Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

1. Preparing the MRSA for Infection (two Days Prior to Infection)

1. Inoculate a loopful of MRSA from a stock culture to a blood agar (Trypticase Soy Agar (TSA)) plate.
2. Check the hemolysis phenotype (a clear zone around each colony) on the blood agar plate.
3. Pick a colony that has a hemolytic phenotype that is consistent with other experiments.
4. Inoculate the colony into 3 mL Todd Hewitt Broth (THB),with the appropriated antibiotic when necessary, in a 15 mL snap-capped tube.
5. Incubate at 37°C overnight with shaking at 220 rpm.

2. Preparing the Mice for Infection (one Day Prior to Infection)

1. Depending on the size of the mice, shave the fur off a 3 x 4 cm area on the back of mice.
2. Apply ~5 mm³ hair remover cream (purchased from a local drug store) to the shaved area.
3. Allow the hair remover cream to incubate on the skin surface for approximately 1 min.
4. Wet paper towels with ddH₂O.
5. Wipe off the hair remover cream with the wet paper towel.

3. Preparing the MRSA for Infection (on the Day of Infection)

1. Dilute the overnight bacterial culture at 1:500 to 1:1000 with 10 mL of pre-warmed THB, in a 50 mL screw-capped tube.
2. Incubate at 37°C for approximately 2.5 hours with shaking at 220 rpm, until A₅₄₀ reaches 2.5.
3. Collect MRSA by centrifugation at 3,225 x g for 10 min at 4°C.
4. Discard the supernatant.
5. Resuspend the bacterial pellet in 10 mL Dulbecco’s phosphate buffered saline (DPBS).
6. Repeat steps 3 and 4.
7. Resuspend the bacterial pellet in DPBS at a desired concentration.
8. Serially dilute the bacterial suspension from 10¹ to 10⁸.
9. Plate the diluted bacterial suspension onto blood agar plates.
10. Incubate the plates at 37°C overnight.
11. Observe and record the hemolytic phenotype of the inocula.
12. Count the Colony Forming Units (CFU) number on the plate.
13. Calculate the inocula based on the CFU number on the plate.

4. Subcutaneous Infection of MRSA (on the Day of Infection and 3 Days Post-infection)

1. Inject subcutaneously 100 μL bacterial suspension in the shaved area.
2. Observe the animals for 3 to 5 hours after injection to make sure the mice are alive.
3. Observe the lesion on the back of the animals daily and record the lesion area daily when it is needed. Lesion observation should include recording of lesion size and morphology. Both skin and muscle lesions are quantitated by multiplying the length and width of the lesion. Irregularly-shaped lesions need to be broken down into smaller symmetrical pieces, and each piece measured by the same method. Lesion measurements may also use a computer-assisted histomorphometric assessment program (ImageJ; open-source available from the NIH at http://rsb.info.nih.gov/ij/) ².
4. Sacrifice the animals on day 3 post-infection by inhalation of isoflurane followed by cervical dislocation.
5. Observe and record the lesion on the skin.
6. Nick the skin with a pair of sterile scissors.
7. Peel the skin off carefully and observe and record the lesion on the muscle.
8. Excise the lesion and approximately 2 – 5 mm of the surrounding area.
9. Collect spleen and kidneys.
10. Homogenize the tissues using a tissue homogenizer or by repeated application of a plunger from a 1 mL syringe to a microfuge tube containing the tissue and 100 μL of DPBS.
11. Add 900 μL of DPBS to each tube.
12. Vortex the samples for 5 min.
13. Serially dilute the suspension with DPBS 10¹ to 10⁶.
14. Plate the diluted suspensions on THA plates.

5. Representative Results:

1. Immediately after each subcutaneous injection, a bleb will be observed on the skin surface when infection is done correctly (Figure 1A). If no bleb is observed on the skin surface, the injection may be too deep, and this may affect the outcome of the infection (lesion size, CFU).
2. The bacterial suspension needs to be serially diluted and the CFU need to be examined on blood agar plates for each experiment. This step will yield important information: 1) whether the inocula have homogeneous phenotype; 2) the exact viable bacterial count for the infection.
3. The lesions and bacterial survival may be assessed at various time points post-infection. Here, we examined the infection on day 3 post-infection. The lesion sizes are analyzed on the skin surface (Figure 1B: 1x 10⁹; Figure 1D: 1 x 10⁷ CFU) and on the muscle surface (Figure 1C: 1x 10⁹; Figure 1E: 1 x 10⁷ CFU). The lesion area equals length (mm) x width (mm) may be measured for assessing the tissue damage (Figs. 2 A and B). This result may help determine the extent of tissue damage caused by a given virulence factor. Additionally, the lesions are excised and homogenized in DPBS, and plated to quantitate the CFU number (Figure 2C), which indicates the viability of the bacteria at the infection site.

Figure 1. The skin and muscle lesions. CD1 mice were inoculated subcutaneous on one flank with MRSA (LAC). Pictures were taken at time 0 and day 3 post-infection. (A) Time 0 post-infection (p.i.), (B)-(G) Day 3 p.i.; B, D. and F: skin lesions; C, E, and G: muscle lesions; B and C: 1x 10⁹ CFU infected; D and E: 1x 10⁷ CFU infected; F and G: mock.

Figure 2. Skin lesion size and viable bacteria count at the infection site on day 3 post-infection. Skin and muscle lesions on MRSA (LAC) infected CD1 mice. (A) Skin lesion size. (B) Muscle lesion size. (C) Total tissue CFU. H: 1 x 10⁹ CFU inocula; L: 1 x 10⁷ CFU inocula. *: p < 0.05, Mann-Whitney test.