Streptococcus pyogenes is an exclusively human pathogen causing a wide range of clinical manifestations from mild superficial infections to severe, life-threatening, invasive diseases. S. pyogenes is consistently susceptible toward penicillin, but therapeutic failure of penicillin treatment has been reported frequently. At the same time, streptococcal resistance to alternative antibiotics, e.g., macrolides, is common. To reduce the application of antibiotics for treatment of S. pyogenes infections, it is mandatory to develop novel therapeutic strategies. Antisense peptide nucleic acids (PNAs) are synthetic DNA derivatives widely applied for hybridization-based microbial diagnostics. They have a high potential as therapeutic agents, because PNA antisense targeting of essential genes was shown to reduce growth of several pathogenic bacterial species. Spontaneous cellular uptake of PNAs is restricted in eukaryotes and in bacteria. To overcome this problem, PNAs can be coupled to cell-penetrating peptides (CPPs) that support PNA translocation over the cell membrane. In bacteria, the efficiency of CPP-mediated PNA uptake is species specific. Previously, HIV-1 transactivator of transcription (HIV-1 TAT) peptide-coupled anti-gyrA PNA was shown to inhibit growth of S. pyogenes. Here, we investigate the effect of 18 CPP-coupled anti-gyrA PNAs on S. pyogenes growth and virulence. HIV-1 TAT, oligolysine (K8), and (RXR)4XB peptide-coupled anti-gyrA PNAs efficiently abolished bacterial growth in vitro. Consistently, treatment with these three CPP-PNAs increased survival of larvae in a Galleria mellonella infection model.

Influenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such as Streptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processes in vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processes in vitro. In this study we analysed VOCs from headspace above (co)-infected human cells by using a customized sampling system. For investigating the influenza A mono-infection and the viral-bacterial co-infection in vitro, we analysed VOCs from Detroit cells inoculated with influenza A virus and S. pyogenes by means of needle-trap micro-extraction (NTME) and gas chromatography mass spectrometry (GC-MS). Besides the determination of microbiological data such as cell count, cytokines, virus load and bacterial load, emissions from cell medium, uninfected cells and bacteria mono-infected cells were analysed. Significant differences in emitted VOC concentrations were identified between non-infected and infected cells. After inoculation with S. pyogenes, bacterial infection was mirrored by increased emissions of acetaldehyde and propanal. N-propyl acetate was linked to viral infection. Non-destructive monitoring of infections by means of VOC analysis may open a new window for infection research and clinical applications. VOC analysis could enable early recognition of pathogen presence and in-depth understanding of their etiopathology.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems.

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