In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

1. Preliminary: Bacterial strains and epithelial cells.

Manipulations of cells and bacteria are performed aseptically, under a laminar flow hood.

1. Freshly isolate the E. coli strains 2787, 2787ΔaidA, and C600 from glycerol stocks on Lysogeny Broth (LB) agar plates (1% tryptone, 0.5% sodium chloride, 0.5% yeast extract, 1.5% agar) and grow at 37°C. To minimize variability in the assay, it is advised to always use freshly plated strains and to keep the strains at 4°C on sealed Petri plates only for a maximum of a couple of weeks.
2. Culture HEp-2 cells (ATCC CCL-23) in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated bovine serum (heat inactivation is performed at 56°C for 30 min). During routine culture we also add 10 U/ml penicillin and 10 μg/ml streptomycin.
3. Grow Hep-2 cells at 37°C in a cell incubator with an atmosphere containing 5% CO₂. Use standard cell culture procedures to maintain the cells, which are grown in 75 cm² flasks and subcultured every time they reach confluence. We use our cultured cells until they reach 30 to 40 passages and then discard them.
4. Prepare an assay when the HEp-2 cells in a 75 cm² flask are nearly reaching confluence and therefore are ready for an adherence assay.

2. DAY 1: Preparing the inoculum and the epithelial cells

1. Wash the HEp-2 cells in the flask once with warm Dulbecco’s phosphate buffered saline (DPBS: 8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH₂PO₄, 0.21 g/L Na₂HPO₄:7H₂O)
2. The cells are incubated with 0.05% trypsin – EDTA for 5 min before adding fresh warm complete medium. After fresh medium is added, the cells are resuspended by pipetting up and down.
3. Centrifuge the cell suspension at 2,000 rpm for 5 min and resuspend the pellets in DPBS and centrifuge again.
4. Resuspend the cells in fresh medium supplemented with 10 % serum but containing no antibiotics, at a concentration of 2×10⁵ cells/ml.
5. Seed one milliliter of cell suspension in three sets of duplicate wells (one for each strain) in the center of a 24-well plate and incubate the plate overnight in a cell culture incubator.
   Note: It is important that the serum be thoroughly decomplemented and that antibiotics are omitted after this stage. Failure to do so might kill the infecting bacteria.
6. Inoculate one isolated colony of each bacterial strain (2787, 2787ΔaidA and C600) in 5 ml of LB broth (1% tryptone, 0.5% sodium chloride, 0.5% yeast extract) and grow overnight at 37°C with vigorous shaking (180 rpm).

3. DAY 2: Infection of cells

1. Inspect the HEp-2 cells under an inverted microscope to ascertain that they are at least 90 % confluent and not contaminated.
2. Washed the cells with warm DPBS and, to each well, add 1 ml of fresh medium supplemented with 10 % serum but containing no antibiotics. Also add fresh medium to three wells without cells. This will be used to determine the total number of bacteria in the inoculum for each strain.
3. Measure the optical density at 600 nm (O.D_.600nm) of the bacterial cultures. Add an aliquot of each bacterial culture to one set of duplicate wells containing Hep-2 cells and to one well not containing cells. Usually, we use a volume of overnight culture corresponding to 10⁶ colony forming units (cfu). This represents a multiplicity of infection (MOI) of 5:1 (bacteria:cells). Although we directly use our overnight cultures, it is sometimes advisable to centrifuge the bacteria and resuspend them in DPBS to avoid deleterious effects of secreted molecules present in the overnight cultures (such as cytotoxins, for instance)
   Note: The MOI can vary between 100:1 and 1:10. Higher MOI yields higher variability and background and bacteria will tend to stick to the plastic of the plate. Lower MOI also yields high variability. Once an MOI is chosen, it is imperative to be consistent and keep this MOI.
4. Incubate the infected cells in the cell culture incubator for 3 hours at 37°C with 5% CO₂.
   Note: It is also possible to centrifuge briefly the plate at low speed (e.g. 1,000 x g for 1-2 min), in order to bring all the bacteria directly in contact with the cells. This has the added benefits of synchronizing the infection, and allowing shorter incubation times (as little as 15-30 min).
5. Remove the medium from the infected cells and wash the cells 3 times with warm DPBS. At this step, adherent bacteria can usually be seen with a standard microscope and we routinely perform this check.
6. To lyse the cells and detach the adhered bacteria, add 100 μl of 1% Triton X-100 to each well containing the cells. Other detergents can be used (such as saponin for instance).
7. Incubate the cells for 10 min at room temperature and then add 900 μl of LB medium.
8. Gently homogenize the suspensions by repeated up-and-down pipetting. The wells without cells containing the inoculum are similarly gently homogenized.
   Note: Some bacteria might be too sensitive to the detergent, in that case we detach the epithelial cells by incubation with 0.05% trypsin – EDTA for 20 min at 37°C. Cells together with adhered bacteria can also be scraped from the wells with a pipette tip.
9. Prepare serial 10-fold dilutions of the suspensions of adhered bacteria and inoculum using LB broth and plate 100 μl from 3 dilutions (usually the 1:1,000, 1:10,000 and 1:100,000 dilutions) on LB agar and incubate overnight at 37°C.

4. DAY 3: cfu counting and data presentation.

1. Count the colonies on the plates and calculate the number of cfu of adhered bacteria and of the inoculum by averaging each series of dilutions. Only plates with between 10-300 colonies should be counted.

5. Representative results :

The following table shows typical results of cfu counting of adhered bacteria and inoculum from 3 experiments performed on different days:

The results can be reported directly as adhered cfu, as shown in Figure 1A. Since the inoculum sizes can vary between strains because of differences in growth rates or pipetting errors, it is often advisable to report the results as percentage of adhered bacteria. The percentage of adhered bacteria can be calculated by dividing the number of cfu of adhered bacteria by the number of cfu of the inoculum, as shown in Figure 1B. By performing a repeated measure ANOVA and Bonferroni post-tests to compare all columns of Figure 1B, we can see that there are significant differences (p<0.05) between 2787 and 2787ΔaidA, as well as between 2787 and C600, but no significant difference between 2787ΔaidA and C600.

Figure 1. Representation of the results as cfu of adhered bacteria (A) or percentage of adhered bacteria (B)

The percentage of adherence observed is often very dependent on the experimental set-up (and, to a lesser extent, on the experimenter). Particularly important are the MOI and the number of washes, so the percentage should not be literally interpreted.

The bacteria in the inoculum can sometimes grow much faster than the bacteria in the presence of cells, skewing the size of the inoculum by comparison with the real total number of bacteria in wells with cells. To alleviate this issue, it is recommended to either: (i) use wells with cells to determine the inoculum: HEp-2 cells are seeded in an additional well and infected. At the end of the assay, instead of discarding the supernatant and washing, 100 μl of 10% Triton X-100 is added to the well. The cells will lyse and the lysate containing adhered and non-adhered bacteria is gently homogenized by repeated up-and-down pipetting.; or (ii) collect the supernatants of infected cells at the end of the assay, as well as the supernatants from the DPBS washes and determine the number of cfu in those pooled supernatants. This will yield the number of cfu of non-adhered bacteria. The percentage of adhered bacteria can then be calculated by dividing the number of cfu of adhered bacteria by the addition of the number of cfu of adhered and non-adhered bacteria.

To illustrate the robustness of the assay, we can compare this protocol with the assay performed with S4074, an adhesive porcine pathogenic strain of Actinobacillus pleuropneumoniae⁷, incubated with cells from a recently established tracheal piglet cell line, NPTr⁸. Aside from differences in the media required for growth of the bacteria and the cells, the only difference is that the bacteria used for infection are from an exponentially growing culture, and not from a stationary-phase overnight culture. With A. pleuropneumoniae it is also very important to respect an MOI of 10:1 and to not exceed 3 hours of infection, otherwise the secretion of toxins will cause cell death and bias the results. In addition, this strain of A. pleuropneumoniae can readily adhere to plastic so it is important to use confluent cells and verify visually that the cells are not sloughing off.