Engineering bright and genetically stable fluorescent Streptococcus suis strains for functional in vitro and in vivo applications

Streptococcus suis is a major cause of streptococcal infections in pigs and an emerging zoonotic pathogen, resulting in substantial economic losses in the swine production industry. The limited efficacy of current vaccine strategies and the rise in antimicrobial resistance have intensified efforts to investigate the biology and pathogenesis of the microorganism as a basis for developing alternative control strategies. In this work, we engineered a genetically stable S. suis strain producing a superfolder green fluorescent protein that may serve to study this pathogen in a variety of in vitro and in vivo assays. Multiple S. suis strains from different genetic backgrounds were successfully transformed, exhibiting strong and stable fluorescence without compromising bacterial growth. Fluorescence intensity remained consistent over 15 serial passages in culture without the need for antibiotic selection, supporting its suitability for long-term experiments. The fluorescent strains were nicely distinguishable by fluorescence microscopy and enabled the detailed study of various biological aspects, including biofilm formation, interactions with eukaryotic cells, and differential growth. In murine infection models, the engineered strains caused streptococcal disease, unlike unencapsulated mutant derivatives, and were detected in internal organs via fluorescence microscopy. Altogether, this work provides a valuable tool for advancing research into S. suis.