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

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.The rates of multiantibiotic resistance among bacteria that infect wounds and burns are constantly on the rise (4, 12). Consequently, rapid control of wound infections and monitoring of therapeutic strategies by optical techniques have recently been proposed. The method of optically monitoring bacterial numbers and viability in real time in living animals by use of genetically engineered bacteria that emit luminescence, together with ultrasensitive photon-counting cameras, has been demonstrated with several models (7,11,16,17,20,21,33).By this technique, quantification of the luminescence images can determine in real time the extent of infection in living animals and can thereby provide both temporal and spatial information about the labeled bacteria and their metabolic activities. Similarly, antibiotic effects can be detected directly, nondestructively, and noninvasively.The study described here aimed to evaluate the effects of an antibiotic in situ by using bioluminescent bacteria. The study was divided into three steps. The first consisted of constructing a plasmid to transform Escherichia coli into a bioluminescent variant. The second step consisted of studying bioluminescent E. coli in vitro as a function of temperature and pH. The third step consisted of determining the MIC and the minimal bactericidal concentration (MBC) of an antibiotic, sulfamethoxazole-trimethoprim (SMX-TMP), for this bioluminescent strain. Finally, the efficacy of SMX-TMP was monitored in vi...

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo

“…To measure this, we used bioluminescent imaging (BLI), which is a method that in recent years, is increasingly being utilized to determine location and strength of luciferase activity in a variety of animal models. [47][48][49][50][51] Luciferase activity is observed following an IP dose of D-luciferin, which results in bioluminescence that is detected in anesthetized mice by a cooled charge-coupled device camera. BLI is now an established method for quantitatively detecting transgene expression in vivo.…”

Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review

“…The use of bioluminiscence for the detection of MAP in a variety of sources (water, milk, blood etc.) and organisms such as M. leprae, M. tuberculosis (Gupta et al, 1997;Martin-Casabona et al, 1997), Saccharomyces cerevisiae, Citrobacter rodentium and Escherichia coli (Jawhara and Mordon, 2004;Cho and Yoon, 2007) using firefly luciferase has been described in the available literature. Recently, studies dealing with the use of bioluminiscence in association with MAP have been published (Williams et al, 1999;Rosseels et al, 2006).…”

Construction of p16S lux , a Novel Vector for Improved Bioluminescent Labeling of Gram-Negative Bacteria

“…Various species of gram-negative bacteria have been rendered luminescent using a variety of approaches. The species include Escherichia coli (3,4,11,12), Salmonella enterica serovar Typhimurium (1, 2), Yersinia enterocolitica (7), Brucella melitensis (9), Pseudomonas aeruginosa (5), and Citrobacter rodentium (13). In E. coli, both firefly luciferase (3, 4) and the bacterial lux system (11) have been used to provide the luminescent signal.…”

“…The species include Escherichia coli (3,4,11,12), Salmonella enterica serovar Typhimurium (1,2), Yersinia enterocolitica (7), Brucella melitensis (9), Pseudomonas aeruginosa (5), and Citrobacter rodentium (13). In E. coli, both firefly luciferase (3,4) and the bacterial lux system (11) have been used to provide the luminescent signal. However, the use of firefly luciferase is somewhat limited in that the substrate for the reaction (luciferin) must be added exogenously.…”