We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. biofilm Initiation <ol><li> Crescere una cultura di biofilm producono organismo in un mezzo ricco di nutrienti. Seminare Staphylococcus aureus ceppo Newman in 10 ml di terreno Mueller-Hinton da uno stock di glicerolo. Eseguire tutti i lavori che coinvolgono la gestione di S. aureus con i guanti e all&#39;interno di un armadio biosicurezza. </li><li> Incubare per 16 ore a 37 ° C su shaker rotativo (100-200 rpm). </li><li> Determinare OD 600 della coltura utilizzando uno spettrofotometro ed una cuvetta standard (lunghezza del percorso: 1cm). </li><li> Calcolare il volume (V) della cultura necessari per preparare 20 ml di inoculo con OD 600 (inoculo) di 0,1 in chelexed mezzi Roswell Park Memorial Institute (CRPMI) 9 utilizzando la formula: [V = 20 ml * 0,1 / OD 600 (cultura )] Nota: Riferimento 9 fornisce dettagli sulla preparazione CRPMI. <ol><li> Aggiungere il volume coltura calcolata (dall&#39;alto) in una provetta da centrifuga e cellule pellet mediante centrifugazione per 1 min a 10.000 x g. per prepare biofilm inoculo, aspirare il mezzo di crescita e risospendere pellet cellulare trascorso in 20 ml CRPMI. </li></ol></li><li> Versare inoculo in un serbatoio 25 ml. </li><li> Rimuovere con cautela il coperchio da una piastra sterile piolo 96 pozzetti tirandola verso l&#39;alto. Nota: Fare attenzione a non contaminare i pioli che pendono dal coperchio. Il coperchio può essere posizionato a testa in giù su una superficie pulita all&#39;interno di un armadio biosicurezza. </li><li> Usando una pipetta microlitri 200 multicanale, aggiungere 125 microlitri inoculo in ciascun pozzetto di righe da A a G della piastra inferiore. </li><li> Riempire media 125 ml sterile CRPMI in ciascun pozzetto di fila H come controllo di sterilità. </li><li> Prendere la peg-coperchio e tenerlo sopra il fondo piatto con i pioli rivolti verso il basso. Allineare con cura i singoli pioli con relativi pozzetti. Evitare il contatto fisico tra la piastra e le spine. Una volta allineati, abbassare il coperchio con cautela verso il basso. Evitare disallineamenti come aggiustamenti eseguiti dopo i picchetti hanno toccato la piastra inferiore può contcontrolli di sterilità aminate in fila H. </li><li> Sigillare il coperchio alla piastra con la pellicola di paraffina per evitare l&#39;evaporazione e mettere in un sacchetto di plastica richiudibile, tenuta saldamente. Mantenere il volume dell&#39;aria nel sacchetto più basso possibile per minimizzare l&#39;evaporazione. </li><li> Incubare senza agitazione a 37 ° C per 48 ore in un incubatore CO 2 5%. Nota: 48 ore di crescita è stato ottimale per S. aureus, ma varierà basato sul organismo utilizzato. Nota: Anche se l&#39;incubazione statico è un buon punto di partenza per ottimizzare il dosaggio, scuotendo leggermente a 150 giri al minuto, un vortex è una delle variazioni che possono migliorare la formazione di biofilm di alcuni S. aureus isolati. </li></ol> 2. Preparazione della piastra biofilm sfida <ol><li> Il giorno della sfida biofilm prendere una piastra a 96 pozzetti fresco e aggiungere 100 ml di CRPMI, equilibrati a RT, a ciascun pozzetto di righe B a H. Per evitare risultati inconsistenti, utilizzare una piastra a 96 pozzetti che può contenere 200 mldei media quando i pioli sono all&#39;interno, e dispone pozzetti con dimensioni identiche a quelle della piastra biofilm iniziazione. </li><li> Diluire fino a 4 composti di prova separatamente in 1,0 ml di CRPMI a 2x la concentrazione finale desiderata, al fine di spiegare una diluizione finale nel passo 2.7. Nota: un buon punto di partenza è di 8 volte superiori alla concentrazione inibitoria delle cellule planctoniche. </li><li> Aggiungere 200 ml di composto 1 a Wells A1 a A3, composto di 2 pozzi in A4 A6, composto di 3 pozzi in A7 a A9 e composto 4 in pozzi A10 a A12. </li><li> In serie diluire i composti di prova utilizzando una pipetta multicanale e trasferire 100 ul dalla fila A alla fila B e mescolare. </li><li> In questo modo, continuare a trasferire 100 ul pozzetto a pozzetto. Mescolare dopo ogni trasferimento e sosta in fila F. </li><li> Dopo la miscelazione, espellere i 100 ml di fila F in un contenitore per rifiuti. Non trasferire alcun liquido alle righe G e H. </li><li> Aggiungere 100 ml di CRPMI a ciascun pozzetto della piastra con una pipetta