This work was supported by BC Lung Association and Mitacs.

1. ceppo batterico e crescita medio <ol><li> Rendere albumina destrosio e soluzione salina magazzino (ADS) solubilizzando 25 g di albumina di siero bovino, 10,0 g di destrosio, e 4,05 g di cloruro di sodio in 460 ml di acqua deionizzata. Filtro-sterilizzare ADS e conservare a 4 ° C. </li><li> Rendere 7H9 brodo aggiungendo 4,7 g di polvere 7H9 e 2 mL di glicerolo a 900 ml di acqua purificata. Autoclavare il brodo 7H9 a 121 ° C per 10 minuti e lasciare raffreddare a temperatura ambiente prima di procedere. Fare 7H9ADST aggiungendo 100 ml di ADS e 0,5 ml di Tween80 a 900 ml di brodo 7H9. Conservare a 4 ° C. </li><li> Pesare 50 mg di kanamicina solfato e sciogliere in 1 ml di acqua deionizzata; la concentrazione finale è di 50 mg / mL. Filtro-sterilizzare e conservare a -20 ° C. Aggiungere 0,5 ml di kanamicina soluzione madre per 1 L di 7H9ADST. NOTA: Questo terreno deve essere fatta fresca, così scalare i volumi modo appropriato secondo la dimensione cultura. </li><li> Crescere M. tuberculosis in 7H9ADST integrato con kanamicina nella cultura in piedi. Agitare la cultura quotidiana e diluirlo prima che la raggiunga OD600 1,0 per evitare grumi. NOTA: Il ceppo M. tuberculosis utilizzato per lo sviluppo di questo metodo è stato trasformato con H37Rv pJAK2.A plasmide 12. pJAK2.A è un plasmide integrativo basato sul vettore pMV361, che permette l&#39;espressione ad alto livello del gene luciferasi di lucciola dal promotore HSP60 e può essere selezionata tramite kanamicina. </li></ol> 2. THP-1 Medium e manutenzione <ol><li> Aggiungere 50 ml di siero inattivato al calore fetale bovino (FBS) e 5 ml di 200 mM di L-glutammina a 500 ml di RPMI 1640 per fare mezzo RPMI incompleta (circa il 10% FBS e 2 mM glutammina). </li><li> Mantenere una coltura cellulare THP-1 secondo protocollo standard 13. Brevemente, crescere cellule THP-1 in mezzo RPMI incompleta mantenendo una densità cellulare da 0,2 a 1 milione per mL di terreno tra passaggi. </li></ol> 3. Screening intracellulare high-throughput Utilizzando luciferasi che esprimono M. tuberculosis H37Rv <ol><li> Misurare la densità ottica di una sospensione batterica attiva crescita in uno spettrofotometro ad una lunghezza d&#39;onda di 600 nm. Calcolare la densità batterica utilizzando il fattore di conversione di 0,1 OD 600 = 3 x 10 7 batteri per mL. </li><li> Pipettare i batteri sufficienti per una molteplicità di infezione (MOI) di 10: 1 in una nuova provetta da centrifuga. Pellet a 3.000 xg per 10 minuti e aspirare il liquido. Aggiungere 50 ml di siero umano a 450 ml di RPMI1640. Scala il volume di appropriarsi dei valori per l&#39;esperimento. </li><li> Per opsonize i batteri, risospendere il pellet ad una densità di 1 x 10 8 batteri per 500 ml di RPMI1640 contenente 10% di siero umano. Lasciare la miscela a incubare a 37 ° C per 30 min. Determinare la THP-1 cellule densità coltura contando con un emocitometro e un microsco invertitape. </li><li> Agglomerare le cellule in provette da centrifuga sterili a 100 xg e 37 ° C per 10 min. Aspirare il surnatante e risospendere le cellule in RPMI incompleta ad una densità di 1 milione di cellule per ml. Aggiungere forbolo-12-miristato-13-acetato (PMA) per un 40 ng ml di concentrazione / finale. NOTA: Questo sarà indicato come il mix di differenziazione. </li><li> Combinare opsonizzati M. tuberculosis con THP-1 mix differenziazione ad una MOI di 10: 1 e aliquota della miscela finale a 100 microlitri per pozzetto in una piastra bianca 96 pozzetti a fondo piatto. Regolarmente agitare la miscela per assicurare