Sappanwood Extract Disarms Staphylococcus aureus by Silencing Virulence and Biofilm Together

A dichloromethane extract of sappanwood, a tropical tree long used in traditional Southeast Asian medicine, has done something that few anti-virulence compounds have managed in the laboratory: it simultaneously suppressed the master virulence regulator of Staphylococcus aureus and weakened the biofilm that shields the bacterium from antibiotics and immune attack. The finding, published in MicrobiologyOpen, matters because the two goals have long been considered difficult to achieve at once. Quorum-sensing inhibitors that silence the accessory gene regulator, or Agr system, frequently push S. aureus in the opposite direction, encouraging the bacterium to abandon its planktonic lifestyle and encase itself in a stubborn biofilm.

S. aureus is a Gram-positive bacterium that lives harmlessly on the skin and in the nasal passages of many healthy people, yet it can cause everything from minor skin infections to life-threatening invasive disease. Methicillin-resistant S. aureus, or MRSA, adds another layer of danger through its resistance to beta-lactam antibiotics, a burden that health systems worldwide continue to struggle with. Beyond resistance genes, the bacterium builds biofilms, structured communities wrapped in a matrix of extracellular polysaccharides, surface proteins, and extracellular DNA. This matrix blocks host immune defenses and blunts the effect of many antibiotics, making biofilm-associated infections notoriously difficult to eradicate.

The Agr system sits at the top of the virulence hierarchy in S. aureus. Activated by a quorum-sensing mechanism in which accumulating autoinducing peptides signal high cell density, Agr switches on a battery of secreted toxins and enzymes while switching off the surface factors needed for attachment and biofilm construction. Because Agr is critical for invasive infection, several Agr inhibitors have advanced to preclinical testing. Yet a complication has dogged the strategy: many clinical isolates carry mutations that disable Agr entirely, and suppressing Agr can promote biofilm formation, as repeatedly reported since early studies two decades ago. An inhibitor that silences Agr but drives cells into biofilms could paradoxically worsen chronic infection.

The new study began with a screen of plant extracts for antibacterial activity. The researchers, working with the dichloromethane extract of Biancaea sappan, formerly Caesalpinia sappan, found that the extract, which they call SWe, behaved unusually. Using S. aureus SH1000, an Agr-positive laboratory strain that builds polysaccharide-dependent biofilms, they measured a minimal inhibitory concentration of roughly 500 micrograms per milliliter. At 250 micrograms per milliliter, a concentration that left viable cell counts essentially unchanged at 95 percent of the untreated control, biofilm formation dropped by 35 percent. Even at 500 micrograms per milliliter, where viability fell to 80 percent, the effect was modest compared with chloramphenicol, which reduced counts to undetectable levels. Crucially, the team separated planktonic cells from biofilm cells and showed that SWe reduced biofilm colony-forming units without reducing the planktonic population, ruling out the common pitfall of mistaking growth inhibition for genuine anti-biofilm activity.

Dissecting the biofilm matrix revealed a striking reshuffling of its components. Poly-N-acetylglucosamine, the main extracellular polysaccharide in this strain, actually increased significantly at 9 and 24 hours of treatment, a result confirmed with a PNAG-specific enzyme probe. Extracellular protein levels stayed flat. The most dramatic change was in extracellular DNA, which fell significantly at every time point tested. Because eDNA in staphylococcal biofilms comes from genomic DNA released by autolysis, the researchers checked autolytic activity and found it reduced as well. Since eDNA acts as a negatively charged glue that binds polysaccharides and proteins into a coherent matrix, its depletion plausibly explains why treated cells drifted loosely in the wells instead of forming firm, adherent biofilms, a difference the authors captured on video.

The extract also silenced Agr. A reporter strain carrying a fluorescent gene under the control of the agr P3 promoter lit up around six hours of growth in untreated cultures and stayed bright, but fluorescence was strongly suppressed in SWe-treated cultures, and microscopy confirmed that fewer individual cells were fluorescent. The suppression was not a side effect of slower growth. Downstream Agr-dependent phenotypes followed: hemolytic activity against sheep red blood cells declined in a dose-dependent manner, colony spreading on soft agar, which depends mainly on phenol-soluble modulins, was curtailed, and the production of staphyloxanthin, the golden carotenoid pigment that helps S. aureus survive oxidative stress, dropped by up to half.

