Name: 视频: 体外持久性测定:一种评估渗透压对细菌持久性影响的方法 - 概念
Uploaded: 2023-04-30T15:03:32.000+00:00
Duration: 2 min 2 s
Description: 129 次观看. 来源:Karki， P. et al. 化合物的高通量筛选以阐明它们对细菌持久性的影响。J. Vis. Exp. （2021 年） 在本视频中，我们演示了一种评估渗透压对细菌持久性形成影响的方法。细菌细胞首先暴露于渗透压下以诱导渗透应激，触发持久性形成，然后用抗生素处理以列举持久性。

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1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks
<ol>
	<li>Regular Luria-Bertani (LB) medium: Add 10 g/L of tryptone, 10 g/L of sodium chloride (NaCl), and 5 g/L of yeast extract in deionized (DI) water. Sterilize the medium by autoclaving.</li>
	<li>LB agar plates: Add 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract, and 15 g/L agar in DI water and sterilize the medium by autoclaving. At the desired temperature (~55 &#176;C), pour ~30 mL of agar medium into square plates (10 x 10 cm). Dry the plates and store them at 4 &#176;C.</li>
	<li>Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in DI water. Sterilize the medium by autoclaving.</li>
	<li>Modified LB medium including an osmolyte: Mix 2x modified LB medium with a 2x osmolyte solution in equal volumes. To prepare 2x modified LB medium, add 20 g/L of tryptone and 10 g/L of yeast extract in DI water and sterilize by autoclaving. To prepare the 2x osmolyte solution, dissolve the osmolyte of interest (2x amount) in DI water, and then filter sterilize the solution.&#160; 
	NOTE: The osmolyte of interest and its concentrations can be determined from the Phenotype Microarray screening assays (see Protocol 4). However, the tested osmolyte and the associated 2x concentration can be adjusted depending on the nature of the research. For example, to study the impact of 1 mM sodium chloride, a 2x osmolyte solution would be a 2 mM sodium chloride solution.</li>
	<li>Ofloxacin stock solution (5 mg/mL): Add 5 mg of ofloxacin (OFX) salt in 1 mL of DI water. Add 10 &#181;L of 10 M sodium hydroxide to increase the solubility of OFX in water, and then filter sterilize the solution. Prepare aliquots and store at -20 &#176;C. 
	NOTE: Ofloxacin is a quinolone antibiotic that has been widely used for both growing and non-growing bacterial cells14,21. The minimum inhibitory concentration (MIC) of OFX for E. coli MG1655 cells is within the range of 0.039&#8211;0.078 &#956;g/mL. Also, note that other antibiotics such as ampicillin and kanamycin are commonly used in persister research. The choice of antibiotics is dependent on the nature of the study.</li>
	<li>E. coli MG1655 cell stocks:&#160; Inoculate 2 mL of regular LB medium with a single colony in a 14 mL test tube (snap-capped) and culture the cells in an orbital shaker at 250 rpm and 37 &#176;C. When the cells reach the stationary phase, mix 500 &#956;L of the cell culture with 500 &#956;L of 50% glycerol (sterile) in a cryogenic vial and store at -80 &#176;C.</li>
</ol>
2. Propagation of cells to eliminate pre-existing persisters
<ol>
	<li>To prepare an overnight culture, scrape a small amount of cells from a frozen cell stock with a sterile pipette tip (do not thaw the glycerol cell stock), and inoculate the cells in 2 mL of modified LB medium in a 14 mL snap-capped test tube. Culture the cells in an orbital shaker at 250 rpm and 37 &#176;C for 12 h.</li>
	<li>First propagation
	<ol>
		<li>After 12 h, transfer 250 &#181;L of overnight culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask covered with sterile aluminum foil.</li>
		<li>Grow the cell culture in an orbital shaker at 250 rpm and 37 &#176;C until cells reach the mid-exponential phase (OD600 = 0.5).</li>
		<li>Measure the optical density at 600 nm (OD600) using a microplate reader every 30 min.</li>
	</ol>
	</li>
	<li>Second propagation
	<ol>
		<li>At OD600 = 0.5, dilute 250 &#181;L of cell culture from the first flask into 25 mL of fresh modified LB medium in a 250 mL baffled flask.</li>
		<li>Grow the second cell culture in an orbital shaker at 250 rpm and 37 &#176;C until OD600=0.5.</li>
		<li>Measure the optical density every 30 min. 
		NOTE: An overnight culture may have a significant amount of persister cells. The dilution/growth cycle method described above can be used to eliminate these pre-existing persisters before transferring the cells to microarrays. Their elimination can be validated by quantifying persister levels of overnight cultures with the assay described in Protocol 3.</li>
	</ol>
	</li>
</ol>
3. Validating the elimination of pre-existing persister cells
<ol>
	<li>After the second propagation (see step 2.3), dilute 250 &#181;L of the cell culture (OD600 = 0.5) in 25 mL of fresh modified LB medium in a 250 mL baffled flask. 
	NOTE: For controls, dilute 250 &#181;L of overnight culture (see step 2.1) in 25 mL of fresh modified LB medium in a 250 mL baffled flask. Before transferring the cells to the flask, the cell density of the overnight culture should be adjusted in a fresh modified LB medium to obtain OD600 = 0.5.</li>
	<li>Add 25 &#181;L of OFX stock solution (5 mg/mL) into the cell suspension and shake the flask gently to make the assay culture homogeneous. The final concentration of OFX is 5 &#181;g/mL in the assay culture.</li>
	<li>Incubate the assay culture in an orbital shaker at 250 rpm and 37 &#176;C.</li>
	<li>At every hour during the treatment (including 0 h, the time point before adding OFX into the assay culture), transfer 1 mL of the assay culture from the flask to a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.</li>
	<li>Carefully remove 950 &#181;L of the supernatant without disturbing the cell pellet.</li>
	<li>Add 950 &#181;L of phosphate-buffered saline (PBS) solution into the microcentrifuge tube.</li>
	<li>Repeat the washing steps 3.5-3.7 for 3x in total until the antibiotic concentration is below the minimum inhibitory concentration (MIC).</li>
	<li>After the final wash, resuspend the cell pellet in 100 &#181;L of PBS solution, resulting in a 10x concentrated sample.</li>
	<li>Take 10 &#181;L of the cell suspension and serially dilute it six times in 90 &#181;L of PBS solution using a 96-well round bottom plate.</li>