uniformità. Permettono la differenziazione e l&#39;infezione di procedere notte a 37 ° C in un incubatore umidificato contenente il 5% di CO 2. </li><li> Lavare i pozzetti due volte con 100 ml di RPMI ciascuno. Aggiungere composti diluiti alle concentrazioni desiderate in RPMI incompleta ed incubare per 3 giorni. </li><li> Aspirare il terreno dai pozzi. Aggiungere 50 ml di reagente saggio luciferasi in ciascun pozzetto. Sigillare le piastre con transparent sigillanti piastra adesivi. Consentire 5 min di incubazione a 22 ° C e quindi ottenere una lettura in un luminometro a 1 s per pozzetto. </li></ol> 4. Analisi citotossicità usando un 3- (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazolio bromuro (MTT) 14 <ol><li> Differenziare le cellule THP-1 in RPMI incompleta completato con 40 ng / ml di PMA in chiaro piastre da 96 pozzetti. Mantenere una densità cellulare di 1 milione per mL e un&#39;aliquota di 100 microlitri per pozzetto. Lasciare differenziazione di procedere notte a 37 ° C in un incubatore umidificato contenente il 5% di CO 2. </li><li> Aspirare il terreno dai pozzetti e lavarli due volte con RPMI 1640. Aggiungere composti diluite in RPMI incompleti ai pozzetti. Incubare per 3 giorni. </li><li> Sciogliere 0.5 g di MTT in 100 mL di tampone fosfato salino (PBS) per ottenere una soluzione stock di 5 mg / mL. filtro sterile e conservare a -20 ° C, al riparo dalla luce; è meglio per rendere questa soluzione fresca. </li><li> 2.5 h before la fine del periodo di incubazione di 3 giorni, aggiungere 25 ml di soluzione di MTT ad ogni pozzetto e completare il periodo di incubazione. </li><li> Preparare 50% N, N-dimetil formammide (DMF) miscelando 250 ml di DMF con 250 mL di acqua deionizzata. </li><li> Preparare tampone di estrazione MTT come segue: Pesare 100 g di SDS in una bottiglia da 500 ml e aggiungere 300 ml di DMF al 50%. Applicare a fuoco basso per permettere al SDS sciogliere. Aggiungere 10 ml di acido acetico puro e 12,5 ml di 1 M HCl. Riempire fino al segno da 500 ml con il 50% DMF; la composizione finale del tampone di estrazione è del 50% DMF, 20% SDS, 2,5% di acido acetico e 2,5% 1 M di acido cloridrico. </li><li> Al termine del periodo di trattamento, aggiungere 100 ml di tampone di estrazione (riscaldato a 45 ° C per sciogliere i cristalli) a ciascun pozzetto. Lasciare la miscela a incubare durante la notte a 37 ° C in un incubatore umidificato contenente il 5% di CO 2. Leggere l&#39;assorbanza a 570 nm. NOTA: Il test di citotossicità migliore viene eseguita in parallelo con un&#39;intracellularischermo lare con persone dello stesso lotto e l&#39;età di cellule THP-1. </li></ol> 5. In brodo di attività di analisi Utilizzando un resazurina Assay 3 <ol><li> Crescere M. tuberculosis in 7H9ADST alla fase di mid-log (~ 0,5-0,8 OD 600). Diluire la cultura con la 7H9ADST a 0,01 OD 600. Diluire i composti in 7H9ADST a 2x le concentrazioni di prova e un&#39;aliquota di 100 microlitri di ciascun composto diluito in ciascun pozzetto. </li><li> Trasferire 100 microlitri della sospensione batterica diluita in ciascun pozzetto. Lasciare le piastre di incubare a 37 ° C in un incubatore umidificato per 5 giorni. Sciogliere 10 mg di resazurina in 100 mL di acqua deionizzata e filtro sterile. </li><li> Aggiungere 30 ml di soluzione di resazurina e monitorare il cambiamento di colore dopo 48 ore; la crescita batterica è indicato da una conversione di colore dal blu al rosa. NOTA: Un&#39;analisi quantitativa può essere eseguita misurando sia la fluorescenza a 590 nm con eccitazione a 530 - 560nm o l&#39;assorbanza a 570 nm e 600 nm 15. </li></ol>

