We would like to thank the members of Orman Lab for their valuable inputs during this study. This study was funded by the NIH/NIAID K22AI125468 career transition award and a University of Houston startup grant.

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 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.&#160;</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/mL19,20. 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 a 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 cells4,5,22. The dilution/growth cycle method5 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. This method has been already validated in a previous study19.</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 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 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 cells19,20. 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 (see below).</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>
5. Validating the identified conditions
<ol>
	<li>Transfer 250 &#181;L of the exponential phase cells from Step 2.3 to 25 mL of fresh modified LB medium containing the osmolyte identified from the microarray screening (see Protocol 4).</li>
	<li>Incubate the flask in an orbital shaker at 250 rpm and 37 &#176;C for 24 h.</li>
	<li>After 24 h, remove the flask from the shaker and transfer 250 &#181;L of the cell culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask.</li>
	<li>Add 25 &#181;L of OFX stock solution (5 mg/mL) in the cell suspension and shake the flask gently to make the assay culture homogenous. Incubate the flask in a shaker at 37 &#176;C and 250 rpm.</li>
	<li>At every hour during the treatment, 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>Remove 950 &#181;L of supernatant and add 950 &#181;L of PBS.</li>
	<li>Repeat the washing steps 5.6 and 5.7 for 3x.</li>
	<li>After the final wash, resuspend the cell pellet in 100 &#181;L of PBS solution.</li>
	<li>Take 10 &#181;L of the cell suspension and serially dilute 6x in 90 &#181;L of PBS solution using a 96 well round bottom plate.</li>
	<li>Spot 10 &#181;L of the diluted cell suspensions on an antibiotic-free fresh agar plate. 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 plate at 37 &#176;C for 16 h, and then count CFUs.</li>
</ol>

