Toxin–antitoxin-stabilized reporter plasmids for biophotonic imaging of Group A streptococcus

Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin–antitoxin-stabilized plasmid. The bacterial luciferin–luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω–ε–ζ toxin–antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.