Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

1. Preparation of Epithelial and Bacterial Cells

1. Thawing of CCL-23 epithelial cells
   1. Prewarm Minimum Essential Media (MEM) containing 10% Fetal Bovine Serum (FBS) in a 37 °C water bath for ~30 min.
   2. Sterilize the tissue culture-approved biosafety cabinet with UV light for at least 10 min before use, and wipe down the work area with 70% ethanol. Wipe the warmed media bottle with 70% ethanol and place in biosafety cabinet.
   3. Using a 25 ml pipette, transfer 14 ml of media into a T-75 flask. Place flask in 37 °C incubator whilst cells are thawing (section 1.1.4).
   4. Remove a vial of CCL-23 cells from liquid nitrogen storage using appropriate safety precautions and warm in a 37 °C water bath until ~80% thawed.
   5. Return the flask prepared in section 1.1.3 to the biosafety cabinet. Wipe outer surface of vial of CCL-23 cells with 70% ethanol and transfer contents (1 ml) into the T-75 flask using a P1000 pipette.
   6. Incubate the flask on its side in a 37 °C, 5% CO₂, 95% relative humidity tissue culture incubator overnight (16-20 hr).
   7. Check the cells under a microscope to confirm a healthy morphology, remove media, and replace with 15 ml fresh, prewarmed MEM + 10% FBS.
2. Maintenance of CCL-23 epithelial cells
   1. Once cells are ~80% confluent (1-2 days), passage by removing media using a 25 ml pipette and gently wash the cells twice with 8-10 ml prewarmed phosphate buffered saline (PBS) using a 10 ml pipette.
   2. Remove PBS and add 1 ml of sterile 0.25% trypsin-EDTA (0.25% (w/v) trypsin, 0.1 mM EDTA). Turn flask on its side, rotate to ensure trypsin covers the confluent layer of cells, and incubate the flask for 2-3 min in the 37 °C, 5% CO₂ incubator.
   3. Return flask to the biosafety cabinet, add 9 ml prewarmed MEM + 10% FBS, and pipette up and down 6-8x to resuspend the cells, ejecting liquid down the side of the flask to dislodge cells.
   4. Split cells 1:15 (1 ml + 14 ml MEM + 10% FBS) or 1:30 (0.5 ml + 14.5 ml MEM + 10% FBS), discard excess cells, and return flask to the 37 °C, 5% CO₂ incubator.
   5. Passage the cells at least twice before use in the adherence assay. Typically cells are split every 3-4 days.
3. Seeding of CCL-23 epithelial cells (one day prior to adherence assay, Protocol 2)
   1. Split confluent cells as described in sections 1.2.1-1.2.3, resuspending in prewarmed MEM + 5% FBS (FBS concentration is lowered to reduce the likelihood of serum components interfering in the assay). Remove 20 μl of cells and count using trypan blue staining and a hemocytometer.
   2. Dilute cells in prewarmed MEM + 5% FBS to a concentration of 1.5 x 10⁵ cells/ml in a sterile 50 ml tube.
   3. Seed 1 ml of cells/well in a tissue-culture treated 24-well plate.
4. Preparation of bacteria
   This procedure can be followed for preparation of pneumococci and S. salivarius. Bacterial growth media is typically kept in a 37 °C incubator overnight to prewarm and test for sterility (generally will appear cloudy if contaminated).
   1. Two days prior to adherence assay, streak bacteria on horse blood agar (HBA) plates and incubate at 35-37 °C and 5% CO₂ for 18-24 hr. Sheep blood agar can be used as an alternative.
   2. The following day, prepare an overnight culture for each bacterial species by pipetting 10 ml Todd-Hewitt Broth with 0.5% yeast extract (THB+YE) medium into a sterile 30 ml tube. Using a 10 μl loop, inoculate approximately 10-12 well separated colonies. Briefly vortex and incubate at 35-37 °C and 5% CO₂ for 14-16 hr. Note that pneumococci will autolyze if left too long in broth culture.

2. Adherence Assay: Addition of Bacteria to Epithelial Cell Monolayers

1. Preparation of mid-log bacterial cultures of pneumococci and S. salivarius
   1. Pipette 10 ml of prewarmed THB+YE into each of two sterile 30 ml tubes, one labeled pneumococci and the other S. salivarius.
   2. To reach mid-log phase, pneumococci require ~3 hr and S. salivarius require ~2 hr incubation (see Figure 1). Start both cultures at the same time. Vortex (3-5 sec) overnight bacterial cultures and add 100 μl pneumococci and 100 μl S. salivarius into corresponding tubes.
   3. Briefly vortex and incubate the cultures at 37 °C for approximately 2 hr for S. salivarius and 3 hr for pneumococci.
2. Preparation of CCL-23 cells for adherence assay
   1. Using an inverted microscope, check the 24-well plate for confluent monolayers with healthy cell morphology. Monolayers should be >80% confluent, without evidence of cell clumping or cytopathic effects such as cell rounding or loss of adherence (see Figure 2).
   2. Remove the media in each well using a transfer pipette, tilting the plate slightly to avoid disturbing the cell monolayer.
   3. To gently wash the cells, add ~500 μl prewarmed PBS into each well using a serological pipette and remove it with a transfer pipette, tilting the plate slightly. Discard the PBS. Repeat wash step once.
   4. Add 500 μl of prewarmed MEM + 5% FBS into each well. Return the 24-well plate to the CO₂ incubator while preparing the bacterial cultures.
      Note: To determine the exact CCL-23 cell number for multiplicity of infection (MOI), two wells can be trypsinized and counted on the day of infection.

3. Adherence Assay

1. Add pneumococci to the CCL-23 cells at a MOI ~10:1. Test the conditions listed in Table 1 in duplicate wells.
   1. Measure the optical density at A₆₀₀ (OD) of 1 ml of S. salivarius culture. OD should be between 0.3-0.6.
   2. Transfer 5 ml of S. salivarius culture into a 10 ml tube and centrifuge at 1,870 x g for 4 min.
   3. Discard the supernatant and based on the OD reading in section 3.1.1, resuspend S. salivarius in 200-1,000 μl of 0.85% NaCl (final concentration ~1.5 x 10⁹ CFU/ml). As a general guideline, an OD of 0.375 will be resuspended in 260 μl. Prepare 1:10 and 1:100 dilutions of S. salivarius using 0.85% NaCl.
      Note: In total, three concentrations (representing high, medium and low doses) of S. salivarius will be tested in the assay. The high dose is a ratio of ~10 S. salivarius : 1 pneumococci, the medium is ~1 S. salivarius : 1 pneumococci, and the low is ~1 S. salivarius : 10 pneumococci.
   4. Take out the 24-well plate from the incubator.
   5. From the heparin control wells, remove 40 μl of media and add 50 μl of heparin (1,000 U/ml stock concentration) for a final concentration of 100 U/ml.
   6. Add 10 μl of 0.85% NaCl to wells containing CCL-23 cells only and wells containing CCL-23 cells and pneumococci (no S. salivarius).
   7. Add 10 ul of undiluted, 1:10 and 1:100 of S. salivarius to respective wells.
      Note: S. salivarius can also be added at the same time as pneumococci (coaddition) or 1 hr after pneumococci (post-addition) to examine the effect of time of probiotic administration.
   8. Centrifuge the 24-well plate at 114 x g for 3 min to promote bacterial contact with the cell layer and incubate for 1 hr at 37° C, 5% CO₂ .
   9. Perform serial dilution of the S. salivarius stock in 0.85% NaCl and plate on duplicate HBA plates for viable counts. First, add 50 μl of stock to 450 μl of 0.85% NaCl to make a 10^-1 dilution, then pulse vortex (5 sec), change tips, and add 50 μl of the 10^-1 dilution to 450 μl of 0.85% NaCl to make a 10^-2 dilution and so forth up to 10^-7.
   10. Plate 100 μl of the 10^-5, 10^-6, and 10^-7 dilutions in duplicate on HBA plates and spread with a sterile spreader. Incubate HBA plates inverted overnight at 37 °C, 5% CO₂.
   11. Towards the end of the 1 hr incubation, repeat sections 3.1.1-3.1.3 for pneumococci. ODs should be between 0.2-0.7. Spin down 1 ml of pneumococcal culture and resuspend the pellet in 300-2,000 μl of 0.85% NaCl (based on the OD reading) to a concentration of ~1.5 x 10⁸ CFU/ml. As a general guideline, an OD of 0.3 will be resuspended in 500 μl.
   12. After 1 hr incubation with S. salivarius, add 10 μl undiluted pneumococci to all wells except negative control wells (containing cells only).
   13. Centrifuge the 24-well plate at 114 x g for 3 min and incubate at 37 °C, 5% CO₂ for 3 hr. Perform serial dilution of the pneumococci stock in 0.85% NaCl as described in section 3.1.9 and plate dilutions 10^-4, 10^-5, and 10^-6 on duplicate HBA plates for viable counts.
2. After 3 hr, check the cells under a microscope to ensure that monolayers look healthy (see Figure 2). Remove the media from each well and gently wash the cells 3x as described in section 2.2.3. using a different pipette for each condition. This step is essential to remove nonadherent pneumococci. The monolayers should not come apart during the washing process; this can be confirmed by checking again under a microscope.

Note: Media removed during this step can be stored at -20 °C for additional analyses (e.g. measurement of cytokine levels).

1. Add 200 μl of 0.1% digitonin (a gentle detergent) to each well and incubate for 7 min at 37 °C, 5% CO₂.
2. Add 800 μl of THB media to each well. Lyse cells by pipetting up and down, and, if necessary, scrape wells 3-4x with the pipette tip to make sure the cell monolayer is completely removed. Change tips between wells. 
3. Remove the contents from each well using a P1000 pipette and transfer into labeled microfuge tubes.

4. Quantification of Adherent Pneumococci

Adherent pneumococci can be quantified by determining viable counts (serial dilution and plating on blood agar) or by quantitative real-time PCR.

1. Viable count method
   This method must be performed immediately after step 3.5 of the adherence assay.
   1. For each assay well, prepare serial dilutions of samples collected in step 3.5 of the adherence assay by adding 50 μl of samples to 450 μl of 0.85% NaCl, pulse vortexing, and serially diluting as described in section 3.1.9 up to the 10^-6 dilution.
   2. Plate 100 μl of the 10^-3, 10^-4, 10^-5, and 10^-6 dilutions in duplicate on blood agar, spread with a sterile spreader, and incubate inverted plates overnight at 37 °C, 5% CO_2. Note that the wells containing CCL-23 cells alone should not contain any bacteria and can be plated neat (100 μl undiluted) as a sterility control.
   3. The following day, determine the pneumococcal inocula by counting the colonies on the appropriate dilution plates (containing 30-300 colonies) prepared in section 3.1.13 of the adherence assay. Calculate the CFU/ml of the inocula using the averaged value from two plates. Similarly, determine the S. salivarius inocula by counting the colonies on the plates prepared in section 3.1.10.
      Note: Plates should contain pneumococci only; the presence of other species indicates that the assay was contaminated and results are invalid.
   4. To determine the pneumococcal adherence levels, count the appropriate dilution plate (containing 30-300 pneumococcal colonies) for each sample prepared in section 4.1.1. Note that S. salivarius colonies will be small and white, whereas pneumococci will be flat and greenish due to α-hemolysis, as shown in Figure 3. To calculate pneumococcal adherence, normalize the number of adherent pneumococci from condition b. CCL-23 cells + pneumococci to 100%, and compare adherent pneumococci from other conditions to this condition.
2. qPCR method
   Samples collected in Adherence Assay step 3.5 can be stored at -20 °C until DNA extraction. DNA extraction is performed using a commercial kit and described in sections 4.2.1-4.2.9 and the qPCR assay in sections 4.2.10-4.2.20.
   1. Prepare Enzymatic Lysis Buffer (50 mM phosphate buffer pH 6.7 containing 1 mg/ml lysozyme, 0.075 mg/ml mutanolysin, and 2 mg/ml proteinase K). The following recipe can be used make 10 ml (enough for 50 extractions). Enzymatic lysis buffer should be freshly prepared prior to use.
      
      7.25 ml of 50 mM Phosphate Buffer solution (pH 6.7)
      1.00 ml of 10 mg/ml lysozyme stock for a final [1 mg/ml]
      0.75 ml of 1 mg/ml mutanolysin stock for a final [0.075 mg/ml]
      1.00 ml of 20 mg/ml Proteinase K stock for a final [2 mg/ml]
       
   2. Thaw the samples on ice or at 4 °C. Vortex and transfer 100 μl aliquots of each sample into labeled microfuge tubes. Return original samples back into the -20°C freezer.
   3. Centrifuge the samples at 6,700 x g for 10 min.
   4. Remove and discard the supernatant. Add 200 μl of enzymatic lysis buffer to the pellet and vortex to resuspend. Incubate the samples at 56 °C for 30 min. Centrifuge briefly (5 sec) at 6,700 x g to collect liquid at the bottom of tube.
   5. Add 10 μl of 20% SDS to complete the cell lysis and mix gently at room temperature for 2 min.
   6. Add 4 μl of RNase A (100 mg/ml stock) and mix gently (on a rocker or by inverting the tubes) at room temperature for 2 min.
   7. Add 200 μl of buffer AL (from kit) and vortex for 15 sec. Incubate samples at 70 °C for 10 min. Centrifuge briefly (5 sec) at 6,700 x g to collect liquid at the bottom of tube.
   8. Add 200 μl of 100% EtOH and vortex for 15 sec. Spin briefly to collect the liquid at bottom of tube, then transfer the mixture (including any precipitate) to the spin column (in a 2 ml collection tube) without wetting the rim. Follow wash steps according of the manufacturer’s protocol (not shown here).
   9. To elute DNA, transfer column into a labeled microfuge tube, add 50 μl of Buffer AE (from kit), and incubate at room temperature for 2 min. Centrifuge at 4,300 x g for 1 min. Repeat once for a final elution volume of 100 μl. DNA can be stored at -20 °C until use.
   10. To perform qPCR, sterilize the hoods with UV light for 15 min prior to start. Preparing a 96-well plate map worksheet to record well locations for each sample is recommended.
   11. Prepare standard curve using a 10 ng/μl stock of genomic pneumococcal DNA (extracted from reference strain ATCC 6305).
       1. Thaw pneumococcal stock DNA and spin down briefly (5 sec) in a tabletop centrifuge.
       2. Perform 1:10 serial dilutions in 6 prelabeled microfuge tubes by adding 3 μl DNA to 27 μl nuclease-free H₂O, changing tips and vortexing between each dilution. The final dilution series consists of 6 tubes: 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001 ng/μl.
          Note: These can be stored at 4 °C for up to one week.
   12. Prepare lytA mastermix as follows in a sterile 15 ml conical tube in a sterile PCR hood. Thaw components on ice and vortex immediately prior to use.

       component	per reaction	x 102 (full plate)
       lytA F	0.25 μl of 10 μM stock	25.5 μl
       lytA R	0.25 μl of 10 μM stock	25.5 μl
       lytA probe	0.5 μl of 10 μM stock	51 μl
       H2O	9.5 μl	969 μl
       Master mix	12.5 μl	1,275 μl
       Final volume	23 μl	2,346 μl

       Note: Some real-time PCR machines require the use of a reference dye, which is contained in some commercial mastermixes or can be added separately. This protocol is designed for running 25 μl reactions in a 96-well plate but can be adjusted to accommodate different real-time PCR machines. The primers and probe sequences were originally published by Carvalho et al.¹⁵ and are shown in the list of materials.
   13. Vortex and load 23 μl/well of the lytA mix into the appropriate wells. Refer to plate map.
   14. Transport plate to template addition area (preferably a sterile cabinet in a separate room) for sample loading.
   15. Use a P2 pipette to load samples, changing tips for each well. All samples are run in duplicate. Refer to plate map for location.
   16. For standard curve wells, add 1 μl/well of the appropriate dilution of standard curve DNA plus 1 μl/well H₂O to bring the final volume to 25 μl. For no template control wells, add 2 μl H₂O.
   17. Add 2 μl/well of sample DNA into appropriate wells.
   18. Cap all wells in use. Make sure caps are closed snugly. Quickly spin the PCR plate (5 sec) before loading the plate into a real-time PCR machine.
   19. Load plate into PCR machine and run qPCR according to the instructions provided by the machine manufacturer. The following hydrolysis probe cycling conditions are recommended:

       Step 1 (1x):	3 min at 95 °C
       Step 2 (40x):	20 sec at 95 °C
       	20 sec at 60 °C

   20. Collect data and quantify pneumococcal DNA using the standard curve. Bacterial loads are calculated as previously described¹⁷, estimating that 1 pg of genomic DNA is equivalent to 447.4 cells, and reported as CFU/ml (assuming one genome per CFU, and one copy of lytA per genome; see Figure 4). For a successful assay, the R² value should be ≥0.98 and all positive results within the range of the standard curve (cT values less than that obtained for the 0.00001 ng/μl standard curve point are considered background/lytA negative).
       Note: Some real-time PCR systems come with analysis software that can be used to construct standard curves and calculate unknowns. Otherwise, these calculations can be performed in Excel or similar programs. An example standard curve is shown in Figure 4.
   21. To calculate pneumococcal adherence, normalize the number of adherent pneumococci from condition b. CCL-23 cells + pneumococci to 100%, and compare adherent pneumococci from other conditions to this condition.