multicanale perportare il volume a 200 ml. Vedere la Figura 1 per il layout piatto finale. </li></ol> 3. biofilm Sfida <ol><li> Aggiungere 200 ml di tampone fosfato salino (PBS) ad un nuovo sterile 96 pozzetti che dispone pozzetti con dimensioni identiche a quelle della piastra biofilm iniziazione. </li><li> Rimuovere il sacchetto di plastica e pellicole di paraffina dalla piastra biofilm iniziazione incubate. </li><li> Sollevare il coperchio verso l&#39;alto. Evitare il contatto tra i picchetti e il fondo piatto come questo può danneggiare il biofilm. Tenere la parte inferiore per la successiva analisi OD 600 (vedi 3.8). </li><li> Risciacquare cellule planctoniche posizionando il peg-coperchio sulla piastra contenente PBS (dal punto 3.1). Immergere pioli per 5 secondi, sollevare dalla PBS, e immergere ancora una volta, facendo attenzione a non colpire i picchetti contro i lati pure. </li><li> Mettere il coperchio peg-risciacquato nella piastra sfida. Evitare il contatto inutili tra i picchetti e piastra di fondo per non danneggiare il biofilm che porta arisultati inconsistenti. </li><li> piastra di tenuta con la pellicola di paraffina e riporre in un sacchetto di plastica richiudibile come descritto al punto 1.10. </li><li> Incubare la piastra senza agitazione per 24 ore a 37 ° C in un incubatore CO 2 5%. </li><li> Prendere parte inferiore della piastra di biofilm iniziazione da 3.3. Risospendere i batteri in ciascun pozzetto utilizzando una pipetta multicanale e misurare il diametro esterno 600 con un lettore di micropiastre adatto per confermare la crescita che si verificano in tutti i pozzetti, ma i controlli di sterilità in fila H. </li></ol> 4. Determinazione biofilm <ol><li> Utilizzare un lettore di piastre dotato di una camera di incubazione e in grado di prendere letture di fluorescenza cinetici per la rilevazione resazurina conversione. Impostare una misurazione della fluorescenza cinetica 24 ore utilizzando una eccitazione 530 nm e 590 nm di emissione. </li><li> Impostare la temperatura della camera a 37 ° C e ha la piastra letto ogni 20 min. Impostare il lettore di piastre per misurare dal fondo della piastra base alle istruzioni del produttorezioni. </li><li> Preparare Lavare la piastra con l&#39;aggiunta di 200 ml di PBS con una pipetta multicanale ad una sterile 96 pozzetti. </li><li> Preparare mezzo di recupero biofilm con l&#39;aggiunta di 400 ml di 0,8 mg / ml resazurina soluzione madre a 20 ml di terreno CRPMI ad una concentrazione finale di 16 mg / ml resazurina. Nota: resazurina è irritante per la pelle conosciuta. Uso di un camice, occhiali anti-schizzo, e guanti durante la manipolazione. CRPMI come supporti di ripristino biofilm è stato usato perché fornisce il segnale di fondo più basso, ma può essere sostituito da mezzi di Mueller Hinton, se del caso. </li><li> Aggiungere 150 pl di mezzo di recupero biofilm con una pipetta multicanale a ciascun pozzetto di una piastra a 96 pozzetti fresco. </li><li> Trasferimento piatto sfida da incubatrice in un armadio biosicurezza e con attenzione rimuovere il sacchetto di plastica e la pellicola sigillante. </li><li> Togliere il peg-coperchio dalla piastra sfida e risciacquare le cellule planctoniche in PBS come descritto in 3.4. Mantenere il fondo della piastra sfida per successiva OD 600 analisi (vedi 4.10). </li><li> Dopo il risciacquo, inserire il piolo-coperchio nella piastra inferiore contenente il mezzo di recupero biofilm. </li><li> Avvolgere il lato della piastra con la pellicola di paraffina, facendo attenzione a non ostacolare il fondo dei pozzetti perimetrali. Subito dopo aver terminato la piastra, collocarlo sul lettore di piastre e iniziare una lettura cinetica in un periodo di 24 ore, avviando il già fatto resazurina protocollo cinetica dal punto 4.1. </li><li> Per quantificare la crescita di cellule planctoniche che possono o non possono verificarsi durante sfida, leggere la OD 600 dell&#39;intera piastra inferiore sfida utilizzando un lettore di micropiastre. Seguire le istruzioni riportate al punto 3.8. Nota: risospendere accuratamente il contenuto di ogni pozzetto come cellule spargimento fuori dal biofilm tendono a crescere in ciuffi sul fondo del pozzo. Eventuali bolle all&#39;interno del pozzo saranno fortemente interferire con OD 600 letture e devono essere evitati o rimossi prima della lettura. </li></ol>