	<li>Spot 10 &#181;L of diluted cell suspensions on antibiotic-free fresh agar plates. To increase the limit of detection, plate the remaining 90 &#181;L of cell suspension on a fresh agar plate.</li>
	<li>Incubate the agar plates at 37 &#176;C for 16 h, and then count the colony-forming units (CFUs). Account for the dilution rates while calculating the total number of CFUs in 1 mL of assay culture. Kill curves are generated by plotting the logarithmic CFU values with respect to the duration of antibiotic treatment. 
	NOTE: A 6 h OFX treatment is sufficient to obtain a biphasic kill curve for E. coli cells. The persister level of propagated cells (as measured by CFU counts at 6 h) should be significantly less than that of the overnight culture. The procedures described in Protocol 2 and 3 can be performed with regular LB depending on the research design.&#160;</li>
</ol>
4. Microarray plate screenings
<ol>
	<li>Preparing microarray-cell cultures
	<ol>
		<li>Transfer 250 &#181;L of exponential-phase cells to 25 mL of fresh modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous. 
		NOTE: The exponential-phase cells (OD600 = 0.5) are obtained from the second propagation step (see step 2.3).</li>
		<li>Transfer the diluted cell suspension into a sterile 50 mL reservoir.</li>
		<li>Using a multichannel pipette, transfer 150 &#181;L of the cell suspension to each well of a microarray, i.e., a 96-well plate containing various osmolytes.&#160; 
		NOTE: Wells that do not have osmolytes serve as controls.</li>
		<li>Cover the microarray with a gas-permeable sealing membrane.</li>
		<li>Incubate the plate in an orbital shaker at 37 &#176;C and 250 rpm for 24 h. 
		NOTE: In these experiments, commercially available plates were used, such as Phenotype Microarrays (PM-9 and PM-10) that include a wide range of osmolytes, pH buffers, and other chemicals in a dried state at various concentrations. These microarrays are in half-area 96 well plate formats. Culture volumes should be adjusted depending on the type of plates being used. Microarrays can also be generated manually.</li>
	</ol>
	</li>
	<li>Manual preparation of microarray plates
	<ol>
		<li>Transfer 75 &#956;L of 2x osmolyte solutions into the wells of a half-area 96 well plate.</li>
		<li>Transfer 500 &#181;L of exponential-phase cells to 25 mL of 2x modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous. 
		NOTE: The inoculation rate was adjusted to be consistent with the microarray screening protocol described in 4.1.&#160;</li>
		<li>Add 75 &#956;L of the cell suspension into each well of the half-area 96-well plate containing 2x osmolyte solutions.</li>
		<li>Incubate the plate in an orbital shaker at 37 &#176;C and 250 rpm for 24 h.</li>
	</ol>
	</li>
	<li>Preparing persister assay plates
	<ol>
		<li>Prepare 25 mL of modified LB medium containing 5 &#181;g/mL of OFX in a 50 mL centrifuge tube and transfer this medium to a sterile reservoir.</li>
		<li>Transfer 190 &#181;L of modified LB medium with OFX from the reservoir into each well of a generic flat-bottom 96 well plate (persister-assay plate) using a multichannel pipette.</li>
		<li>Remove the microarray from the shaker (after culturing for 24 h) and transfer 10 &#181;L of cell cultures from the microarray to the wells of the persister-assay plate, containing modified LB medium with OFX.</li>
		<li>Take 10 &#181;L of cell suspensions from the persister-assay plate and serially dilute three times in 290 &#181;L of PBS solution using a round-bottom 96 well plate and a multichannel pipette.</li>
		<li>Following the serial dilution, spot 10 &#181;L of all serially diluted cell suspensions on antibiotic-free fresh agar plates using a multichannel pipette.&#160;</li>
		<li>Incubate the persister-assay plate (prepared in step 4.3.3) in an orbital shaker at 37 &#176;C and 250 rpm for 6 h after covering the plate with a gas-permeable sealing membrane.</li>
		<li>After 6 h incubation in a shaker, take the persister-assay plate out and repeat the steps 4.3.4-4.3.5.</li>
		<li>Incubate the agar plates for 16 h at 37 &#176;C, and then count CFUs. The CFU levels before and 6 h after the antibiotic treatment enable to calculate the persister fraction in each well. The CFU counts before the OFX treatment also help assess the effects of osmolytes and on E. coli viability.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>14-ml test tube</td><td>Fisher Scientific</td><td>14-959-1B</td><td></td></tr><tr><td>Flat-bottom 96-well plate</td><td>USA Scientific</td><td>5665-5161</td><td></td></tr><tr><td>Gas permeable sealing membrane</td><td>VWR</td><td>102097-058</td><td>Sterilized by gamma irradiation and free of cytotoxins</td></tr><tr><td>Half-area flat-bottom 96-well plate</td><td>VWR</td><td>82050-062</td><td></td></tr><tr><td>LB agar</td><td>Fisher Scientific</td><td>BP1425-2</td><td>Molecular genetics grade</td></tr><tr><td>Ofloxacin salt</td><td>VWR</td><td>103466-232</td><td>HPLC &amp;ge;97.5</td></tr><tr><td>Phenotype microarray (PM-9 and PM-10)</td><td>Biolog</td><td>N/A</td><td>PM-9 and PM-10 plates contained various osmolytes and buffers respectively</td></tr><tr><td>Round-bottom 96-well plate</td><td>USA Scientific</td><td>5665-0161</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-500</td><td>Certified ACS grade</td></tr><tr><td>Sodium nitrate</td><td>Fisher Scientific</td><td>AC424345000</td><td>ACS reagent grade</td></tr><tr><td>Sodium nitrite</td><td>Fisher Scientific</td><td>AAA186680B</td><td>98% purity</td></tr><tr><td>Square petri dish</td><td>Fisher Scientific</td><td>FB0875711A</td><td></td></tr><tr><td>Tryptone</td><td>Fisher Scientific</td><td>BP1421-500</td><td>Molecular genetics grade</td></tr><tr><td>Varioskan lux multi mode microplate reader</td><td>Thermo Fisher Scientific</td><td>VLBL00D0</td><td>Used for optical density measurement at 600 nm</td></tr><tr><td>Yeast extract</td><td>Fisher Scientific</td><td>BP1422-100</td><td>Molecular genetics grade</td></tr></tbody></table>

