Il lavoro è stato realizzato con il sostegno finanziario del National Institute of Health e la American Lung Association.

1. Biofilm crescente di M. tubercolosi in una bottiglia con tappo a vite 250mL <ol><li> Preparazione Media: sciogliere 0,5 g di KH 2 PO 4, 0,5 g di MgSO 4, 4 g di L-asparagina, 2 g di acido citrico, 0,05 g di citrato ferrico di ammonio, 60 ml di glicerolo in 900 ml di acqua. Regolare il pH a 7,0 con NaOH. Autoclave, fresco e appena prima di iniziare l&#39;esperimento, ZnSO 4 aggiungere sterile ad una concentrazione finale di 0,1% w / v Dal 7000 mc 2 è un auxotroph pantotenato questo sforzo richiede anche l&#39;acido pantotenico al 10μg/mL di concentrazione finale. </li></ol> Nota: Questa è una composizione standard dei supporti utilizzati per la Sauton di M. tuberculosis. Tuttavia, se richiesto altri media specializzati possono essere utilizzati anche per altre specie micobatteriche. <ol start="2"><li> Inoculums preparazione: Grow M. tubercolosi in 7H9OADC con 0,05% Tween-80 per una settimana, o OD 600 di 0,7 1,0. Il culture può essere direttamente utilizzata come inoculo in una diluizione 1:100. </li><li> Distribuire 25mL di media Sauton ad un Flacone da 250 ml con tappo a vite di polistirene (Corning). Aggiungere 250μl di inoculo al mezzo, tappo della bottiglia molto strettamente e collocarlo in un indisturbato umidificata a 37 ° C incubatore per 3 settimane. Osservare la bottiglia una volta al giorno per assicurarsi che non vi è alcuna contaminazione. </li><li> Alla fine della terza settimana, svitare il tappo della bottiglia per consentire l&#39;ulteriore crescita di M. tubercolosi all&#39;interfaccia. Se il tappo non è allentato in questa fase quindi la concentrazione di ossigeno insufficiente nel contenitore ritardare l&#39;ulteriore crescita di batteri. </li></ol> 2. La crescita di M. biofilm tubercolosi a 12 pozzetti <ol><li> Preparare la media e inoculo di mc 2 7000, come descritto nelle sezioni A1 e A2. </li><li> Mescolare 60ml di media con 600μl di inoculo. Dispensare 4,5 mL della miscela in ciascun pozzetto della piastra. Coprire la piastra con il coperchio. Avvolgere la piastra più volte con parafilm. Incubare la piastra indisturbato in incubatore umidificato a 37 ° C per 5 settimane. </li></ol> 3. Determinare la frequenza di persistenti droga tolleranti in M. la tubercolosi biofilm <ol><li> Grow M. biofilm tubercolosi nell&#39;arco di 12 pozzetti, come descritto nella sezione B. Questo richiederà un totale di circa 5 settimane. </li><li> Una volta che i biofilm sono maturati (dopo 5 settimane di incubazione) iniettare la vostra scelta di antibiotici a concentrazione desiderata nei media sotto i biofilm utilizzando una micropunta in una pipetta. </li></ol> Nota: Il volume dei supporti sotto le pellicole riduce a circa 3.0mL. Così gli investigatori dovrebbero di conseguenza calcolare la quantità di farmaco. <ol start="3"><li> Agitare delicatamente la piastra in modo che l&#39;antibiotico viene accuratamente diffuso nel mezzo. Per ottenere risultati statisticamente significativi, iniettare il antibioticiotic in quattro pozzi. Parallelamente, iniettare lo stesso volume di solvente in cui l&#39;antibiotico è stato sciolto in altre quattro pozzetti, e lasciare gli ultimi quattro pozzetti della piastra intatto. Coprire la piastra con il coperchio e mettere nuovi strati di parafilm intorno alla piastra. Mettere di nuovo in incubatrice per un periodo di tempo desiderato. </li><li> Al termine dell&#39;incubazione, aprire la piastra e aggiungere Tween-80 alla concentrazione finale dello 0,1% (volume / volume) in ciascuno dei pozzetti. Agitare gentilmente l&#39;intera piastra di dispersione uniforme del detersivo. Incubare la piastra a temperatura ambiente per 15 minuti. Mescolare il contenuto di ciascun pozzetto con pipette volte in modo che l&#39;intero contenuto può essere uniformemente trasferiti in un tubo conico 15mL. </li><li> Rallenta il contenuto del tubo a 4000rpm per 10 minuti a temperatura ambiente. Risospendere il pellet in 5 ml di tampone di lavaggio fresca (PBS con 10% di glicerolo e 0,05% Tween-80). Ripetere il lavaggio tre volte. Risospendere il pellet in 5 ml di tampone di lavaggio. Keep it sul bilanciere per overnight a 4 ° C. </li></ol> Nota: Anche se a bassa temperatura è stato originariamente sviluppato per M. smegmatis (per ridurre