In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R² = 0.98) or transcutaneously in the abdominal region of whole animals (R² = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩP_amiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.Among the wide variety of bacteria that colonize the gastrointestinal tracts of mammals, Escherichia coli is the most abundant facultative anaerobe of the human intestinal microflora. Aside from being part of the normal flora, E. coli is also a versatile organism capable of causing a variety of intestinal and extraintestinal diseases (18). The mechanisms that allow commensal E. coli to colonize the intestine and survive successfully in this niche remain poorly characterized. Conventional mice display natural resistance to colonization by commensal E. coli, but oral administration of streptomycin, which alters the intestinal microflora, allows colonization of the mouse large intestine by this species (25). The streptomycin-treated mouse model has been used extensively to study the factors of gram-negative bacteria implicated in the intestinal colonization process. However, this model is limited to the viable plate counts of bacteria in the feces and misses some critical information, such as the kinetics of colonization, the fate of the bacterial cells across the digestive tract, and the site of colonization. A better understanding of colonization would be facilitated by direct in vivo follow-up of this process.Bioluminescence imaging (BLI) technology is emerging as a powerful tool for the study of a wide range of biological processes in live animals, including real-time monitoring of infections (16). Bioluminescence systems emit visible light due to the luciferase-mediated oxidation of a luciferin substrate. A variety of luciferin-luciferase systems with different peak emissions have been identified in nature from numerous species (14). The luciferase of the soil bacterium Photorhabdus luminescens has been expressed successfully in gram-negative and gram-positive bacteria. This system emits blue-green light, with an emission maximum of approximately 490 nm, and does not require the addition of an exogenous substrate since the luciferase operon contains the genes required for synthesis of the substrate. Therefore, this luciferase has been used extensively to monitor bacterial infections in the living mouse. One of the first investigations with Salmonella enterica serovar Typhimurium transformed with the lux operon of P. luminescens evaluated the tissue distribution and the virulence of various S. Typhimurium strains (9). Subsequent modification of the lux operon led to the generation of highly bioluminescent Staphylococcus aureus and allowed the monitoring of infections due to this species in living mice (11). The modified lux operon was engineered into a lux-kan transposon cassette for chromosomal integration in gram-positive bacteria, such as S. aureus, Streptococcus pneumoniae, group A Streptococcus, and Listeria monocytogenes (16). Replication of L. monocytogenes in the lumen of the gall bladder was demonstrated for the first time by BLI (13).Bioluminescent E. coli was used in the neutropenic mouse thigh model of infection to evaluate the in vivo activity of antimicrobial agents (29). Bioluminescence was as indicative of therapeutic efficacy as CFU counts but, in addition, allowed real-time monitoring of the infection and of treatment efficacy in the same animal; however, only short-term monitoring (12 h) could be performed.Because luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. After oral administration of bioluminescent Salmonella to susceptible mice, the bioluminescent signal recorded in the abdominal region was greatly enhanced after air exposure (9). It was therefore assumed that direct bioluminescence imaging of intestine-colonizing microorganisms would not be optimal unless oxygen was provided exogenously or as the result of the close interaction between cells and the bacteria (9). However, the bacterial luciferase was used to trace in real time the colonization dynamics by Citrobacter rodentium of the gastrointestinal tracts of living animals, demonstrating that the gut represents a semianaerobic environment that allows the study of bacterial colonization by BLI (33).Factors essential for colonization are best studied in cocolonization experiments (7, 17). There are several luciferases with distinct emission spectra (34) that could be used in competition experiments to trace simultaneously two bacterial populations in the same living animal. However, in order not to impose additional and different metabolic burdens on the bacteria under study, the exogenous luciferases ideally have to be similar to allow comparison between strains. The thermostable luciferase variants PpyRE-TS and PpyGR-TS, derived from wild-type luciferase from the North American firefly Photinus pyralis, emit red (612 nm) and green (552 nm) light, respectively, at 37°C and are encoded by single genes of 1,650 bp, differing by only 9 bp (4). Bioluminescence color is determined by the Ser₂₈₄Thr (PpyRE-TS) and Val₂₄₁Ile, Gly₂₄₆Ala, and Phe₂₅₀Ser (PpyGR-TS) amino acid changes (5, 34). By use of optical filters, the emission spectra are readily distinguishable (4, 5). Five additional mutations provide enhanced thermostability at 37°C (4), improving the compatibility of the enzymes with bacterial culture conditions and BLI in animal models.While the luciferase mutants and all firefly luciferases use as substrates firefly luciferin and ATP to produce light, in vivo imaging is commonly performed with endogenous ATP and requires only exogenous administration of the luciferase substrate.The aim of this study was to develop a dynamic mouse model using in vivo bioluminescence imaging systems to monitor bacterial colonization in situ and in real time in whole living animals. Various strains of E. coli harboring the genes for the bacterial luciferase from P. luminescens or the firefly luciferase mutants (PpyRE-TS and PpyGR-TS) from P. pyralis have been engineered and used to follow bacterial intestinal colonization in mice. BLI was found to be well adapted to compare the intestine-colonizing capacities of various E. coli strains and to monitor cocolonization in vivo by use of dual bioluminescence emission.