<ol>
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The authors declare no competing financial interests for this work.

L&#39;obiettivo di questo studio era quello di creare un metodo semplice e conveniente HTS utilizzando un modello di infezione intracellulare umana per M. tuberculosis. La tubercolosi è una malattia umana caratterizzata da infezione di macrofagi alveolari di M. tuberculosis. A causa di problemi di biosicurezza, ricerca che coinvolgono modelli biologici sia del batterio e le cellule ospiti è stata utilizzata in passato. Tuttavia, è stato dimostrato che l&#39;uso di batteri surrogate e modelli non uma...

Mycobacterium tuberculosis, l&#39;agente eziologico della tubercolosi (TB), è una delle principali cause di morbilità e mortalità in tutto il mondo. TB Drug-sensibile è una malattia curabile che richiede più antibiotici per un periodo di 6 mesi. Pur essendo una malattia curabile, la mortalità TB è stato stimato a 1,5 milioni nel 2015 1. Negli ultimi 10 anni, ci sono stati crescenti preoccupazioni sulla prevalenza della farmaco-resistente M. tuberculosis. Multiresistente TB (MDR-TB) è definita come TBC che è resistente ad almeno isoniazide (INH) e rifampicina (RMP), e la maggior parte dei ceppi MDR-TB sono anche resistenti per selezionare farmaci di seconda linea TB, limitando così le opzioni di trattamento . Gli effetti della resistenza ai farmaci creano una sfida maggiore per il trattamento di pazienti co-infettati con il virus dell&#39;immunodeficienza umana (HIV); 400.000 pazienti in tutto il mondo sono morte di tubercolosi associata ad HIV nel 2015 1. Purtroppo, gli Stati Uniti Food and Drug Administrazione ha approvato un solo nuovo farmaco contro la TB MDR-TB, bedaquiline, negli ultimi 40 anni 2. I progressi nella ricerca di una migliore e più brevi terapie TB sono urgentemente necessarie al fine di rimanere in testa nella lotta contro la tubercolosi e la MDR-TB. Tradizionalmente, TB droga schermi vengono eseguite in vitro in condizioni di crescita in terreno di coltura, per cui i composti vengono aggiunti ad una coltura in crescita e la loro efficacia nel sradicare i microrganismi sono determinati dal conteggio delle unità formanti colonie (CFU) su terreno solido. Come conta CFU sono alta intensità di lavoro, che richiede tempo, e costoso, vari metodi indiretti sono stati sviluppati per alleviare questo problema. Tali metodi includono la vitalità dosaggio Alamar Blue 3, la determinazione della fluorescenza 4 dalla proteina verde fluorescente (GFP) o luminescenza 5 dai batteri luciferasi che esprimono, e la stima di adenosina trifosfato totale (ATP) 6, 7. TB tipica è caratterizzata da un&#39;infezione da M. tuberculosis del polmone, in cui i batteri risiedono e replicano all&#39;interno dei fagosomi di macrofagi alveolari 8. Lo schermo semplice in brodo fenotipica può soddisfare patogeni extracellulari; tuttavia, in prospettiva storica, ha colpito composti contro M. tuberculosis individuati con questo metodo spesso non riescono a essere all&#39;altezza delle aspettative durante le fasi di validazione a valle in modelli di infezione. Proponiamo che la tubercolosi farmaco è meglio eseguita in un modello di infezione cellula ospite intracellulare. Tuttavia, i modelli intracellulari possiedono molte barriere tecnologiche e biologiche di screening (HTS) sviluppo ad alta produttività. Un grande ostacolo è la complessità del processo di infezione, esemplificato da numerosi passaggi e la rimozione elaborato di batteri extracellulari da in-tra lavaggio. Un secondo ostacolo principale è il tempo lungo rigenze, il rilevamento crescita, normalmente svolto da CFU contando su piastre di coltura, è un processo che richiede più di 3 settimane per completare. Una soluzione per sostituire una conta è stata fornita mediante microscopia a fluorescenza automatizzata in combinazione con i batteri fluorescenti. Tuttavia, questa soluzione richiede un investimento iniziale che apparecchiatura è fuori portata per molti laboratori di ricerca. Un semplice, a basso costo, e il metodo HTS malattie rilevanti sarebbero migliorare notevolmente il processo di scoperta di nuovi farmaci. In questo studio, riportiamo un nuovo sistema modulare HTS che mira a fornire un rapido e altamente scalabile, ma economica, saggio atto a determinare l&#39;attività dei composti contro M. tuberculosis intracellulare. Questo sistema è composto da tre moduli: (i) intracellulari, (ii) citotossicità, e (iii) Prove in brodo. Il risultato finale combinato fornisce una descrizione completa delle proprietà dei componenti, con ulteriori informazioni riguardanti la modalità potenziale di azione. Questo scSistema reening è stato utilizzato in diversi progetti con varie librerie di composti modalità di azione che colpiscono, compresa l&#39;analisi di sinergia farmaco 9, la stimolazione dell&#39;autofagia 10, e l&#39;inibizione di M. tuberculosis -secreted fattore di virulenza (non pubblicata). I composti di modalità sconosciuta di azione sono stati studiati 11. Una versione modificata di questo metodo è stato adottato anche dal nostro partner industriale come il metodo di screening primario di identificare nuovi composti contro intracellulare M. tuberculosis 11.

