Sitafloxacin is a 4th generation fluoroquinolone antibiotic with broad activity against a wide range of Gram-negative and Gram-positive bacteria. It is approved in Japan and used to treat pneumonia and urinary tract infections (UTIs) as well as other upper and lower respiratory infections, genitourinary infections, oral infections and otitis media. Compared to other fluoroquinolones, sitafloxacin displays a low minimal inhibitory concentration (MIC) for many bacterial species but also activity against anaerobes, intracellular bacteria, and persisters. Furthermore, it has also shown strong activity against biofilms of P. aeruginosa and S. aureus in vitro, which was recently validated in vivo with murine models of S. aureus implant-associated bone infection. Although limited in scale at present, the published literature supports the further evaluation of sitafloxacin in implant-related infections and other biofilm-related infections. The aim of this review is to summarize the chemical-positioning-based mechanisms, activity, resistance profile, and future clinical potential of sitafloxacin.

Diderm bacteria use contact-dependent Type VI secretion systems (T6SS) to gain a competitive advantage within bacterial communities and during infection. Whereas the structural core assembly of T6SS is well characterized, the role of diverse accessory proteins during assembly remains under investigation. One well-conserved accessory protein is TagF, a post-translational inhibitor of T6SS dynamics, which was previously characterized in the context of a kinase-phosphatase signaling relay. Here, we identify a subset of T6SS clusters in which TagF occurs in a distinct regulatory context and without known binding partners. Investigating the role of TagF in the constitutively active Acinetobacter baylyi T6SS, we show that though TagF can suppress assembly dynamics, it primarily acts as an assembly coordinator. Using structured illumination microscopy, we show that TagF coordinates the transition from baseplate assembly to sheath elongation by preventing premature and nonproductive sheath assembly in absence of the VgrG spike. Direct interactions with the conserved T6SS tube protein Hcp suggested that TagF blocks sheath elongation by binding and disrupting Hcp hexamers and thus preventing tube formation. Finally, we show that TagF activity depends on crosstalk with TagZ, a previously uncharacterized membrane associated accessory protein that recruits TagF to the cell periphery, and demonstrate that this interaction can be specifically disrupted by expression of an artificial TagF-binding protein. Together, our findings establish a novel role of TagF as a checkpoint protein that controls for spike-insertion into the baseplate to ensure effective T6SS assembly.

The sugar fructose has previously been shown to increase efficacy of aminoglycoside antibiotics against stationary phase bacteria. The sugar may achieve this by activating metabolism, membrane potential and growth rate. Whether this effect is also seen for other antibiotics more active against biofilm or Staphylococcus abscess communities (SAC), such as the fluoroquinolone sitafloxacin (sita), has not been tested to date. Combining the antibiotic and the sugar into a carrier-free nanodrug (ND) may further increase antibacterial activity, however, the optimal combination, their efficacy against Staphylococcus aureus, and ability to assemble into a ND has not been previously described.S. aureus USA300 was grown in Tryptic soya broth (TSB) to logarithmic or stationary growth phase, to biofilm for 48 hours on titanium discs with a media change after 24 hours and to SAC in a collagen gel supplemented with human plasma. They were treated with sita for 24 hours at 100× minimal inhibitory concentration (MIC), alone or in combination with 0.2% glucose (glu), fructose (fru) or mannose (man). Surviving bacteria were quantified by colony forming unit (CFU) counts and microscopy using Live/dead staining. Sugar-antibiotic combinations were assembled into NDs using sequential precipitation in a mixed solvent (Dimethylsulfoxid, Dimethylformamide and ethanol) and water. The reaction was confirmed by nuclear magnetic resonance (NMR) and ND stability observed using dynamic light scattering (DLS). The ND size and morphology was determined by scanning electron microscopy (SEM).Sita was shown to be the most effective antibiotic against stationary phase S. aureus with a 3 log reduction in CFU. Its efficacy was increased by at least 1 log when combined with 0.2 % glucose, fructose or mannose. Sita showed good activity against biofilm and SAC. ND composed of sita and glu, fru or man were successfully synthesized and tested with NMR and SEM, which showed an average size of 200nm and spherical morphology. DLS confirmed stability of the ND for up to 16 days at room temperature and solubility in water.To target stationary phase S. aureus, we combined sita as potent antibiotic with either one of the sugars glu, fru and man, which achieved increased bacterial killing activity.

Staphylococcus epidermidis , a dominant human skin commensal from early life, can transition to an opportunistic pathogen, including invasive, biofilm-associated infections linked to medical devices. Neonatal exposure to skin commensals induces a lifelong immunological imprint in the skin, characterized by immunoregulatory responses. We therefore hypothesized that early life exposure to S. epidermidis influences immune responses to invasive biofilm-associated infections later in life. Using a mouse model of biofilm-related S. epidermidis bone infection, we show that neonatal and adult skin colonization altered the immune response to the subsequent infection in adulthood. Neonatal colonization led to increased NK cells and neutrophils compared to no colonization, along with reduced Tregs and Th1 cells, and consistent increase in immune checkpoint receptor PD-1 ⁺ Tregs, T effector, Th1 and Th2 cells across infected bone marrow, blood and spleen. These PD-1-related immune modulations were absent in the adult-colonized group, which had the highest numbers of Tregs, Th1 and Th2 cells of all groups. These findings reveal that early exposure to commensal bacteria strongly impacts the response to invasive infection later in life. Notably, the response depends on the timing of previous exposure. Neonatal colonization drives T cell modulation, resembling neonatal immunity, while adult-colonization increases specific T cell abundance. These differences highlight the essential role of skin colonization in shaping the quality of pathogen immunity to protect against invasive, biofilm-associated infection later in life, emphasizing that immunological studies using uncolonized animal models may not fully capture human immune dynamics.