Bacterial pathogens such as Staphylococcus aureus and Staphylococcus epidermidis can survive in different types of cells including professional phagocytes, causing intracellular infections. Antibiotic treatment of intracellular infections is often unsuccessful due to the low efficacy of most antibiotics inside cells. Therefore, novel techniques which can improve intracellular activity of antibiotics are urgently needed. We aimed to use photochemical internalization (PCI) to enhance cytosolic release of antibiotics from endocytic vesicles after internalization. Our results show that PCI indeed caused cytosolic release of gentamicin and significantly increased its efficacy against S. epidermidis in vitro in mouse macrophages. Upon illumination for 15 min, the killing of intracellular S. epidermidis in RAW 264.7 cells by 10 or 30 μg/ml gentamicin was increased to 1 or 3 CFU log, respectively, owing to the use of PCI, whereas no killing by gentamicin only without PCI was observed. Moreover, survival of S. aureus-infected zebrafish embryos was significantly improved by treatment with PCI-gentamicin. PCI improved the therapeutic efficacy of gentamicin at a dose of 0.1 ng per embryo to a level similar to that of a dose of 0.4 ng per embryo, indicating that PCI can lower the antibiotic dose required for treating (intracellular) staphylococcal infection. Thus, the present study shows that PCI is a promising novel approach to enhance the intracellular efficacy of antibiotics via cytosolic release, allowing them to reach intracellular bacteria. This will expand their therapeutic window and will increase the numbers of antibiotics which can be used for treatment of intracellular infections.

Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment.

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A₂ (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including <i>S. aureus</i>. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of <i>lspA</i>, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the <i>lspA</i> mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, <i>lspA</i> deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of &#x3e;26,000 <i>S. aureus</i> genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since <i>Streptococcus mutans</i> and <i>Enterococcus faecalis</i> were also more hGIIA-susceptible after <i>lspA</i> deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.