Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct

Abstract

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.

FIG. 1

Schematic diagram of the plasmid pMK4 luxABCDE. Nucleotide sequences of the lux genes, ordered as shown, are as given in GenBank (accession number M90093) flanked by the relevant sequences shown in Table 1. This plasmid can be used to generate constitutive bioluminescent strains by ligating genomic DNA (partially digested by 4-base cutter) at the unique BamHI or SmaI site and selecting for light in the gram-positive bacterium from which the DNA was derived.

FIG. 2

Comparison of bioluminescence from S. aureus and E. coli containing the native luxCDABE operon versus that in strains containing the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), S. aureus RN4220 pMK4 luxCDABE P1 (-▴-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥) and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were diluted across black 96-well microtiter plates in doubling dilutions (−0.3 log) and monitored for light over a period of 30 min using the ICCD camera. The content of each well was then plated to allow the number of CFU to be compared to the level of bioluminescence (RLU).

FIG. 3

Temperature stability of the modified luxABCDE operon. Exponential cultures of S. aureus RN4220 pMK4 luxABCDE P1 (-■-), E. coli DH5α pMK4 luxABCDE P1 (‥■‥), and E. coli DH5α pMK4 luxCDABE P1 (‥▴‥) were grown to approximately 1 × 10⁷ CFU/ml at 30°C and 1-ml volumes of each were placed in heating blocks set at 31, 33, 35, 37, 39, 41, 43, 45, and 47°C. After 1 h at each of these elevated temperatures, the nine heating blocks were sequentially placed inside a dark chamber and light from each of the three cultures was recorded for a period of 1 min using the ICCD camera. Shown are the number of RLU at each of the temperatures, with this data expressed as a percentage of the maximum (Max) bioluminescence attained and adjusted for variation in the number of CFU.

FIG. 4

Monitoring the effects of amoxicillin on bioluminescent S. aureus in mice. Exponential cultures of the bioluminescent bacteria were injected intramuscularly into the left anterior tibialis (8 × 10⁶ CFU) of 24 anesthetized mice, 12 mice with S. aureus 8325-4 pMK4 luxABCDE P1 and 12 mice with S. aureus 8325-4 pMK4 luxABCDE P2. Six mice from each group were treated with a single oral dose of amoxicillin (10 mg/kg) immediately prior to anesthesia, with the remaining six mice from each group serving as untreated controls. Shown are three treated and three untreated S. aureus 8325-4 pMK4 luxABCDE P1-infected mice, imaged ventrally for 5 min at 0, 8, and 24 h postinfection using the ICCD camera.

FIG. 5

Bioluminescence data recorded from S. aureus 8325-4 pMK4 luxABCDE P1 (A)- and S. aureus 8325-4 pMK4 luxABCDE P2 (B)-infected mice. Each data set represents the mean number of RLU from six mice either untreated (■) or treated (⧫) with amoxicillin (10 mg/kg), imaged both dorsally and ventrally for 5 minutes at 0, 4, 8, and 24 h postinfection using the ICCD camera. Error bars, standard errors of the means.