<ol>
	<li>Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+insights+from+biofilm+biology+to+drug+development+-+can+a+new+approach+be+developed.">Applying insights from biofilm biology to drug development - can a new approach be developed.</a> Nat Rev Drug Discov. 12, 791-808 (2013).</li><li>Song, Z., 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=Prosthesis+infections+after+orthopedic+joint+replacement:+the+possible+role+of+bacterial+biofilms.">Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms.</a> Orthop Rev (Pavia). 5, 65-71 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases).">Bacterial biofilms: from the natural environment to infectious diseases).</a> Nat Rev Microbiol. 2, 95-108 (2004).</li><li>Bordi, C., de Bentzmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hacking+into+bacterial+biofilms:+a+new+therapeutic+challenge.">Hacking into bacterial biofilms: a new therapeutic challenge.</a> Ann Intensive Care. 1, 19 (2011).</li><li>Fux, C. A., Shirtliff, M., Stoodley, P., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+laboratory+reference+strains+mirror+'real-world'+pathogenesis?.">Can laboratory reference strains mirror 'real-world' pathogenesis?.</a> Trends Microbiol. 13, 58-63 (2005).</li><li>Kwasny, S. M., Opperman, 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=Static+biofilm+cultures+of+Gram-positive+pathogens+grown+in+a+microtiter+format+used+for+anti-biofilm+drug+discovery.">Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.</a> Curr Protoc Pharmacol. Chapter 13, Unit 13A 18 (2010).</li><li>Ceri, 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=The+MBEC+Assay+System:+multiple+equivalent+biofilms+for+antibiotic+and+biocide+susceptibility+testing.">The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing.</a> Methods Enzymol. 337, 377-385 (2001).</li><li>Ceri, 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=The+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J Clinical Microbiol. 37, 1771-1776 (1999).</li><li>Baker, 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=Copper+stress+induces+a+global+stress+response+in+Staphylococcus+aureus+and+represses+sae+and+agr+expression+and+biofilm+formation.">Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.</a> Appl Environ Microbiol. 76, 150-160 (2010).</li><li>Haeili, 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=Copper+complexation+screen+reveals+compounds+with+potent+antibiotic+properties+against+methicillin-resistant+Staphylococcus+aureus.">Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 3727-3736 (2014).</li><li>O'Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> Eur J Biochem. 267, 5421-5426 (2000).</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=Establishing+the+minimal+bactericidal+concentration+of+an+antimicrobial+agent+for+planktonic+cells+(MBC-P)+and+biofilm+cells+(MBC-B).">Establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (MBC-P) and biofilm cells (MBC-B).</a> J Vis Exp. (83), e50854 (2014).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li><li>Shah, D., 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=Persisters:+a+distinct+physiological+state+of+E.+coli.">Persisters: a distinct physiological state of E. coli.</a> BMC Microbiol. 6, 53 (2006).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45, 999-1007 (2001).</li></ol>

