A pleuromutilin-ciprofloxacin hybrid exhibits potent activity against Streptococcus suis.

UNLABELLED: The emergence and dissemination of antimicrobial resistance in Streptococcus suis, an important zoonotic pathogen, have created an urgent demand for new therapeutic agents with improved antibacterial efficacy. To address this challenge, we previously designed and synthesized a novel hybrid molecule, PHC, by linking ciprofloxacin to a pleuromutilin derivative via an L-homocysteine bridge. Although preliminary evaluations indicated favourable antibacterial activity, the antimicrobial effect of PHC against S. suis and its mechanism of action have not yet been systematically characterized. Therefore, the present study aimed to investigate the antibacterial activity of PHC against S. suis and elucidate its underlying antibacterial mechanisms. METHODS: The antibacterial efficacy of PHC was assessed through time-kill kinetics and a murine infection model. Molecular docking was conducted to predict the interactions between PHC and its potential molecular targets, and RT-qPCR analysis was performed to evaluate the effects of PHC on genes associated with DNA replication, ribosomal function, oxidative stress, and energy metabolism in S. suis. Biofilm formation was quantified using crystal violet staining. In addition, DAPI/PI staining and DiSC3(5) fluorescence assays were employed to assess bacterial membrane integrity and membrane potential, respectively. Changes in intracellular ATP levels and ROS production were further measured to characterize the antibacterial mode of action of PHC. RESULTS: PHC exhibited potent antibacterial activity against S. suis both in vitro and in vivo. Molecular docking and transcriptional analyses suggested that PHC may interfere with bacterial DNA replication and protein synthesis by affecting topoisomerase- and ribosome-associated pathways. In addition, PHC disrupted membrane integrity and membrane potential, altered cellular energy metabolism and promoted oxidative stress responses. Furthermore, PHC demonstrated favourable therapeutic efficacy and biosafety in a murine infection model. CONCLUSION: PHC exhibits potent antibacterial activity against S. suis through interference with DNA replication- and protein synthesis-related processes, accompanied by enhanced bacterial stress responses and disruption of membrane integrity and cellular homeostasis. This study provides a theoretical basis for the further development of PHC and offers valuable insights for the design of novel hybrid antibacterial agents against S. suis.