<table><tbody><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R5886</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Middlebrook 7H9</td><td>Becton, Dickinson and Company</td><td>271210</td><td></td></tr><tr><td>Tween80</td><td>Fisher Scientific</td><td>T164</td><td></td></tr><tr><td>Albumin, Bovine pH7</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>BP350</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>kanamycin sulfate</td><td>Fisher Scientific</td><td>BP906</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Fisher Scientific</td><td>D131</td><td></td></tr><tr><td>1 M Hydrocholoric acid (HCl)</td><td>Fisher Scientific</td><td>351279212</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>351269</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Resazurin</td><td>Alfa Aesar</td><td>B21187</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229</td><td></td></tr><tr><td>THP-1</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; H37Rv</td><td></td><td></td><td></td></tr><tr><td>96-well flat bottom white plate</td><td>Corning</td><td>3917</td><td></td></tr><tr><td>95-well flat bottom clear plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Transparent plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Biomate 3</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Biotek</td><td>Epoch</td><td></td></tr><tr><td>luminometer</td><td>Applied Biosystems</td><td>Tropix TR717</td><td></td></tr></tbody></table>

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.

High-Throughput Screening intracellulare con M. tuberculosis che esprime il gene della luciferasi Figura 2A e 1 contengono i dati grezzi raccolti dal luminometro, espressi in unità luminescenti relativa (RLU), che mostra l&#39;effetto di un aumento di concentrazione di TB rifampicina farmaco su M. tuberculosis all&#39;interno cellule THP-1. La figura 2A è un d...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

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

Abbiamo sviluppato un sistema di screening ad alto rendimento modulare per scoprire nuovi composti contro Mycobacterium tuberculosis, il targeting intracellulare e in brodo coltura di batteri e citotossicità contro la cellula ospite di mammifero.

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 ...

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Research

JoVE Journal

Immunology and Infection

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

8.5K Views.  University of British Columbia.  Abbiamo sviluppato un sistema di screening ad alto rendimento modulare per scoprire nuovi composti contro Mycobacterium tuberculosis, il targeting intracellulare e in brodo coltura di batteri e citotossicità contro la cellula ospite di mammifero. 

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

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) ...

Ricerca

Immunologia e infezioni

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Un test fenotipico microscopico per la quantificazione dei micobatteri intracellulari adattati per lo screening ad alta produttività / alto contenuto

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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 ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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 ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Un sistema di coltura automatizzato da utilizzare nei test preclinici di terapie dirette all'ospite per la tubercolosi

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

<meta charset="utf8"></head><body>Saggio di screening ad alto rendimento per Mycobacterium abscessus utilizzando doppi reporter

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>Autore in primo piano: Protocollo scalabile di screening farmacologico per la scoperta efficiente dei trattamenti con M. abscessus

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Sviluppo di saggi per lo screening di farmaci ad alto rendimento contro i micobatteri

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Accelerazione della determinazione della tubercolosi con il metodo Micro-CFU

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Autore in primo piano: Ottimizzazione della determinazione delle CFU per una valutazione efficiente dell'efficacia del vaccino contro la tubercolosi e dell'analisi della presentazione dell'antigene 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Sistema per l'efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare

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Preparazione del campione di

Un test fenotipico microscopico per la quantificazione dei micobatteri intracellulari adattati per lo screening ad alta produttività / alto contenuto

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

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi

Un sistema di coltura automatizzato da utilizzare nei test preclinici di terapie dirette all'ospite per la tubercolosi

Sviluppo di saggi per lo screening di farmaci ad alto rendimento contro i micobatteri

Sviluppo di saggi per lo screening di farmaci ad alto rendimento contro i micobatteri

Sviluppo di saggi per lo screening di farmaci ad alto rendimento contro i micobatteri

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi