Name: targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay
Uploaded: 2016-05-05T13:00:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

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>

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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><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></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

13.1K 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

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensori per il rilevamento di antibiotici batteri di stafilococco resistenti

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioingegneria

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 ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Test di suscettibilità agli antibiotici indotti dallo stress su un chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Una piattaforma di test anti-biofilm Adatto per l&#39;esplorazione del Biblioteche composto naturale

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Saggi standardizzati in vitro per visualizzare e quantificare le interazioni tra neutrofili umani e biofilm di Staphylococcus aureus

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Un saggio di riduzione solubile a base di tetrazolio per valutare l'effetto degli anticorpi sui biofilm di Candida tropicalis

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Test di protezione enzimatica migliorato per studiare l'internalizzazione dello Staphylococcus aureus e l'efficacia intracellulare dei composti antimicrobici

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biologia

In Vitro Biofilm Synthesis by Staphylococcus aureus

Source: Rana, P. S. et al., <a href="https://app.jove.com/v/63773">Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

Sintesi di biofilm in vitro da parte di Staphylococcus aureus

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

Targeting biofilm Associated

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