Noninvasive in vivo imaging to evaluate immune responses and antimicrobial therapy against Staphylococcus aureus and USA300 MRSA skin infections

Abstract

Staphylococcus aureus skin infections represent a significant public health threat because of the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). As greater understanding of protective immune responses and more effective antimicrobial therapies are needed, a S. aureus skin wound infection model was developed in which full-thickness scalpel cuts on the backs of mice were infected with a bioluminescent S. aureus (methicillin sensitive) or USA300 community-acquired MRSA strain and in vivo imaging was used to noninvasively monitor the bacterial burden. In addition, the infection-induced inflammatory response was quantified using in vivo fluorescence imaging of LysEGFP mice. Using this model, we found that both IL-1α and IL-1β contributed to host defense during a wound infection, whereas IL-1β was more critical during an intradermal S. aureus infection. Furthermore, treatment of a USA300 MRSA skin infection with retapamulin ointment resulted in up to 85-fold reduction in bacterial burden and a 53% decrease in infection-induced inflammation. In contrast, mupirocin ointment had minimal clinical activity against this USA300 strain, resulting in only a 2-fold reduction in bacterial burden. Taken together, this S. aureus wound infection model provides a valuable preclinical screening method to investigate cutaneous immune responses and the efficacy of topical antimicrobial therapies.

Figure 1. Mouse model of Staphylococcus aureus skin wound infection

Three 8-mm in length, parallel, full-thickness scalpel wounds on the backs of mice were inoculated with 2 × 10⁵, 2 × 10⁶, or 2 × 10⁷ colony-forming units (CFUs) per 10 µl of S. aureus or no bacteria (none) (n = 12 mice per group). (a) Mean total lesion size (cm²) ± 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 S. aureus bioluminescence (mean total flux (photons per second) ± SEM) (logarithmic scale). (d) Representative in vivo S. aureus bioluminescence on a color scale overlaid on top of a grayscale image of mice. *P<0.05; ^†P<0.01; ^‡P<0.001, S. aureus-infected mice versus none (Student’s t-test).

Figure 2. In vivo bioluminescence highly correlated with ex vivo bacterial colony-forming unit (CFU) counts

Bacteria present within the infected skin lesions that were inoculated with 2 × 10⁵, 2 × 10⁶, and 2 × 10⁷ CFUs per 10 µl of Staphylococcus aureus (n = 5 mice per group) were harvested from mice on postinoculation day 1 and CFUs were determined after overnight culture. (a) Representative bacterial culture plates after overnight culture with or without bioluminescence. (b) Mean CFUs of S. aureus ± SEM recovered from 8-mm lesional punch biopsies on day 1. (c) Correlation between in vivo bioluminescence signals and total CFUs harvested from the infected skin lesions. The logarithmic trendline (blue line) and the correlation coefficient of determination (R²) between in vivo bioluminescence signals and total CFUs are shown.

Figure 3. In vivo fluorescence imaging to measure the infection-induced inflammation

Three 8-mm in length, parallel scalpel wounds on the backs of (a) C57BL/6 mice or (b–e) LysEGFP mice were inoculated with 2 × 10⁶ colony-forming units (CFUs) per 10 µl of Staphylococcus aureus or no bacteria (none). (a) Representative photomicrographs (1 of 3, with similar results) of sections from 8-mm punch biopsies taken at 1 day after wounding ± S. aureus infection labeled with hematoxylin and eosin (H&E) stain, anti-Gr-1 mAb (neutrophil marker), and Gram stain. Scale bars = 150 µm. (b) In vivo S. aureus burden as measured by in vivo bioluminescence imaging (mean total flux (photons per second) ± SEM) (logarithmic scale). (c) Infection-induced inflammation (enhanced green fluorescence protein (EGFP)-neutrophil infiltration) as measured by in vivo fluorescence imaging (mean total flux (photons per second) ± SEM). (d) Representative photographs of in vivo S. aureus bioluminescence. (e) Representative photographs of in vivo EGFP-neutrophil fluorescence.

Figure 4. Contribution of IL-1α and IL-1β to IL-1R-mediated host defense against Staphylococcus aureus skin infection

IL-1α-, IL-1β-, and IL-1R-deficient mice and wild-type (wt) mice (n = 12 mice per group) were inoculated with (a) 2 × 10⁶ colony-forming units (CFUs) per 10 µl of S. aureus in the superficial S. aureus skin infection model or with (b) an intradermal injection of 2 × 10⁶ CFUs per 100 µl of S. aureus. (Left panels) Mean total lesion size (cm²) ± SEM. (Right panels) In vivo bacterial counts as measured by mean total flux (photons per second) ± SEM. *P<0.05; ^†P<0.01; ^‡P<0.001, IL-1α-, IL-1β- or IL-1R-deficient mice versus wt mice (Student’s t-test).

Figure 5. In vivo efficacy of mupirocin and retapamulin topical therapy against USA300, a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain

Three 8-mm in length, parallel scalpel wounds on the backs of LysEGFP mice were inoculated with 2 × 10⁶ colony-forming units (CFUs) per 10 µl of USA300. (a–c) Mupirocin 2% ointment, (d–f) retapamulin 1% ointment, or the corresponding vehicle ointment (polyethylene glycol (mupirocin) and white petrolatum (retapamulin)) (n = 6 mice per group) were topically applied to the infected skin (0.1 ml volume per treatment) at 4 hours after inoculation followed by twice-daily (every 12 hours) application for the next 7 days. (a, d) Mean total lesion size (cm²) ± SEM. (b, e) Bacterial counts as measured by in vivo USA300 bioluminescence (mean total flux (photons per second) ± SEM) (logarithmic scale). (c, f) Infection-induced inflammation (enhanced green fluorescence protein (EGFP)-neutrophil infiltration) as measured by in vivo fluorescence (total flux (photons per second) ± SEM). *P<0.05; ^†P<0.01; ^‡P<0.001, antibiotic ointment versus vehicle ointment (Student’s t-test).