In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

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Abstract
A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed. This study was divided into three steps: (i) construction of a plasmid to transform Escherichia coli into a bioluminescent variant, (ii) study of the bioluminescent E. coli in vitro as a function of temperature and pH, and (iii) determination of the MIC and the minimal bactericidal concentration of sulfamethoxazole-trimethoprim (SMX-TMP). Finally, the efficacy of SMX-TMP was monitored in vivo in a cutaneous wound model (hairless rat) infected with this bioluminescent bacterium by using a bioluminescence imaging system. E. coli was transformed by electroporation with a shuttle vector (pRB474) containing the firefly ( Photinus pyralis ) luciferase gene, resulting in a bioluminescent phenotype. It was found that pH 5.0 was optimal for incorporation of the susbstrate d -luciferin for the luciferase reaction. In vitro, when the agar dilution method, standard turbidity assays, and the bioluminescence imaging system were used, E. coli (pRB474) proved to be susceptible to SMX-TMP. In vivo, at 4 h, SMX-TMP treatment was already efficient compared to no treatment ( P = 0.034). At 48 h, no bioluminescence was detected in the wound, demonstrating the susceptibility of E. coli to SMX-TMP. In conclusion, this study points out the advantage of using bioluminescence imaging to evaluate the effects of antibiotics for the treatment of acute infections in vivo in a nondestructive and noninvasive manner.

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