Group B Streptococcus (GBS; Streptococcus agalactiae) is a globally disseminated opportunistic pathogen that causes meningitis, sepsis, pneumonia and soft tissue infections in neonates and healthy or immunocompromised adults (1-4). GBS is the leading cause of neonatal mortality due to bacterial infection, and in developing countries up to 3 babies per 1000 live births develop GBS infection, with a fatality rate as high as 15% (1, 2). GBS is also a prominent veterinary pathogen of bovine mastitis (5), and extensive use of tetracycline facilitated transmission of distinct clones of GBS into humans (6). Many studies have examined GBS physiology, virulence and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been primarily limited to antibody-based methods or non-specific stains that bind DNA or protein, and use dead cells and/or tissues.
In this study, we developed and validated a green fluorescence protein (GFP)-expressing plasmid, pGU2664 that can be applied as a marker to visualize GBS in experimental studies. The synthetic, constitutively active CP25 promoter drives strong and stable expression of GFP in GBS carrying pGU2664. GBS maintains GFP activity across different phases of growth. Application of fluorescence polarization enables easy discrimination of GBS GFP activity from the auto-fluorescence of culture media commonly used to grow GBS, such as THB. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFP in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualise phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking the control of global virulence regulator CovR in live adhesion assays. The system for GFP-expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.