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Antimicrobial Agents and Chemotherapy, September 2004, p. 3436-3441, Vol. 48, No. 9
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.9.3436-3441.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

UPRES EA 2689-INSERM IFR 114, Lille University Hospital, Lille, France

Received 8 December 2003/ Returned for modification 8 February 2004/ Accepted 16 May 2004

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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