<ol>
	<li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persister+cells,+dormancy+and+infectious+disease.">Persister cells, dormancy and infectious disease.</a> Nature Reviews Microbiology. 5 (1), 48-56 (2007).</li><li>Hobby, G. L., Meyer, K., Chaffee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+Mechanism+of+Action+of+Penicillin.">Observations on the Mechanism of Action of Penicillin.</a> Experimental Biology and Medicine. 50 (2), 281-285 (1942).</li><li>Bigger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+staphylococcal+infections+with+penicillin+by+intermittent+sterilisation.">Treatment of staphylococcal infections with penicillin by intermittent sterilisation.</a> The Lancet. 244 (6320), 497-500 (1944).</li><li>Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+persistence+as+a+phenotypic+switch.">Bacterial persistence as a phenotypic switch.</a> Science. 305 (5690), 1622-1625 (2004).</li><li>Keren, I., Kaldalu, N., Spoering, A., Wang, Y., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persister+cells+and+tolerance+to+antimicrobials.">Persister cells and tolerance to antimicrobials.</a> FEMS Microbiology Letters. 230 (1), 13-18 (2004).</li><li>Keren, I., Minami, S., Rubin, E., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+transcriptome+analysis+of+mycobacterium+tuberculosis+persisters.">Characterization and transcriptome analysis of mycobacterium tuberculosis persisters.</a> mBio. 2 (3), (2011).</li><li>Mulcahy, L. R., Burns, J. L., Lory, S., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+Pseudomonas+aeruginosa+Strains+Producing+High+Levels+of+Persister+Cells+in+Patients+with+Cystic+Fibrosis.">Emergence of Pseudomonas aeruginosa Strains Producing High Levels of Persister Cells in Patients with Cystic Fibrosis.</a> Journal of Bacteriology. 192 (23), 6191-6199 (2010).</li><li>LaFleur, M. D., Kumamoto, C. A., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilms+produce+antifungal-tolerant+persister+cells.">Candida albicans biofilms produce antifungal-tolerant persister cells.</a> Antimicrobial Agents and Chemotherapy. 50 (11), 3839-3846 (2006).</li><li>Allison, K. R., Brynildsen, M. P., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite-enabled+eradication+of+bacterial+persisters+by+aminoglycosides.">Metabolite-enabled eradication of bacterial persisters by aminoglycosides.</a> Nature. 473 (7346), 216-220 (2011).</li><li>Lechner, S., Lewis, K., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+persisters+tolerant+to+bactericidal+antibiotics.">Staphylococcus aureus persisters tolerant to bactericidal antibiotics.</a> Journal of Molecular Microbiology and Biotechnology. 22 (4), 235-244 (2012).</li><li>Barrett, T. C., Mok, W. W. K., Murawski, A. M., Brynildsen, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antibiotic+resistance+development+from+fluoroquinolone+persisters+after+a+single+exposure+to+antibiotic.">Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic.</a> Nature Communications. 10 (1), 1177 (2019).</li><li>Windels, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+persistence+promotes+the+evolution+of+antibiotic+resistance+by+increasing+survival+and+mutation+rates.">Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates.</a> ISME Journal. 13 (5), 1239-1251 (2019).</li><li>D&#246;rr, T., Lewis, K., Vuli&#263;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOS+response+induces+persistence+to+fluoroquinolones+in+Escherichia+coli.">SOS response induces persistence to fluoroquinolones in Escherichia coli.</a> PLoS Genetics. 5 (12), 1000760 (2009).</li><li>V&#246;lzing, K. G., Brynildsen, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stationary-phase+persisters+to+ofloxacin+sustain+DNA+damage+and+require+repair+systems+only+during+recovery.">Stationary-phase persisters to ofloxacin sustain DNA damage and require repair systems only during recovery.</a> mBio. 6 (5), (2015).</li><li>Grant, S. S., Kaufmann, B. B., Chand, N. S., Haseley, N., Hung, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+bacterial+persisters+with+antibiotic-generated+hydroxyl+radicals.">Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals.</a> Proceedings of the National Academy of Sciences. 109 (30), 12147-12152 (2012).</li><li>Gerdes, K., Maisonneuve, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Persistence+and+Toxin-Antitoxin+Loci.">Bacterial Persistence and Toxin-Antitoxin Loci.</a> Annual Review of Microbiology. 66 (1), 103-123 (2012).</li><li>Orman, M. A., Brynildsen, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+stationary+phase+respiration+impairs+persister+formation+in+E.+coli.">Inhibition of stationary phase respiration impairs persister formation in E. coli.</a> Nature Communications. 6 (1), 7983 (2015).</li><li>Korch, S. B., Henderson, T. A., Hill, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+hipA7+allele+of+Escherichia+coli+and+evidence+that+high+persistence+is+governed+by+(p)ppGpp+synthesis.">Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis.</a> Molecular Microbiology. 50 (4), 1199-1213 (2003).</li><li>Karki, P., Mohiuddin, S. G., Kavousi, P., Orman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+effects+of+osmolytes+and+environmental+ph+on+bacterial+persisters.">Investigating the effects of osmolytes and environmental ph on bacterial persisters.</a> Antimicrobial Agents and Chemotherapy. 64 (5), 02393 (2020).</li><li>Mohiuddin, S. G., Hoang, T., Saba, A., Karki, P., Orman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Metabolic+Inhibitors+to+Reduce+Bacterial+Persistence.">Identifying Metabolic Inhibitors to Reduce Bacterial Persistence.</a> Frontiers in Microbiology. 11, 472 (2020).</li><li>Brooun, A., Liu, S., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dose-response+study+of+antibiotic+resistance+in+Pseudomonas+aeruginosa+biofilms.">A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms.</a> Antimicrobial Agents and Chemotherapy. 44 (3), 640-646 (2000).</li><li>Luidalepp, H., J&#245;ers, A., Kaldalu, N., Tenson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+of+inoculum+strongly+influences+persister+frequency+and+can+mask+effects+of+mutations+implicated+in+altered+persistence.">Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence.</a> Journal of Bacteriology. 193 (14), 3598-3605 (2011).</li><li>Baba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+The+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection.</a> Molecular Systems Biology. 2, 0008 (2006).</li><li>Zaslaver, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+library+of+fluorescent+transcriptional+reporters+for+Escherichia+coli.">A comprehensive library of fluorescent transcriptional reporters for Escherichia coli.</a> Nature Methods. 3 (8), 623-628 (2006).</li><li>Hajmeer, M., Ceylan, E., Marsden, J. L., Fung, D. Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+sodium+chloride+on+Escherichia+coli+O157:H7+and+Staphylococcus+aureus+analysed+using+transmission+electron+microscopy.">Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy.</a> Food Microbiology. 23 (5), 446-452 (2006).</li></ol>

The authors have nothing to disclose.

