Assessing Intracellular Bacterial Persistence and Antibiotic Resistance in a Mouse Model

Streszczenie

Source: Zhang, C., et al., Development and Assessment of Intracellular Infection Models for Staphylococcus aureus. J. Vis. Exp. (2025)

The video demonstrates the isolation of kidneys containing Staphylococcus aureus-infected macrophages, followed by tissue homogenization to release intracellular bacteria. It quantifies bacterial persistence within macrophages in control and antibiotic-treated groups by counting colony-forming units.

Protokół

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 

1. Intracellular bacterial infection in mice
   1. Randomly divide 20 Balb/c mice into four groups (n=5, Figure 1A).
   2. For Group I, inject 3 x 10⁶ CFU (Colony-Forming Unit) planktonic MRSA252 per mouse by intravenous injection. For Group II, inject 3 x 10⁶ CFU planktonic MRSA252 (Methicillin-Resistant Staphylococcus aureus 252) and vancomycin (110 mg/kg; see Table of Materials) per mouse by intravenous injection. For Group III, inject the intracellular bacteria Staphylococcus aureus (S. aureus) by intravenous injection. For Group IV, inject the intracellular bacteria S. aureus and vancomycin (110 mg/kg) per mouse by intravenous injection.
   3. Place the mice under the infrared physiotherapy lamp until their tail veins become dilated.
   4. Intravenously inject 3 x 10⁶ CFU planktonic MRSA252 per mouse of Group I and Group II. Intravenously inject the intracellular bacteria into the mice of Groups III and IV.
      NOTE: When using a syringe to collect liquid containing intracellular bacteria, it is recommended to first gently mix the liquid evenly with a pipette to ensure consistent infection levels across all mice.
   5. After 30 min, intravenously inject vancomycin into Group II and Group IV per mouse.
2. Evaluation of in vivo intracellular infection model
   1. Evaluate the intracellular infection model of mice by counting the colonization of bacteria in the kidneys. After 24 h of injection, anesthetize the mice with 3% isoflurane and sacrifice them by cervical dislocation. Disinfect them in 75% ethyl alcohol.
   2. Immobilize the mice, lift the abdominal skin with tweezers with one hand, cut the abdominal skin of the mouse with ophthalmic scissors with the other hand, find the kidneys in the abdominal cavity, and completely strip the kidneys. Put them in PBS (Phosphate-Buffered Saline).
   3. Add 1 mL of PBS to the tissue grinding tube. Transfer the kidneys from each mouse into separate grinding tubes and grind until no solid tissue remains. Pour the homogenized tissue into individual Eppendorf tubes and label each tube with the corresponding mouse identification.
   4. Use PBS to serially dilute the tissue homogenate. Spot each dilution onto separate TSA (Tryptic Soy Agar) plates and incubate overnight at 37 °C. Count the colonies and analyze the data (Figure 1B).

Wyniki

Figure 1: Intracellular infection with MRSA252 (Methicillin-Resistant Staphylococcus aureus 252) is resistant to vancomycin killing. (A) Graphical abs...

Materiały

Lista materiałów użytych w tym artykule

Nazwa	Firma	Numer katalogowy	Komentarze
Beef extract powder	BBI, UK	A600114-0500
Biohazard safety equipment	Heal force, China	VS-1300L-u
Cell incubator	ESCO, Singapore	CCL-170B-8
Centrifuge M1416R	RWD, China	M1416R
Centrifuge tube	Guanghou Labselect, China	CT-002-50A
Gentamycin	Shanghai Sangon, China	B540724-0010
Incubator	Shanghai Hengzi, China	HDPF-150
Methicillin-Resistant Staphylococcus aureus 252	Third Military Medical University, China	null
Penicillin and Streptomycin	Shanghai Beyotime, China	C0222
Phosphate Buffer Solution	Shanghai Beyotime, China	ST476
Saline	Sichuan Kelun, China	null
Tryptic Soy Agar (TSA) plates	Beijing AOBOX Biotechnology Co., LTD,China	02-130
Tryptic Soy Broth (TSB) medium	Beijing AOBOX Biotechnology Co., LTD,China	02-102K
Tryptone	OXOID, UK	LP0042B
Vancomycin	Shanghai Beyotime, China	ST2807-250mg