato. I batteri sono seminate nelle camere e esposti a filtrati espettorato. Dopo 48 ore di crescita, biofilm sono macchiate di un biofilm kit commerciale viabilità prima di microscopia e analisi confocale. A seguito di acquisizione delle immagini, le proprietà biofilm possono essere valutati utilizzando piattaforme software differenti.Questo metodo ci permette di visualizzare le proprietà principali della crescita di biofilm in presenza di sostanze diverse, tra cui antibiotici.

Il disegno complessivo di questo esperimento è rappresentata in figura 1. L&#39;uso di questo protocollo fornisce un metodo conveniente per visualizzare le variazioni di biofilm coltivate per diversi periodi di tempo (ad esempio, 24, 48 o 72 hr). È importante sottolineare che i segnali esogeni, quali espettorato filtrati, possono essere aggiunti per visualizzare i cambiamenti nello sviluppo biofilm. Come si vede nella figura 2, la presenza del...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

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

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

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 ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

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Immunology and Infection

8.3K Views.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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Immunologia e infezioni

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

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

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.

Crescita Mycobacterium tuberculosis I biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso di un terreno dell&#39;espettorato artificiale per testare l&#39;efficacia degli antibiotici contro<em&gt; Pseudomonas aeruginosa</em&gt; In condizioni più rilevanti al polmone Fibrosi Cistica

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologie per lo studio<em&gt; B. subtilis</em&gt; I biofilm come modello per Caratterizzare piccole molecole inibitori biofilm

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Il WinCF Modello - Un Microcosmo Economico e Tractable di un muco bronchiale Plugged per studiare la Microbiologia del polmone Infezioni

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

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

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

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 ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Test di efficacia antibiotica in un modello ex vivo di Pseudomonas aeruginosa e Staphylococcus aureus Biofilms nel polmone della fibrosi cistica

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

<meta charset="utf8"></head><body>Modello CF adattabile per test antimicrobici e analisi della dinamica microbica

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>Sviluppo di un modello di biofilm di colonia polimicrobica per testare gli antimicrobici nella fibrosi cistica

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Sviluppo di un modello di biofilm di colonia polimicrobica per testare gli antimicrobici nella fibrosi cistica

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

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Co-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

Crescita Mycobacterium tuberculosis I biofilm

Uso di un terreno dell'espettorato artificiale per testare l'efficacia degli antibiotici contro

Metodologie per lo studio

Il WinCF Modello - Un Microcosmo Economico e Tractable di un muco bronchiale Plugged per studiare la Microbiologia del polmone Infezioni

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

Test di efficacia antibiotica in un modello ex vivo di Pseudomonas aeruginosa e Staphylococcus aureus Biofilms nel polmone della fibrosi cistica

Sviluppo di un modello di biofilm di colonia polimicrobica per testare gli antimicrobici nella fibrosi cistica

Sviluppo di un modello di biofilm di colonia polimicrobica per testare gli antimicrobici nella fibrosi cistica