Fast and reliable testing for pathogenic bacteria like Stapylococcus aureus (S. aureus) is highly desirable in the clinic as it can cause deep-seated infections such as osteomyelitis and endocarditis and is the major cause for hospital acquired infections of surgical wounds or indwelling medical devices. S. aureus is known for its ability to become resistant to antibiotics with the Methicillin-resistant S. aureus (MRSA) as the most prominent example causing epidemic waves of hospital-acquired and community-associated infections. To prevent the spread of resistant strains of S. aureus, rapid and widespread testing of patients and hospital environments is of utter importance.
Researchers at the Karolinska Institute in Sweden have used an EbbaBiolight-like Molecule as fluorescent label for Staphylococci in mixed liquid cultures. Added to a mixed species sample containing Staphylococcus aureus and Salmonella Enteritidis, Staphylococcus epidermidis and Escherichia coli or Staphylococcus aureus and Enterococcus faecalis, Staphylococci are immediately distinguished by a bright fluorescent label.
As background fluorescence is almost non-existent using the EbbaBiolight-like Molecule, washing steps are obsolete and, importantly, quantitative studies of live, growing bacteria are feasible as the number of bacteria linearly correlates with the fluorescence intensity. This feature was used in a high throughput approach where a transposon mutagenesis library with >2000 strains was used to determine the binding target of the EbbaBiolight-like Molecule by tracking bacterial growth by means of optical density (OD) measurement and by analyzing binding of the EbbaBiolight-like Molecule by detecting relative fluorescence intensity.
The results from the high-throughput assay indicated that the EbbaBiolight-like Molecule likely binds to cell envelope structures. Further analysis confirmed that cell wall components lipoteichoic acid (LTA), peptidoglycan (PGN) as well as wall teichoic acids (WTA) act as epitopes for the EbbaBiolight-like Molecule. Super-resolution microscopy using an Airyscan detector was used to confirm localization of the EbbaBiolight-like Molecule in the cell wall including the division septum of Staphylococcus epidermidis.
Interestingly, selective binding of S. aureus was achieved, even when mixed with E. faecalis which also carries an exposed cell envelope. This was explained by hydrophobic interactions playing a major role in binding of the EbbaBiologht-like. At pH5, when cell envelope components are protonated and electrostatic repulsion is reduced the EbbaBiolight-like Molecule indeed binds to gram-positive S. aureus and E. faecalis but not gram-negative S. Enteritidis.