in vitro persister assay: a method to evaluate the effect of osmolytes on bacterial persistence

Source: Karki, P. et al. <a href="https://app.jove.com/v/61597">High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a method to evaluate the effect of osmolytes on the formation of bacterial persisters. The bacterial cells are first exposed to osmolytes to induce osmotic stress, triggering persister formation, then treated with antibiotics to enumerate the persisters.

In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

Source: Karki, P. et al. High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence. J. Vis. Exp. (2021)
In this ...

On exposure to harmful environment conditions, a subset of bacteria enter a state of low metabolic activity, evading the detrimental effects. These bacteria, called persisters, can tolerate transient exposure to high concentrations of antibiotics.
To study the effect of osmolytes - small organic solutes - on bacterial persistence, first, obtain an exponential phase bacterial cell culture with pre-existing persisters below the limit of detection, suspended in media.
Next, transfer the suspension into osmolyte-containing wells of a microarray plate. Keep a set of wells without osmolyte as control. Seal the plate with a gas-permeable membrane to allow rapid gas diffusion for cell growth and incubate.
The osmolytes in the medium at appropriate concentrations induce osmotic stress, triggering some cells to reprogram their cellular metabolism from rapid to slow growth, forming persisters.
Now, transfer the osmolyte-treated and control cells to an antibiotic-containing persister assay plate. Seal the persister assay plate and incubate.
In the presence of antibiotics, normal cells undergo cell death, whereas persisters - being antibiotic-tolerant - survive. Last, collect a small fraction of the cells, serially dilute with buffer, and spot onto an antibiotic-free agar plate. Incubate the plate.
In the absence of antibiotics, the persisters resume growth and proliferate, forming colonies. Count the number of colonies on the agar plate.
A higher number of colonies than no-osmolyte control post-antibiotic treatment suggests a positive effect of osmolytes on persister formation.

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129 次观看. 来源:Karki， P. et al. 化合物的高通量筛选以阐明它们对细菌持久性的影响。J. Vis. Exp. （2021 年） 在本视频中，我们演示了一种评估渗透压对细菌持久性形成影响的方法。细菌细胞首先暴露于渗透压下以诱导渗透应激，触发持久性形成，然后用抗生素处理以列举持久性。 

视频: 体外持久性测定:一种评估渗透压对细菌持久性影响的方法 - 概念

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.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

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

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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.

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系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

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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.

In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

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In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence