Materials and Reagents

Sterile specimen containers (Fisher Scientific, catalog number: 16-320-730 )
12-well tissue culture plates (Corning, Falcon^®, catalog number: 351143 )
Sterile culture tubes (4 ml) (Fisher Scientific, catalog number: 14-956-3D )
Microcentrifuge tubes (1.7 ml) (Fisher Scientific, catalog number: S348903 )
Glass scintillation vials (20 ml) (Sigma-Aldrich, catalog number: Z253081 )
Sterile culture tubes (15 ml) (Fisher Scientific, catalog number: 14-956-6D )
Desired bacterial strain(s) (e.g., Streptococcus mutans OMZ175)
Extirpated heart tissues
EGM-MV Bullet Kit (Lonza, catalog number: CC-3125 )
Gentamicin (Sigma-Aldrich, catalog number: G1397 )
Brain heart infusion medium (BHI) (BD, Bacto^TM, catalog number: 237500 )
Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14025092 )
Erythromycin (Sigma-Aldrich, catalog number: E5389 )
Kanamycin (Sigma-Aldrich, catalog number: K1377 )
Sodium chloride (NaCl) (Avantor^® Performance Materials, J.T. Baker^®, catalog number: 3628-01 )
Potassium chloride (KCl) (Avantor^® Performance Materials, J.T. Baker^®, catalog number: 3045-01 )
Sodium phosphate dibasic (Na₂HPO₄) (Avantor^® Performance Materials, J.T. Baker^®, catalog number: 3827-01 )
Potassium dihydrogen phosphate (KH₂PO₄) (Avantor^® Performance Materials, J.T. Baker^®, catalog number: 3246-01 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Glutaraldehyde (Sigma-Aldrich, catalog number: 340855 )
Sodium cacodylate trihydrate (Sigma-Aldrich, catalog number: C0250 )
Agar (Fisher Scientific, catalog number: BP1423 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0392 )
Hydrocortisone
Bovine brain extract
Human recombinant epidermal growth factor
1x phosphate buffer solution (PBS) (see Recipes)
Fixative solution (see Recipes)

Equipment

Biosafety cabinet class 2 (Nuaire, model: Labgard ES Energy Saver Class II, Type A2 , catalog number: NU-425-600)
3 mm skin biopsy punch (Acuderm, catalog number: P325 )
Stainless steel forceps (Sigma-Aldrich, catalog number: Z168696 )
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM Multifuge^TM 1 S-R )
Vortex
Motorized pestle (Kimble Chase Life Science and Research Products, catalog number: 7495400000 )
pH meter
Rocker (Reliable Scientific, model: 55D )
CO₂ incubator (VWR; model: 2325 )
Zeiss-Auriga focused ion beam field emission scanning electron microscope (FIB-FE-SEM)
Gatan Erlangshen digital camera

Procedure

Sample collection and preparation

Aortic valve tissues that would otherwise be discarded should be aseptically removed from patients undergoing aortic valve replacement by a cardiac surgery specialist.
Immediately after surgery, tissues should be placed in a sterile specimen container, kept on ice and immediately taken to the laboratory for processing.

Tissue preparation

In a biosafety cabinet, open the vial containing the heart tissue and assess the level of calcification, inflammation and damage prior to further processing. Ideally, valve sections should be obtained from smooth areas with no calcification, signs of inflammation or residual blood (Figure 1).
With a biopsy punch, press firmly on the selected areas of the tissue to cut through and remove the section from the rest of the tissues (Figure 1).

Figure 1. Selection of tissue from aortic valve tissues. After primary tissues are obtained, they should be immediately taken to the laboratory for evaluation and preparation. Only areas with no signs of calcification or inflammation are selected for sectioning with a 3 mm biopsy punch.
Use sterile forceps to collect the section and place it in individual wells of a 12-well plate containing 1 ml of supplemented EGM + gentamicin 300 µg ml^-1.
The number of sections that can be effectively obtained depends on the quality and the size of the tissue as well as on the type of experiment to be performed (see below).
Incubate the 12-well plate containing the valve sections at 37 °C in a 5% CO₂ atmosphere overnight (18-20 h).

Bacterial attachment to aortic valve tissues

On the same day of valve tissue processing, grow overnight bacterial cultures in triplicate. To grow S. mutans OMZ175, add 2 ml of BHI in a 4 ml sterile culture tube and inoculate by picking a single isolated colony from a freshly streaked plate.
Incubate cultures at 37 °C in a 5% CO₂ atmosphere overnight (18-20 h) without aeration.
On the following day, wash the valve sections twice at room temperature with HBSS buffer to remove residual antibiotics. For this, add 1 ml of HBSS into two clean empty wells of the same plate and transfer the sections using sterile tweezers. Gently rock plate containing valve sections for 5 min and set the rocker to a setting of 40-50 motions per minute for each wash step.
In the meantime, centrifuge the overnight cultures at 3,500 x g for 10 min at 4 °C, wash twice with sterile PBS and resuspend cultures with the initial culture volume.
For each strain replicate, add 2.7 ml of fresh EGM without antibiotics to a clean 4 ml sterile culture tube and 300 µl of the bacterial culture making a 1:10 dilution of the bacterial suspension (approximately 1 x 10⁷ CFU ml^-1).
Transfer washed valve sections to a new 12-well plate and add 1 ml of the bacterial suspension in EGM. In addition, take 100 µl of each replicate for serial dilution and plating of the initial culture CFU (T₀).
Incubate plate at 37 °C in a 5% CO₂ atmosphere with gentle rocking for 90 min.
After incubation, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec. This process should be performed three times in order to remove loosely bound bacteria.
After washing, place each valve section in a 4 ml sterile culture tube containing 500 µl of PBS and homogenize the tissue for 2 min with a motorized pestle to detach tightly adhered bacteria.
Take 100 µl aliquot from the homogenized valve suspension for serial dilution and plating of the final culture on BHI medium plates to determine the CFU (T_F).
Incubate inoculated plates at 37 °C in a 5% CO₂ atmosphere. After 48 h, count colonies for CFU determination.

Tissue preparation for analysis of adhesion by scanning electron microscopy (SEM)

To visualize bacterial adhesion to heart valve sections by SEM, follow the inoculation protocol described above (steps 3a-3g).
After incubation of tissue with bacteria, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec once.
Note: It is important not to perform too many washes at this stage as the SEM sample preparation includes several washing steps.
Upon washing, transfer the valve section to a labeled glass scintillation vial containing 15 ml of fixative solution.
After the sample is fixed at 4 °C with gentle rocking for 48 h, it is ready for SEM analysis.

Competition analysis of bacterial attachment

On the same day of valve tissue processing, grow overnight bacterial cultures in triplicate. Unlike monoculture inoculations, this protocol requires marked strains so that they can be distinguished in CFU determination.
For this study, S. mutans OMZ175 containing an erythromycin resistance cassette (Erm^R) and S. mutans OMZ175 with the collagen binding protein gene (cnm) interrupted by a kanamycin resistance cassette (Kan^R) were used.
To grow bacterial cultures, add 2 ml of BHI + Erm 10 µg ml^-1 or BHI + Kan 1 mg ml^-1 in a 4 ml sterile culture tube and inoculate by picking colonies from freshly streaked plates.
Incubate cultures at 37 °C in a 5% CO₂ atmosphere overnight (18-20 h) without aeration.
On the following day, wash the valve sections twice at room temperature with HBSS buffer to remove residual antibiotics. For this, add 1 ml of HBSS into two clean empty wells of the same plate and transfer the sections using sterile tweezers. Gently rock plate containing valve sections for 5 min and set the rocker to a setting of 40-50 motions per minute for each wash step.
In the meantime, centrifuge the overnight cultures at 3,500 x g for 10 min at 4 °C, wash twice with sterile PBS and resuspend cultures with the initial culture volume.
For each strain replicate, add 2.7 ml of fresh EGM without antibiotics to a clean 4 ml sterile culture tube and 300 µl of the bacterial culture making a 1:10 dilution of the bacterial suspension (approximately 1 x 10⁷ CFU ml^-1).
Transfer washed valve sections to a new 12-well plate and add 500 µl of the S. mutans OMZ175 (Erm^R) and 500 µl of the S. mutans ∆cnm (Kan^R) bacterial suspensions in EGM. In addition, take 100 µl of each mixed bacterial replicate for serial dilution and plating of the initial culture CFU (T₀).
Incubate the inoculated valve sections at 37 °C in a 5% CO₂ atmosphere with gentle rocking for 90 min.
After incubation, transfer each valve section into a 1.7 ml microcentrifuge tube containing 1 ml of HBSS and then vortex at medium intensity for 5 sec. This process should be repeated three times in order to remove loosely bound bacteria.
After washing, place each valve section in a 4 ml sterile culture tube containing 500 µl of PBS and homogenize the tissue for 2 min with a motorized pestle to detach tightly adhered bacteria.
Take 100 µl aliquot from the homogenized valve suspension for serial dilution and plating of the final culture on both BHI + Erm 10 µg ml^-1 and BHI + Kan 1 mg ml^-1 plates to determine the CFU (T_F).
Incubate inoculated plates at 37 °C in a 5% CO₂ atmosphere. After 48 h, count colonies for CFU determination.

Data analysis

For visualization of bacterial adherence, tissues should be thoroughly examined by SEM (Figure 2). The top and bottom parts of the tissues, which are the areas of the heart valve continuously exposed to arterial flow and are generally undamaged, are referred to as smooth surfaces. The edge of tissues, which are displaying collagen fibers due to rupture by the biopsy punch, is referred to as rough surfaces.

Figure 2. Adhesion of S. mutans OMZ175 to human aortic valve sections. Adhesion of S. mutans to human aortic valve sections analyzed by SEM. Center picture is a low magnification (25x) image of the valve section (scale bar = 200 µm). Representative images of attached bacteria to rough (A) and smooth surfaces (B) were taken at a 5,000x magnification (scale bars = 1 µm). The ex vivo adhesion of S. mutans OMZ175 (black arrows) to human valve sections requires the collagen-binding protein Cnm as no bacteria is detected in the ∆cnm strain.
To calculate the competitive index (CI), CFUs must be determined and applied in the following formula:

where, Inoculum = T₀; Substrate = T_F.
Values are then plotted on a column scatter plot and analyzed using one-way ANOVA with Bonferroni post hoc comparisons to determine substrate preferences among strain pairs. A CI value of 1 represents no difference in adherence between strains; a CI value lower than 1 represents less binding by test strain compared to the reference strain; a CI value higher than 1 represents more binding by the test strain compared to the reference strain.
A representative graph showing the competitive index (CI) of S. mutans OMZ175 and its ∆cnm counterpart is shown below. Each point in the graph represents an individual experiment using different tissue samples. The variability comes from the nature of the tissues since specimens are not exactly the same for every patient (Figure 3).

Figure 3. CI analysis of S. mutans OMZ175 and its ∆cnm counterpart. The CI values were calculated from CFU at T₀ and T_F for both strains using the CI formula described above. The mean CI was higher than 1 indicating that S. mutans OMZ175 outcompetes its ∆cnm counterpart. Expression of Cnm in OMZ175 enhances the bacterial capacity to adhere human aortic valve tissues.

Notes

All protocols involving human subject must be previously approved by an Institutional Review Board. This protocol was established using discarded tissues from patients undergoing cardiac surgery for aortic valve replacement due to calcific stenosis. Patients younger than 18 years old, pregnant women and HIV-positive patients were excluded from the original study.
All tissues should be individually and thoroughly evaluated prior to the ex vivo binding analysis. This is due to the different levels of calcification, inflammation and tissue damage in each patient.
Tissues should be processed within 6 h of obtaining them. If required, tissues may be processed and placed in EGM + gentamicin 300 µg ml^-1 for up to 72 h before performing colonization assay.
For this study, tissue visualization by SEM was performed by the University of Rochester Medical Center Electron Microscopy Core. Samples were analyzed using a Zeiss-Auriga focused ion beam field emission scanning electron microscope (FIB-FE-SEM) and the images were captured using the attached Gatan Erlangshen digital camera.
The number of input bacteria (total number of bacterial cells added to the tissues) for the competition experiment was previously determined to be approximately similar for S. mutans OMZ175 and its ∆cnm counterpart. Similarly, CFU input should be determined for any bacterial strains to be tested prior to competition analysis. To maintain reproducibility, we suggest adjusting bacterial cultures to specific OD₆₀₀ nm to match the number of CFUs between strains.

Recipes

1x phosphate buffer solution (PBS)
137 mM NaCl
2.7 mM KCl
10 mM Na₂HPO₄
2 mM KH₂PO₄
Fixative solution
4% paraformaldehyde
2.5% glutaraldehyde
0.1 M sodium cacodylate
EGM-MV Bulletkit
500 ml EGM complete medium
5% fetal bovine serum
500 mg hydrocortisone
6 mg bovine brain extract
0.1% human recombinant epidermal growth factor

Acknowledgments

We would like to thank Michael Swartz and Peter Knight from the University of Rochester for sample collection. This work was supported by the NIH-NIDCR (R01 DE022559). A.A.-R. was supported by NIH-NHLBI (F31 HL124951). I.A.F. was supported by the São Paulo Research Foundation (FAPESP, Brazil, 2013/25080-7). This protocol is a modified version from a study by Chuang-Smith et al., 2009.

References

Banas, J. A. (2004). Virulence properties of Streptococcus mutans. Front Biosci 9: 1267-1277.
Chuang-Smith, O. N., Wells, C. L., Henry-Stanley, M. J. and Dunny, G. M. (2010). Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization. PLoS One 5(12): e15798.
Freires, I. A., Aviles-Reyes, A., Kitten, T., Simpson-Haidaris, P. J., Swartz, M., Knight, P. A., Rosalen, P. L., Lemos, J. A. and Abranches, J. (2016). Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence. Virulence 3: 1-12.
Que, Y. A. and Moreillon, P. (2011). Infective endocarditis. Nat Rev Cardiol 8: 322-336.
Yetkin, E. and Waltenberger, J. (2009). Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 135: 4-13.