al minimo la sua crescita durante la dispersione) e utilizzati per M. tuberculosis pure, le specie a lenta crescita può molto probabilmente essere cullato a temperatura ambiente senza alcun impatto sul risultato. Dondolo a temperatura ambiente può essere necessario se si lavora in BSL-3 struttura. <ol start="6"><li> Preparare una siringa sterile dotato di micropunta (2-200 pl), tagliando la sua estremità ampia per la dimensione appropriata, adattandosi alla siringa e avvolgendolo con parafilm. Passare tutto il contenuto del tubo attraverso la punta attrezzata siringa e raccogliere in un tubo fresco 15ml. Ripetere questo passaggio da 5 a 6 volte fino ad osservare una sospensione abbastanza omogenea. </li><li> Preparare diluizioni seriali della sospensione e le diluizioni piastra sulla piastra 7H11OADC per determinare il numero di colonie vitali in ciascun pozzetto. Incubare le piastre per tre settimane a 37 ° C incubatore. Determinare la frequenficienza di persistenti nella popolazione biofilm calcolando il rapporto tra numero di colonie ottenute nel trattamento antibiotico a quelli ottenuti solvente lastre trattate. </li></ol> 4. Risultati rappresentativi Quando coltivate in una bottiglia, crescita di M. tuberculosis può essere visto alla base della bottiglia dalla fine della prima settimana. Alla fine della seconda settimana, la crescita di batteri chiazze sulla interfaccia aria-media può essere visto, anche se la crescita all&#39;interfaccia aria-media interfaccia è sempre visibile alla fine della terza settimana (Fig. 1A). In questo momento l&#39;attacco dei batteri alla parete del contenitore è anche osservato. Da questo punto in poi la crescita della coltura avviene principalmente sulla interfaccia aria-media. Il liquido sotto la superficie di crescita è chiara. Tipicamente, la struttura matura dalla fine della quinta settimana (Fig. 1B). Se incubazione è prolungata, le strutture inizierà a depositarsi sul fondo del contenitore. Curiosamente,serraggio del tappo fino alla fine della terza settimana è un passo importante nel processo, e per ragioni sconosciute un loose-flacone con tappo ritarda notevolmente l&#39;inizio della crescita sull&#39;interfaccia 9. Nella 12-pozzetti, uno biofilm robusta all&#39;interfaccia aria-media interfaccia è visto in ciascuno dei pozzetti alla fine di cinque settimane (Fig. 2A). Se le piastre non sono avvolte completamente quindi crescita biofilm differenziale viene osservata. Nel caso peggiore, significativa evaporazione media possono ritardare la crescita dei batteri (Fig. 2B). Così, avvolgimento della piastra è necessaria sia per evitare l&#39;evaporazione e per fornire un ambiente per la formazione di biofilm (vedi paragrafo precedente). Il numero di bacilli vitali in biofilm determinati con questa tecnica è molto riproducibile. Risposta di M. tubercolosi biofilm varia con la natura degli antibiotici. Per un antibiotico battericida, come la rifampicina, perdita di vitalità segue un bipha<html> sic trend 9. Una rapida riduzione della redditività durante i primi tre o quattro giorni seguita da una fase persistente in cui una piccola percentuale della popolazione restano completamente recalcitrante agli antibiotici indipendentemente dalla concentrazione di antibiotico o il tempo di esposizione. La Figura 3 mostra il numero di bacilli vitali in biofilm maturi dopo 7 giorni di esposizione a 50μg/mL (50 volte superiore alla MIC) di rifampicina. <img alt="Figura 1A." src="/files/ftp_upload/3820/3820fig1A.jpg" /> Figura 1A. Comparsa precoce di M. bacilli tubercolosi sulla interfaccia aria-media di batteri dopo 3 settimane di incubazione. <img alt="Figura 1B." src="/files/ftp_upload/3820/3820fig1B.jpg" /> Figura 1B. Stagionato biofilm di M. tubercolosi in onda-Media Interface dopo cinque settimane di incubazione. 820/3820fig2A.jpg &quot;/&gt; Figura 2A. 5 settimane biofilm vecchi M. tubercolosi cresciuta in 12-pozzetti. <img alt="Figura 2B." src="/files/ftp_upload/3820/3820fig2B.jpg" /> Figura 2B. Un tentativo fallito di crescere biofilm M. tuberculosis in 12 pozzetti senza parafilm. <img alt="Figura 3." src="/files/ftp_upload/3820/3820fig3.jpg" /> Figura 3. Un grafico rappresentante che mostra la frequenza di persistenti droga tolleranti in M. tubercolosi biofilm cresciuto in 12 pozzetti ed esposte a 50μg/mL di rifampicina per sette giorni.

