Antibacterial and antibiofilm activities of ZnO-NPs against Streptococcus suis serotype 9 in vitro

ABSTRACT Streptococcus suis is an important swine pathogen and an emerging zoonotic threat, with serotype 9 (SS9) increasingly recognized as an important pathogenic serotype. Biofilm formation contributes to the persistence, antimicrobial tolerance, and pathogenicity of S. suis , yet effective strategies for controlling biofilm-associated infections caused by SS9 remain limited. In this study, we evaluated the antibacterial and antibiofilm activities of zinc oxide nanoparticles (ZnO-NPs) against SS9 and investigated whether their effects were associated with oxidative stress, biofilm formation, and alterations in virulence-associated genes. The minimum inhibitory concentration and minimum biofilm eradication concentration of ZnO-NPs against SS9 were both 49 μg/mL. ZnO-NPs exhibited potent antibacterial and antibiofilm activities, significantly inhibiting biofilm formation and reducing biofilm viability in a dose-dependent manner. Crystal violet staining demonstrated a marked reduction in biofilm biomass, while SEM analysis revealed substantial disruption of bacterial morphology and biofilm architecture following ZnO-NPs treatment. In addition, ZnO-NPs markedly increased intracellular reactive oxygen species levels, indicating the induction of oxidative stress in SS9. ZnO-NPs treatment also reduced the major extracellular matrix components of the biofilm, including extracellular proteins and extracellular DNA. Furthermore, RT-qPCR analysis showed that ZnO-NPs downregulated the expression of biofilm- and virulence-associated genes, including ef , fbps , gdh , and luxS . Collectively, these findings indicate that ZnO-NPs exert antibacterial and antibiofilm effects against SS9, accompanied by oxidative stress induction, disruption of biofilm architecture and extracellular matrix, and modulation of biofilm- and virulence-associated genes. ZnO-NPs may, therefore, represent a promising candidate for controlling SS9-associated infections. IMPORTANCE Streptococcus suis serotype 9 (SS9) is an important pathogen associated with disease in pigs and represents a potential zoonotic threat. The increasing prevalence of antimicrobial resistance highlights the need for alternative approaches to control SS9 infections. In this study, zinc oxide nanoparticles (ZnO-NPs) showed antibacterial and antibiofilm activity against SS9, including disruption of established biofilms. These findings provide evidence for further exploring ZnO-NPs as a non-antibiotic strategy for controlling SS9 infections and support future studies evaluating their safety and efficacy in animals.