RNA sequencing at 3, 9, and 24 hours painted a transcriptomic portrait of this unusual cellular state. Roughly 100 to 150 genes were differentially expressed at each time point, and the agrBDCA operon itself was downregulated at 3 hours, while Agr-regulated toxin genes, including those encoding alpha-hemolysin, delta-hemolysin, and phenol-soluble modulins, remained suppressed through 24 hours, results confirmed by quantitative PCR. Among the downregulated genes were a suite of immune-evasion and adhesion proteins: the complement inhibitors Efb, Ecb, and SCIN-B, the immunoglobulin-binding protein Sbi, the extracellular adherence protein Eap, and several MSCRAMM adhesins such as SdrC and SdrD. Fibronectin binding, a classic adhesin-mediated function, fell below the detection limit in treated cells. Many of these factors are controlled by the SaeRS two-component system, and its expression was also reduced, suggesting that SWe suppresses both major virulence regulators at once. The downregulation of capsule biosynthesis genes, which compete with PIA synthesis for the same UDP-GlcNAc precursor, may partly explain the paradoxical rise in polysaccharide production.

The strain breadth of the effect strengthened the case. In MW2, a methicillin-resistant clinical strain with intact Agr function but weak biofilm formation, SWe suppressed the RNA-III reporter signal and reduced hemolysis, colony spreading, and pigmentation, all without pushing biofilm upward. In ATCC25923, a strain lacking Agr activity altogether, the extract still significantly reduced biofilm without affecting viability. Follow-up experiments showed that the anti-biofilm effect persisted in a strain deleted for agr and in SA113, which carries a mutation in the SigB regulatory pathway, indicating that neither system is strictly required for the extract’s action, though SigB activity, reflected in the asp23 marker gene, was dampened by treatment.

The significance of the work lies less in immediate clinical application than in proof of concept. The authors note that no previous report had clearly demonstrated inhibition of both biofilm and Agr without affecting growth. Existing Agr inhibitors tell a cautionary tale: staquorsin, designed to interfere with AgrA, promotes biofilm in the Newman strain, and a fruit extract of Schinus terebinthifolia along with an oleanolic-acid-family compound increase biofilm at low concentrations. By contrast, SWe appears to hold cells in a planktonic, low-virulence state rather than letting them retreat into a biofilm. The nearest mechanistic parallel is fenoprofen, an anti-inflammatory drug that inhibits SaeR and loosens biofilm structure, though it leaves PIA production unchanged and was not active against PIA-dependent biofilms, unlike SWe.

Considerable questions remain before sappanwood chemistry could inform therapy. The active compounds in the dichloromethane extract are unidentified, though their low polarity is suggested by the fact that methanol and ethanol extracts of the same wood showed bactericidal activity without anti-quorum-sensing effects. Sappanwood contains brazilin, chalcones, coumarins, xanthones, and homo isoflavonoids, and brazilin has previously been reported to affect biofilm through a different regulatory route, while fatty acids in the extract could contribute anti-biofilm activity of their own. The extract also altered metabolic gene expression and raised intracellular reactive oxygen species, connections whose roles in the virulence-suppressed state are unexplored. And as the authors emphasize, in vitro results under limited conditions do not necessarily translate to the behavior of S. aureus during infection. Still, the study provides the first documented example of a cellular status in which Agr activity is reduced and biofilm is impaired simultaneously, offering a template for anti-virulence strategies that avoid the biofilm rebound that has undermined quorum-sensing approaches so far.

Subject of Research: In vitro suppression of Staphylococcus aureus virulence and biofilm by a sappanwood extract

Article Title: Multifaceted Suppression of Staphylococcal Virulence Phenotypes: In Vitro Study of a Cellular Status With Reduced Agr Activity and Impaired Biofilm

Article References: Nguyen, N. B., Ushijima, Y., Sekiya, R., Morikawa, K., & Nguyen, L. T. T. (2026). Multifaceted Suppression of Staphylococcal Virulence Phenotypes: In Vitro Study of a Cellular Status With Reduced Agr Activity and Impaired Biofilm. MicrobiologyOpen, 15(5), Article e70380. https://doi.org/10.1002/mbo3.70380

Image Credits: AI Generated

DOI: 10.1002/mbo3.70380

Keywords: Staphylococcus aureus, MRSA, Agr quorum sensing, biofilm, sappanwood, Biancaea sappan, virulence factors, SaeRS system, extracellular DNA, anti-virulence, natural products, quorum-sensing inhibition