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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 curve^4,5  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.  

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 subpopulations^11,12. Substantial efforts in this field have revealed that persistence mechanisms are highly complex and diverse; both stochastic and deterministic factors associated with the SOS response^13,14, 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’ 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 phenotype^19,20. 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.

Protokół

1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks

1. 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.
2. 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 °C), pour ~30 mL of agar medium into square plates (10 x 10 cm). Dry the plates and store at 4 °C.
3. Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in DI water. Sterilize the medium by autoclaving. 
4. 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.  
   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.
5. Ofloxacin stock solution (5 mg/mL): Add 5 mg of ofloxacin (OFX) salt in 1 mL of DI water. Add 10 µL 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 °C.
   NOTE: Ofloxacin is a quinolone antibiotic that has been widely used for both growing and non-growing bacterial cells^14,21. The minimum inhibitory concentration (MIC) of OFX for E. coli MG1655 cells is within the range of 0.039–0.078 μg/mL^19,20. 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.
6. E. coli MG1655 cell stocks:  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 °C. When the cells reach the stationary phase, mix 500 μL of the cell culture with 500 μL of 50% glycerol (sterile) in a cryogenic vial and store at -80 °C.

2. Propagation of cells to eliminate pre-existing persisters

1. 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 °C for 12 h.
2. First propagation
   1. After 12 h, transfer 250 µL of overnight culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask covered with a sterile aluminum foil.
   2. Grow the cell culture in an orbital shaker at 250 rpm and 37 °C until cells reach the mid-exponential phase (OD₆₀₀ = 0.5).
   3. Measure the optical density at 600 nm (OD₆₀₀) using a microplate reader every 30 min.
3. Second propagation
   1. At OD₆₀₀ = 0.5, dilute 250 µL of cell culture from the first flask into 25 mL of fresh modified LB medium in a 250 mL baffled flask.
   2. Grow the second cell culture in an orbital shaker at 250 rpm and 37 °C until OD₆₀₀=0.5.
   3. Measure the optical density every 30 min.
      NOTE: An overnight culture may have a significant amount of persister cells^4,5,22. 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.

3. Validating the elimination of pre-existing persister cells

1. After the second propagation (see step 2.3), dilute 250 µL 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 µL 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.
2. Add 25 µL 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 µg/mL in the assay culture.
3. Incubate the assay culture in an orbital shaker at 250 rpm and 37 °C.
4. 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.
5. Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.
6. Carefully remove 950 µL of the supernatant without disturbing the cell pellet.
7. Add 950 µL of phosphate-buffered saline (PBS) solution into the microcentrifuge tube.
8. Repeat the washing steps 3.5-3.7 for 3x in total until the antibiotic concentration is below the minimum inhibitory concentration (MIC).
9. After the final wash, resuspend the cell pellet in 100 µL of PBS solution, resulting in a 10x concentrated sample.
10. Take 10 µL of the cell suspension and serially dilute six times in 90 µL of PBS solution using a 96 well round bottom plate.
11. Spot 10 µL of diluted cell suspensions on antibiotic-free fresh agar plates. To increase the limit of detection, plate the remaining 90 µL of cell suspension on a fresh agar plate.
12. Incubate the agar plates at 37 °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^19,20. 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.  

4. Microarray plate screenings

1. Preparing microarray-cell cultures
   1. Transfer 250 µL 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).
   2. Transfer the diluted cell suspension into a sterile 50 mL reservoir.
   3. Using a multichannel pipette, transfer 150 µL of the cell suspension to each well of a microarray, i.e., a 96 well plate containing various osmolytes.  
      NOTE: Wells that do not have osmolytes serve as controls.
   4. Cover the microarray with a gas-permeable sealing membrane.
   5. Incubate the plate in an orbital shaker at 37 °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).
2. Manual preparation of microarray plates
   1. Transfer 75 μL of 2x osmolyte solutions into the wells of a half-area 96 well plate.
   2. Transfer 500 µL 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.  
   3. Add 75 μL of the cell suspension into each well of the half-area 96 well plate containing 2x osmolyte solutions.
   4. Incubate the plate in an orbital shaker at 37 °C and 250 rpm for 24 h.
3. Preparing persister assay plates
   1. Prepare 25 mL of modified LB medium containing 5 µg/mL of OFX in a 50 mL centrifuge tube and transfer this medium to a sterile reservoir.
   2. Transfer 190 µL 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.
   3. Remove the microarray from the shaker (after culturing for 24 h) and transfer 10 µL of cell cultures from the microarray to the wells of the persister-assay plate, containing modified LB medium with OFX.
   4. Take 10 µL of cell suspensions from the persister-assay plate and serially dilute three times in 290 µL of PBS solution using a round-bottom 96 well plate and a multichannel pipette.
   5. Following the serial dilution, spot 10 µL of all serially diluted cell suspensions on antibiotic-free fresh agar plates using a multichannel pipette.  
   6. Incubate the persister-assay plate (prepared in step 4.3.3) in an orbital shaker at 37 °C and 250 rpm for 6 h after covering the plate with a gas-permeable sealing membrane.
   7. After 6 h incubation in a shaker, take the persister-assay plate out and repeat the steps 4.3.4-4.3.5.
   8. Incubate the agar plates for 16 h at 37 °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.

5. Validating the identified conditions

1. Transfer 250 µL 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).
2. Incubate the flask in an orbital shaker at 250 rpm and 37 °C for 24 h.
3. After 24 h, remove the flask from the shaker and transfer 250 µL of the cell culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask.
4. Add 25 µL 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 °C and 250 rpm.
5. At every hour during the treatment, transfer 1 mL of the assay culture from the flask to a 1.5 mL microcentrifuge tube.
6. Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.
7. Remove 950 µL of supernatant and add 950 µL of PBS.
8. Repeat the washing steps 5.6 and 5.7 for 3x.
9. After the final wash, resuspend the cell pellet in 100 µL of PBS solution.
10. Take 10 µL of the cell suspension and serially dilute 6x in 90 µL of PBS solution using a 96 well round bottom plate.
11. Spot 10 µL of the diluted cell suspensions on an antibiotic-free fresh agar plate. To increase the limit of detection, plate the remaining 90 µL of cell suspension on a fresh agar plate.
12. Incubate the agar plate at 37 °C for 16 h, and then count CFUs.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
14-ml test tube	Fisher Scientific	14-959-1B
E. coli strain MG1655	Princeton University		Obtained from Brynildsen lab
Flat-bottom 96-well plate	USA Scientific	5665-5161
Gas permeable sealing membrane	VWR	102097-058	Sterilized by gamma irradiation and free of cytotoxins
Half-area flat-bottom 96-well plate	VWR	82050-062
LB agar	Fisher Scientific	BP1425-2	Molecular genetics grade
Ofloxacin salt	VWR	103466-232	HPLC ≥97.5
Phenotype microarray (PM-9 and PM-10)	Biolog	N/A	PM-9 and PM-10 plates contained various osmolytes and buffers respectively
Round-bottom 96-well plate	USA Scientific	5665-0161
Sodium chloride	Fisher Scientific	S271-500	Certified ACS grade
Sodium nitrate	Fisher Scientific	AC424345000	ACS reagent grade
Sodium nitrite	Fisher Scientific	AAA186680B	98% purity
Square petri dish	Fisher Scientific	FB0875711A
Tryptone	Fisher Scientific	BP1421-500	Molecular genetics grade
Varioskan lux multi mode microplate reader	Thermo Fisher Scientific	VLBL00D0	Used for optical density measurement at 600 nm
Yeast extract	Fisher Scientific	BP1422-100	Molecular genetics grade