The high throughput persister assay described here was developed to elucidate the effects of various chemicals on E. coli persistence. In addition to commercial PM plates, microarrays can be constructed manually as described in step 4.2. Moreover, the protocol presented here is flexible and can be used to screen other microarrays, such as drug panels and cell libraries, that are in 96 well plate formats. The experimental conditions including the growth phase, inoculation rate and medium can be adjusted to test t...

Bacterial cultures contain a small subpopulation of persister cells that are temporarily tolerant to unusually high levels of antibiotics. Persister cells are genetically identical to their antibiotic-sensitive kins, and their survival has been attributed to transient growth inhibition1. Persister cells were first discovered by Gladys Hobby2 but the term was first used by Joseph Bigger when he identified them in penicillin-treated Staphylococcus pyogenes cultures3. A seminal study published by Balaban et al.4 discovered two persister types: type I variants that are primarily formed by passage through the stationary phase, and type II variants that are continuously generated during the exponential growth. Persisters are detected by clonogenic survival assays, in which culture samples are taken at various intervals during antibiotic treatments, washed, and plated on a typical growth medium to count the surviving cells that can colonize in the absence of antibiotics. The existence of persisters in a cell culture is assessed by a biphasic kill curve4,5 &#160;where the initial exponential decay indicates the death of antibiotic-sensitive cells. However, the killing trend decreases over time, eventually leading to a plateau region which represents the surviving persister cells. &#160;
Persister cells have been associated with various diseases such as tuberculosis6, cystic fibrosis7, candidiasis8 and urinary tract infections9. Almost all microorganisms tested so far were found to generate persister phenotypes, including highly pathogenic Mycobacterium tuberculosis6, Staphylococcus aureus10, Pseudomonas aeruginosa7 and Candida albicans8. Recent studies also provide evidence of the rise of multidrug-resistant mutants from persister subpopulations11,12. Substantial efforts in this field have revealed that persistence mechanisms are highly complex and diverse; both stochastic and deterministic factors associated with the SOS response13,14, reactive oxygen species (ROS)15, toxin/antitoxin (TA) systems16, autophagy or self-digestion17 and ppGpp-related stringent response18 are known to facilitate persister formation.
Despite significant progress in understanding the persistence phenotype, the effects of osmolytes on bacterial persistence have not been fully understood. Since the maintenance of optimal osmotic pressure is a necessity for cells&#8217; growth, proper functioning and survival, an in-depth study of osmolytes could lead to potential targets for anti-persister strategies. Although laborious, high-throughput screening is a very effective approach for identifying metabolites and other chemicals that play a crucial role in the persistence phenotype19,20. In this work, we will discuss our published method19, where we have used microarrays, i.e., 96 well plates containing various osmolytes (e.g., sodium chloride, urea, sodium nitrite, sodium nitrate, potassium chloride), to identify osmolytes that significantly influence E. coli persistence.

<table><tbody><tr><td>14-ml test tube</td><td>Fisher Scientific</td><td>14-959-1B</td><td></td></tr><tr><td>E. coli strain MG1655</td><td>Princeton University</td><td></td><td>Obtained from Brynildsen lab</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>

high-throughput screening of chemical compounds to elucidate their effects on bacterial persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

Figure 1 describes our experimental protocol. The dilution/growth cycle experiments (see Protocol 2) were adapted from a study conducted by Keren et al.5 to eliminate the persisters originating from the overnight cultures. Figure 2A is a representative image of agar plates used to determine CFU levels of cell cultures before and after OFX treatment. In these experiments, cells were cultured in modified LB medium with osmolytes in half-are...

Watch this Scientific Journal Video about High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence at JoVE.com

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

high-throughput-screening-chemical-compounds-to-elucidate-their

Research

JoVE Journal

Immunology and Infection

4.0K Views.  University of Houston. Het hier beschreven protocol maakt het mogelijk om omstandigheden te identificeren die een aanzienlijke invloed kunnen hebben op de bacteriële persistentie op een manier met hoge doorvoer. Deze methode is gemakkelijk aan te passen aan het scherm voor verschillende gebieden, zoals medicijnpanelen, genbibliotheken en meer. Omdat de methode verschillende substappen omvat, die tegelijkertijd moeten worden uitgevoerd, kan visuele demonstratie onderzoekers helpen om hun timing te beheren om nauwke...

Video: High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Cancer Research

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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.

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

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.

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

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Biology

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

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.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

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.

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

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Samenvatting

Meer video's verkennen