<ol>
	<li>Saltini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+and+diagnosis+of+tuberculosis.">Chemotherapy and diagnosis of tuberculosis.</a> Respir. Med. 100, 2085-2097 (2006).</li><li>Hall-Stoodley, L., 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=Biofilm+formation+and+dispersal+and+the+transmission+of+human+pathogens.">Biofilm formation and dispersal and the transmission of human pathogens.</a> Trends Microbiol. 13, 7-10 (2005).</li><li>Costerton, J. W., Stewart, P. S., Greenberg, E. 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:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Blankenship, J. R., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Curr Opin Microbiol. 9, 588-594 (2006).</li><li>Henke, J. M., Bassler, B. 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La tubercolosi (TB), causata da l&#39;infezione di Mycobacterium tuberculosis, rimane una grave minaccia per la salute pubblica globale. Quasi un terzo della popolazione mondiale è stimata essere asintomatica infettati dal patogeno, circa 9 milioni di nuovi casi appaiono in clinica ogni anno con sintomi di TB attiva e circa 1,7 milioni muoiono di infezione ogni anno 11. L&#39;enorme peso della malattia è principalmente contribuito la mancanza di un vaccino e di una chemioterapia altamente complessa...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

Mycobacterium tuberculosis, l&#39;agente eziologico della tubercolosi umana, ha una straordinaria capacità di sopravvivere contro gli stress ambientali, compresi gli antibiotici. Sebbene la tolleranza allo stress di M. la tubercolosi è uno dei contributori probabili a 6 mesi di chemioterapia lunga serie di tubercolosi 1, i meccanismi molecolari alla base di questo fenotipo caratteristico del patogeno rimangono poco chiari. Molte specie microbiche sono evoluti per sopravvivere in ambienti stressanti con l&#39;auto-assemblaggio in altamente organizzato, superficie attaccata, e matrice di strutture incapsulate chiamata biofilm 2-4. La crescita nelle comunità sembra essere una strategia di sopravvivenza preferito di microbi, e si ottiene attraverso componenti genetiche che regolano la superficie di applicazione, le comunicazioni intercellulari, e la sintesi di sostanze polimeriche extracellulari (EPS) 5,6. La tolleranza allo stress ambientale è probabilmente facilitata da EPS, e forse dal fisiologicoadattamento gico di bacilli individuale microambienti eterogenei all&#39;interno della complessa architettura dei biofilm 7. In una serie di lavori recenti abbiamo stabilito che M. smegmatis Mycobacterium tuberculosis e hanno una forte propensione a crescere in strutture organizzate pluricellulari, chiamata biofilm, che può tollerare più di 50 volte le concentrazioni minime inibenti dei farmaci antitubercolari isoniazide e rifampicina 8-10. M. la tubercolosi, però, richiede intrigante condizioni specifiche per formare biofilm maturi, in particolare il rapporto di 9:01: spazio di testa dei media così come limitato scambio d&#39;aria con l&#39;atmosfera 9. Requisiti di specializzati condizioni ambientali potrebbe essere legato al fatto che M. la tubercolosi è un patogeno obbligato umana e quindi si è adattata agli ambienti dei tessuti. In questa pubblicazione si dimostra metodi di coltura M. tubercolosibiofilm in una bottiglia e un 12-pozzetti piastra, che è conveniente per studi batteriologici nonché genetica. Abbiamo descritto il protocollo per un ceppo attenuato di M. la tubercolosi, mc 2 7000, con l&#39;eliminazione nei due loci, i panCD e RD1, che sono fondamentali per la crescita del patogeno vivo 9. Questo ceppo può essere tranquillamente utilizzato in un BSL-2 di contenimento per la comprensione della biologia di base del patogeno della tubercolosi evitando così l&#39;esigenza di un costoso impianto di BSL-3. Il metodo può essere esteso, con le opportune modifiche in media, a crescere biofilm di altre specie coltivabili micobatteri. Nel complesso, un protocollo uniforme di biofilm coltura dei micobatteri aiuterà i ricercatori interessati a studiare le caratteristiche di base di micobatteri resistenti. Inoltre, un metodo chiaro e conciso di biofilm che crescono micobatteriche aiuterà anche i inv clinico e farmaceuticoestigators per testare l&#39;efficacia di un potenziale farmaco.

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forma biofilm droga tolleranti, quando coltivate in determinate condizioni. Qui si descrivono i metodi per la coltura M. biofilm tubercolosi e che determinano la frequenza di persistenti droga tolleranti. Questi protocolli saranno utili per ulteriori studi sui meccanismi di tolleranza al farmaco in M. tubercolosi.

Crescita Mycobacterium tuberculosis I biofilm

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

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Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

23.6K Views.  University of Pittsburgh. Mycobacterium tuberculosis forma biofilm droga tolleranti, quando coltivate in determinate condizioni. Qui si descrivono i metodi per la coltura M. biofilm tubercolosi e che determinano la frequenza di persistenti droga tolleranti. Questi protocolli saranno utili per ulteriori studi sui meccanismi di tolleranza al farmaco in M. tubercolosi.

Video: Growth of Mycobacterium tuberculosis Biofilms

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Test di suscettibilità antimicrobica del complesso Mycobacterium tuberculosis delle droghe prima e seconda linea per diluizione brodo in un formato micropiastra

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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Immunologia e infezioni

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Un nuovo approccio Microdissezione per recuperare<em&gt; Mycobacterium tuberculosis</em&gt; Trascrizioni specifici da formalina paraffina fisso incorporato Lung granulomi

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Imaging Raziometrico del pH extracellulare nel dentale biofilm

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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 ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...