Amplified by the decline in antibiotic discovery, the rise of antibiotic resistance has become a significant global challenge in infectious disease control. Extraintestinal Escherichia coli (ExPEC), known to be the most common instigators of urinary tract infections (UTIs), represents such a global threat. Novel strategies for more efficient treatments are therefore desperately needed. These include silver nanoparticles, which have been used as antimicrobial surface coatings on catheters to eliminate biofilm-forming uropathogens and reduce the risk of nosocomial infections. AGXX is a promising silver-ruthenium coating that presumably kills bacteria through the generation of reactive oxygen species (ROS). However, neither AGXX’s mode of action is fully understood, nor have its effects on Gram-negative bacteria or bacterial response and defense mechanisms toward AGXX been studied in detail. Here, we report that the bactericidal effects of AGXX are primarily based on ROS formation, as supplementation of the media with a ROS scavenger completely abolished AGXX-induced killing. We further show that AGXX impairs the integrity of the bacterial cell envelope and causes substantial protein aggregation and DNA damage already at sublethal concentrations. ExPEC strains appear to be more resistant to the proteotoxic effects of AGXX compared to non-pathogenic E. coli, indicating improved defense capabilities of the uropathogen. Global transcriptomic studies of AGXX-stressed ExPEC revealed a strong oxidative stress response, perturbations in metal homeostasis, as well as the activation of heat shock and DNA damage responses. Finally, we present evidence that ExPEC counteracts AGXX damage through the production of the chaperone polyphosphate, protecting cells from protein aggregation. IMPORTANCE: The rise in drug-resistant bacteria, together with the decline in antibiotic development, requires new strategies for infectious disease control. Gram-negative pathogens are particularly challenging to combat due to their outer membrane. This study highlights the effectiveness of the silver-containing antimicrobial AGXX against the Gram-negative bacterium Escherichia coli . AGXX effectively reduces bacterial survival by interfering with the membrane integrity and causing DNA damage and protein aggregation, which is likely a consequence of uncontrolled generation of oxidative stress. Our findings emphasize AGXX’s potential as an antimicrobial surface coating and shed light on potential targets to reduce bacterial resistance to AGXX.

Background/Objectives. The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen, causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit the growth or impair the survival of bacterial pathogens. Given their large structural and chemical diversity, as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against Gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin-resistant S. aureus (MRSA) and the multi-drug-resistant community-acquired strain USA300LAC. Methods. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Results/Conclusions. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 μg/mL), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane-associated mechanism of action.

The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit growth or impair survival of bacterial pathogens. Given their large structural and chemical diversity as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin resistant S. aureus (MRSA) and the multi drug-resistant community acquired strain USA300LAC. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 mg/ml), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane associated mechanism of action.