Antibacterial and antibiofilm activities of Zn²⁺ against Staphylococcus aureus and Streptococcus mutans

Staphylococcus aureus (S aureus) and Streptococcus mutans (S mutans) are pathogenic bacteria associated with denture-related stomatitis. Zinc (Zn), a widely used metallic antibacterial agent, can induce the production of reactive oxygen species (ROS) in cells, thereby inhibiting microbial growth and proliferation; studies, however, are lacking.

The purpose of this in vitro study was to determine the antibacterial and antibiofilm function of Zn²⁺ against S aureus and S mutans and assess gene expression changes to clarify its inhibitory mechanisms.

Zn²⁺ at one-fourth minimum inhibitory concentration (MIC), one-half MIC, and MIC was co-cultured with the bacteria. Membrane integrity was evaluated by alkaline phosphatase (AKP) activity and live-dead staining. The oxidative stress mechanism was assessed by intracellular reactive oxygen species (ROS) and lipid peroxidation levels. Deoxyribonucleic acid (DNA) integrity was evaluated using agarose gel electrophoresis. Ribonucleic acid (RNA) sequencing combined with gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) bioinformatics analyses was performed to identify Zn²⁺-affected molecular pathways. The effect of Zn²⁺ on key gene expression in S aureus and S mutans was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

In S aureus, Zn²⁺ upregulated ROS and malondialdehyde (MDA) levels and disrupted oxidative phosphorylation, which indicated the induction of oxidative stress and caused bacterial death. In S mutans, Zn²⁺ interfered with metabolic pathways by targeting 2-component systems (TCS), weakening acid resistance, impairing biofilm adhesion, and disrupting cell wall peptidoglycan synthesis, ultimately resulting in bacterial death. The qRT-PCR results were consistent with those of GO and KEGG analyses.

Differentially expressed genes (DEGs) in S aureus were primarily enriched in pathways related to DNA replication, mismatch repair, and oxidative phosphorylation, suggesting that Zn²⁺ co-culture significantly affected genetic stability and redox-associated cellular functions. In contrast, DEGs in S mutans were predominantly involved in microbial metabolism, molecular systems, and bioadhesion pathways, with no significant changes in oxidative stress markers.