Streptococcus mutans (S. mutans) is a common cariogenic bacterium that secretes glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs) and plays an important role in plaque formation. Propolis essential oil (PEO) is one of the main components of propolis, and its antibacterial activity has been proven. However, little is known about the potential effects of PEO against S. mutans. We found that PEO has antibacterial effects against S. mutans by decreasing bacterial viability within the biofilm, as demonstrated by the XTT assay, live/dead staining assay, LDH activity assay, and leakage of calcium ions. Furthermore, PEO also suppresses the total of biofilm biomasses and damages the biofilm structure. The underlying mechanisms involved may be related to inhibiting bacterial adhesion and GTFs activity, resulting in decreased production of EPSs. In addition, a CCK8 assay suggests that PEO has no cytotoxicity on normal oral epithelial cells. Overall, PEO has great potential for preventing and treating oral bacterial infections caused by S. mutans.

Combining antibiotics with propolis is an effective method to combat bacterial drug resistance. Nanoparticles are of interest in the antimicrobial field because of their higher drug stability, solubility, penetration power, and treatment efficacy. In this study, propolis nanoparticles (PNPs) were synthesized, and their antibacterial and anti-biofilm activities against methicillin-resistant Staphylococcus aureus (MRSA) in combination with ampicillin sodium (AS) were analyzed. The PNPs had an average particle diameter of 118.0 nm, a polydispersity index of 0.129, and a zeta potential of −28.2 mV. The fractional inhibitory concentration indices of PNPs and AS against tested MRSA strains highlighted this synergy, ranging between 0.375 and 0.5. Crystal violet staining showed that combined PNPs and AS significantly inhibited biofilm formation and reduced existing biofilm biomass. We then discovered that PNPs inhibited bacterial adhesion, extracellular polysaccharide synthesis, and mecR1, mecA, blaZ, and icaADBC gene expression. These results indicated that PNPs exerted a synergistic antibacterial effect with AS by inhibiting mecR1, mecA, and blaZ gene expressions to reduce the drug resistance of MRSA. Meanwhile, PNPs weakened bacterial adhesion and aggregation by suppressing icaADBC gene expression, allowing antibiotics to penetrate the biofilm, and exhibiting significant synergistic anti-biofilm activity. In summary, PNPs are promising candidates for combating MRSA-related diseases.

Within the global epidemiological landscape, methicillin-resistant Staphylococcus aureus (MRSA) stands out as a major contributor to infectious disease burden. The persistent public health crisis it presents arises from a dual challenge: intrinsic multidrug resistance coupled with a high rate of healthcare-associated infections. Recent studies have shown that propolis has unique advantages in bacterial infection prevention and treatment. The present study revealed that propolis ethanolic extract (PEE) exhibited notable antibacterial activity against both MRSA ATCC 43300 and MRSA CI2, with a minimum inhibitory concentration (MIC) of 128 μg/mL for each strain. Crystal violet (CV) staining and XTT sodium reduction assays were employed to evaluate the anti-biofilm efficacy of PEE. CV staining revealed that PEE significantly inhibited biofilm formation and reduced the biomass of pre-formed biofilm. Additionally, the XTT sodium reduction assay demonstrated a substantial reduction in the metabolic activity of the biofilm-embedded. Scanning electron microscopy and bacterial adhesion experiments revealed that PEE significantly reduced bacterial adhesion and aggregation. Furthermore, experiments on the synthesis of extracellular polysaccharides and proteins showed that PEE inhibits the production of water-soluble and alkali-soluble polysaccharides and extracellular proteins. Real-time quantitative Polymerase Chain Reaction (RT-qPCR) analysis revealed that PEE inhibited the expression of icaADBC, fnbAB, clfAB, and sarA. These results revealed that PEE inhibits biofilm formation and development by inhibiting the expression of sarA, icaADBC, fnbAB, and clfAB, thereby reducing the synthesis of extracellular polysaccharides and proteins to attenuate the adhesion capacity of MRSA. In summary, this study provides experimental evidence for the development of PEE as a potential antimicrobial agent for the prevention and treatment of MRSA-associated infections. Future work will focus on identifying its key active monomers and investigating its therapeutic effects and mechanisms of action in animal models.

Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl₂. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.