Murine MRSA-infected wound model

A previously reported [28,49] murine skin infection model with modifications, was used to validate the in vivo efficacy of the ZnPcE-PDT treatment against MRSA. All animal experiments were conformed to the university guidelines and approved by the Animal Experimentation Ethics Committee (Ref. no.16/176/MIS) of The Chinese University of Hong Kong. 4–6 weeks old male BALB/c mice (17–21 g) were supplied by Laboratory Animal Services Centre (LASEC), The Chinese University of Hong Kong. They were housed in individual ventilated cages (IVC) under the conditions of 22–25°C and a 12 h light–dark cycle, with free access to chow and tap water. MRSA RN4220/pUL5054 was grown in MHB under aerobic conditions at 37°C with 100 rpm orbital shaking for overnight. The overnight culture was diluted with fresh MHB and re-incubated for 2–3 h until the mid-log growing phase. The desired suspension was centrifuged at 1000× g for 15 min to harvest the cell pallet. The cell pallet was re-suspended and diluted in MHB to achieve 0.6 optical density at 600 nm (OD₆₀₀) corresponded to 1 × 10⁸ CFU/mL, and used for wound inoculations.

On Day 0, mice were anaesthetized by an intraperitoneal (i.p.) injection of ketamine (40 mg/kg) and xylazine (8 mg/kg), with the hair of the back shaved, and the skin cleansed with 10% povidone-iodine solution. A circular full-thickness excision wound (4–5 mm in diameter) was established through puncher on the back subcutaneous tissue of each animal. The lesion overlaid with gauze was dressed up with adhesive bandage. Buprenorphine, commercially available as Temgesic^®, at a dosage of 0.05 mg/kg was administrated subcutaneously to the mice 12 hourly for 24 h after wound induction to relieve pain.

Two days after the wound induction (Day 2), mice were anaesthetized with ketamine/xylazine cocktail and in all animals, the adhesive bandage were removed. A 20 μL aliquot was drawn from the 1 × 10⁸ CFU/mL suspension of MRSA in MHB and spread evenly over the wound area using a micropipette. A dressing (Tegaderm^TM film, 3M, USA) was applied to cover the wound immediately. The mice with infected wound were equally divided into five cohorts (n = 6 per each group) as listed in Table 1.

The first treatment was carried out 30 min after MRSA inoculation on Day 2. A 50 µL of 7.8 µM ZnPcE solutions, Fucidin (2% w/w) cream or distilled water was injected under the dressing (Tegaderm^TM film) by syringe and allowed to spread over the wound.

For Group C and E, photoactivation (Biolitec group, Bonn, Germany) was initiated immediately. Single dosage of laser at 1W was delivered for 60 s by optical fibre 2 mm in diameter, corresponding to 60 J/wound. After each treatment, the mice were returned to IVC and Groups D and E were placed in dark. The second, third and fourth treatments were carried out on Day3, Day 5 and Day 9, respectively (Figure 2).

Timeline for in vivo aPDT study. Four treatment cycles were performed at Day 2, 3, 5 and 9. After the 4th treatment, the mice were sacrificed for the CFU count.

On Day 9, animals were killed with an overdose of dorminal pentobarbital solution after the last treatment. The wound (5 × 10 mm) was then excised aseptically. Each skin sample was homogenized in 0.5 mL of PBS solution for bacterial viability counts. Quantification of viable bacteria was performed by culturing serial dilutions (10 μL) of the bacterial suspension on blood agar plates. For this purpose, all plates were incubated at 37°C for 24 h and the bacteria counts in colony-forming units (CFU) were enumerated.