In vivo bioluminescence imaging to evaluate systemic and topical antibiotics against community-acquired methicillin-resistant Staphylococcus aureus-infected skin wounds in mice

Abstract

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) frequently causes skin and soft tissue infections, including impetigo, cellulitis, folliculitis, and infected wounds and ulcers. Uncomplicated CA-MRSA skin infections are typically managed in an outpatient setting with oral and topical antibiotics and/or incision and drainage, whereas complicated skin infections often require hospitalization, intravenous antibiotics, and sometimes surgery. The aim of this study was to develop a mouse model of CA-MRSA wound infection to compare the efficacy of commonly used systemic and topical antibiotics. A bioluminescent USA300 CA-MRSA strain was inoculated into full-thickness scalpel wounds on the backs of mice and digital photography/image analysis and in vivo bioluminescence imaging were used to measure wound healing and the bacterial burden. Subcutaneous vancomycin, daptomycin, and linezolid similarly reduced the lesion sizes and bacterial burden. Oral linezolid, clindamycin, and doxycycline all decreased the lesion sizes and bacterial burden. Oral trimethoprim-sulfamethoxazole decreased the bacterial burden but did not decrease the lesion size. Topical mupirocin and retapamulin ointments both reduced the bacterial burden. However, the petrolatum vehicle ointment for retapamulin, but not the polyethylene glycol vehicle ointment for mupirocin, promoted wound healing and initially increased the bacterial burden. Finally, in type 2 diabetic mice, subcutaneous linezolid and daptomycin had the most rapid therapeutic effect compared with vancomycin. Taken together, this mouse model of CA-MRSA wound infection, which utilizes in vivo bioluminescence imaging to monitor the bacterial burden, represents an alternative method to evaluate the preclinical in vivo efficacy of systemic and topical antimicrobial agents.

Fig 1

Mouse model of CA-MRSA wound infection. Three 8 mm in length, parallel, full-thickness scalpel wounds on the backs of C57BL/6 mice were inoculated with 2 × 10⁵, 2 × 10⁶, or 2 × 10⁷ CFU of the bioluminescent CA-MRSA strain, USA300 LAC::lux, or no bacteria (none)/10 μl (n = 5 mice per group). (A) Mean total lesion size (cm²) ± the standard error of the mean (SEM). (B) Representative photographs of the lesions of the entire dorsal back (upper panels) and close-up photographs of the lesions (lower panels) are shown. (C) Bacterial counts as measured by in vivo bioluminescence imaging (mean total flux [photons/s] ± the SEM) (logarithmic scale). (D) Representative in vivo bioluminescent signals on a color scale overlaid on top of a grayscale image of mice. *, P < 0.05; †, P < 0.01; ‡, P < 0.001 (USA300 LAC::lux-infected mice versus none) (Student's t test [two-tailed]).

Fig 2

In vivo bioluminescent signals of CA-MRSA-infected wounds highly correlate with ex vivo bacterial CFU counts. Scalpel wounds on the backs of mice were inoculated with USA300 LAC::lux (2 × 10⁶ CFU/10 μl). Correlation between in vivo bioluminescent signals (mean total flux [photons/s] ± the SEM) (logarithmic scale) from mice (n = 6 per group) imaged at 4 h and on days 1, 3, 5, and 7 and total ex vivo CFU (logarithmic scale) recovered from 8-mm lesional punch biopsies performed on euthanized mice (n = 6 per group) at the same time points. The trendline and the correlation coefficient of determination (R²) between in vivo bioluminescent signals and total CFU are shown.

Fig 3

Efficacy of systemic (subcutaneous and oral) antibiotics against CA-MRSA-infected wounds. Scalpel wounds on the backs of mice were inoculated with USA300 LAC::lux (2 × 10⁶ CFU/10 μl). Mice (n = 5 to 10 mice per group) were treated with subcutaneous vancomycin (110 mg/kg twice daily), daptomycin (50 mg/kg once daily), linezolid (60 mg/kg twice daily), or sterile saline (sham control) (top panels) or oral (via gavage) linezolid (60 mg/kg twice daily), clindamycin (100 mg/kg three times a day), doxycycline (100 mg/kg twice daily), TMP/SMX (320/1600 mg/kg once daily), or sterile saline (sham control) (middle and bottom panels). Antibiotic treatment was initiated at 4 h after CA-MRSA inoculation and continued for the first 7 days. (A, C, and E) Mean total lesion size (cm²) ± the SEM. (B, D, and F) Bacterial counts as measured by in vivo bioluminescence imaging (mean total flux [photons/s] ± the SEM) (logarithmic scale). *, P < 0.05; †, P < 0.01; ‡, P < 0.001 (antibiotic treatment versus sham treatment [saline]) (Student's t test [two-tailed]).

Fig 4

Efficacy of topical antibiotics against CA-MRSA-infected wounds. Scalpel wounds on the backs of mice were inoculated with USA300 LAC::lux (2 × 10⁶ CFU/10 μl). Mice (n = 5 mice per group) were treated with topical mupirocin 2% ointment, retapamulin 1% ointment, or the corresponding vehicle ointment (polyethylene glycol [mupirocin] and white petrolatum [retapamulin]). Antibiotic treatment was initiated at 4 h after CA-MRSA inoculation and continued twice daily for the first 7 days. (A) Mean total lesion size (cm²) ± the SEM. (B) Bacterial counts as measured by in vivo bioluminescence imaging (mean total flux [photons/s] ± the SEM) (logarithmic scale). *, P < 0.05; †, P < 0.01; ‡, P < 0.001 (antibiotic treatment versus sham treatment [saline]) (Student's t test [two-tailed]).

Fig 5

Efficacy of subcutaneous vancomycin, daptomycin, and linezolid against a CA-MRSA wound infection in diabetic mice. Scalpel wounds on the backs of NONcNZO10/LtJ diabetic mice were inoculated with USA300 LAC::lux (2 × 10⁶ CFU/10 μl). Mice (n = 5 to 10 mice per group) were treated with subcutaneous vancomycin (110 mg/kg twice daily), daptomycin (50 mg/kg once daily), linezolid (60 mg/kg twice daily), or sterile saline (sham control) beginning at 4 h after CA-MRSA inoculation and continued for the first 7 days. (A) Mean total lesion size (cm²) ± the SEM. (B) Bacterial counts as measured by in vivo bioluminescence imaging (mean total flux [photons/s] ± the SEM) (logarithmic scale). *, P < 0.05; †, P < 0.01; ‡, P < 0.001 (antibiotic treatment versus sham treatment [saline]) (Student's t test [two-tailed]).