The authors have nothing to disclose.

Qui, abbiamo descritto un saggio biofilm modificato per determinare l&#39;attività di inibitori testati su S. biofilm aureus concentrandosi sullo stato metabolico delle cellule biofilm associato. Mentre il biofilm iniziazione descritto e procedure di contestazione per lo più imitate raccomandazioni del produttore, l&#39;uso di resazurina colorante per rilevare e quantificare le cellule biofilm associato che sopravvivono una sfida inibitore 24 ore semplifica notevolmente la procedura raccomandata di r...

Microbi patogeni possono formare biofilm in vivo che portano a infezioni croniche non acute 1. Infezione biofilm-associata è un grave fattore di rischio di procedure mediche che coinvolgono l&#39;impianto di corpi estranei (ad esempio, la sostituzione di ossa artificiali, le protesi mammarie) o l&#39;installazione di tubi tracheali o cateteri urinari 2. In questi contesti, terapia anti-infettiva è quasi sempre necessario come infezioni biofilm-based sono raramente cancellati da soli, anche in individui immunocompetenti. Staphylococcus aureus è uno dei patogeni più frequentemente osservati implicati nella complicazioni biofilm correlati che si verificano durante l&#39;uso di dispositivi medici invasivi 3. Purtroppo, la natura stessa del biofilm da barriera protettiva rende più resistenti al trattamento delle cellule planctoniche 1,4, e la valutazione della efficacia clinica predetto è una parte critica sia inizialelo sviluppo di farmaci così come la sorveglianza della resistenza ai farmaci. Vi è un crescente riconoscimento che condizioni di laboratorio, si è concentrata sulle culture planctonici, potrebbero non rappresentare fedelmente la malattia del mondo reale 5. Ulteriori aggravando il problema, la replica di fenotipi biofilm è difficile, e modelli biofilm esistenti sono noioso e soffrono di alta inter- e intra-assay variabilità 6. Così, molti ricercatori, per necessità, di default per saggi cellulari planctonici di sensibilità ai farmaci, quindi potenzialmente trascurare un aspetto importante della virulenza batterica e la malattia. Qui si descrive il protocollo per il saggio di batteri, in particolare S. aureus, in biofilm pre-grown utilizzando una base di 7,8 sistema di biofilm ben 96. Mentre il protocollo per la formazione di biofilm e la sfida segue essenzialmente la raccomandazione del costruttore, presentiamo una metodologia ricca di informazioni alternative per quantificare la fattibilità del BIOFilm dopo sfida. Brevemente, i batteri sono coltivate in piastra piolo, dove biofilm formano sui pioli sporgenti collegati al coperchio della piastra. Dopo la formazione di biofilm, i pioli sono delicatamente immersi in pozzetti di una piastra fresca ripiena con PBS per rimuovere le cellule planctoniche. Il peg-coperchio, con biofilm allegate, viene trasferito in una nuova piastra sfida, contenente varie concentrazioni di antibiotici da dosare. Dopo una seconda incubazione, coperchi sono nuovamente rimosso, lavato, e trasferiti ad una piastra di recupero contenente resazurina tintura, dove subiscono una incubazione finale. conversione resazurina può essere registrata cineticamente o preso come una lettura finale dopo un periodo di recupero definito. Questo metodo di tintura a base di quantificare la vitalità del biofilm si differenzia notevolmente dalle noiose CFU (unità formanti colonie) metodologia basata su conteggio descritto nel protocollo originale 7. OD 600 misurazioni della piastra sfida droga e resazurina cinetiche di conversione fungono redditività letturaout di cellule planctonici e biofilm, rispettivamente, offrendo un test veloce, affidabile e tecnicamente semplice ricco di informazioni per la sopravvivenza biofilm.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Il saggio resazurina è abbastanza sensibile per rilevare in modo affidabile pochissime cellule vitali in grado di replicare in terreno privo di droga dopo la sfida. In questa metodologia, definiamo la concentrazione di biofilm sradicare minima come la concentrazione più bassa alla quale nessuna conversione resazurina è visto entro 24 ore. Il test si basa su resazurina molecole ossidanti presenti nelle cellule metabolicamente attivi che convertono il colore della tintura dal blu al ros...

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Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Targeting biofilm Associated<em&gt; Staphylococcus aureus</em&gt; Uso resazurina base Assay farmaco-sensibilità

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

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Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

12.9K Views.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

Video: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtitolazione Dish Biofilm saggio Formazione

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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Immunologia e infezioni

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

L&#39;uso del flusso gocciolamento e Reattori disco rotante per Staphylococcus aureus Analisi Biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Sottocutaneo infezione di Staphylococcus aureus meticillino resistente (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endocardite modello sperimentale di meticillino resistente<em&gt; Staphylococcus aureus</em&gt; (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus crescita con emoglobina umana come fonte di ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR per la tipizzazione di stafilococco cassette cromosomiche Mec Tipi I a V, resistente alla meticillina<em&gt; Staphylococcus aureus</em

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Lo studio delle interazioni di<em&gt; Staphylococcus aureus</em&gt; Con neutrofili mediante citometria a flusso e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

La genotipizzazione di<em&gt; Staphylococcus aureus</em&gt; Per ribosomiale Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

Visualizzazione del carbone Agar resazurina Assay per semi-quantitativa, medio-throughput enumerazione dei micobatteri

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodologia per lo studio del orizzontale Gene Transfer in<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...