As we have mentioned in an earlier article, Pterostilbene is a methoxylated derivative of the perhaps more well known supplement resveratrol. It has been known for some time now that resveratrol and other related compounds have antibacterial properties but it was unknown until recently if pterostilbene could be effective against drug-resistant Staphylococcus aureus bacteria and for treating skin bacteria.
The staphylococcus aureus (S. aureus) bacteria is the main cause of bacterial infections in hospitals and healthcare facilities. In fact S. aureus leads to more deaths than the total sum of deaths from viral hepatitis, human immunodeficiency virus, tuberculosis, and influenza combined in the United States(1).
The current generation of antibiotics are able to combat S. aureus infections, however, there are drug-resistant strains of S. aureus such as the methicillin-resistant S. aureus (MRSA) which are becoming an increasing health risk due to their ability to resist treatment.
What is MRSA?
Believe it or not MRSA lives on the skin of around 1 in 30 people and is generally harmless. It is typically found in the armpits, nose, groin or buttock areas of the body and those carrying it likely have no idea. MRSA can be transmitted touching someone who has it, sharing towels, sheets or clothes with someone who has MRSA on their skin or even from surfaces or objects that have MRSA on them.
Having MRSA on your skin is no cause for alarm, it will not make you ill and likely will go away in a few weeks or months without you even noticing. However, the danger is that if it can penetrate deeper into the body and cause an infection, this is why it is so dangerous in the hospital setting where patients are vulnerable to the bacteria getting into the body. Patients who have had surgery, who are wounded or have other breaches in their defences are at risk of MRSA infection.
Each year, around 90,000 Americans suffer from MRSA infections and around 20,000 die as a result. Healthcare-related infections from MRSA and other drug resistant bacterias also cause a significant increase in mortality(2).
MRSA has also made treating skin infections more difficult and it can often be present in the skin where it can lead to cellulitis, folliculitis, and the formation of abscesses(3). The problem with infections associated with biofilm and MRSA are harder to deal with due to being resistant to antibiotics and also to the host’s immune system and one reason for that resistance is the biofilm.
The bacterial biofilm
As human beings we live in communities creating shelter to help protect ourselves from external threats. We construct homes to protect our family and larger buildings that protect the wider community. Coming together as a community and using these places of shelter improves our chances of survival and so it should hardly come as a surprise that bacteria has had the same idea. .
When we encounter bacteria it is rarely as single entities and instead as communities. Pathogens that cause acute infection are typically free floating bacteria also known as planktonic bacteria that can remain active for decades and in doing so have evolved ways to form into communities. They do this for the same reason we humans do, to improve survival odds. When bacteria form communities they are better at dealing with our immune system which is trying to destroy them.
So it turns out that a large number of pathogens we are host to form communities called biofilms. A bacterial biofilm can be thought of as a structured community of bacterial cells surrounded by a secreted polymeric matrix that sticks to an inert or living surface. In short, that means that bacteria can group together on essentially any surface and create a protective matrix around their community. This matrix is made of polymers – substances composed of molecules with repeating structural units that are connected by chemical bonds.
The biofilm around bacterial communities is a problem as it prevents the penetration of antibiotics and also causes the bacteria to become resistant to treatment due to them being alive for long periods in the host(4). This means the need to find new antibacterial compounds that can overcome this resistance has become very important.
The search for novel antibacterial agents
Resveratrol, a compound occurring naturally in grapes and other berries and plant sources has been shown to inhibit the growth of some bacteria and fungi(5). Pterostilbene is closely related to resveratrol and so it is logical that some researchers thought that it might have similar potential for combating bacteria.
Pterostilbene is a methoxylated variant of resveratrol that is found primarily in blueberries and as we have discussed elsewhere on this website, it has anticancer, anti-inflammatory and neuroprotective properties(6). There have also been some studies suggesting pterostilbene improves the anti-MRSA potential of oxacillin and could be effective against the biofilm of Candida albicans, an opportunistic fungus (or form of yeast) that is the cause of Candida(7-8).
There have been some other reports that suggest pterostilbene could be an antibacterial compound, however there is little in the way of data for in vivo efficacy or the mechanism of action.
A new study set out to evaluate resveratrol and pterostilbene for its potential to combat planktonic, biofilm and intracellular bacteria as well as their potential for treating skin infections(9).
Comparing the antibacterial activity of Resveratrol and Pterostilbene
The researchers created a panel of MRSA, Pseudomonas aeruginosa (P. aeruginosa), and other drug-resistant types to test the antimicrobial activity of both compounds. The research team then tested resveratrol and pterostilbene for their antibacterial activity against this panel.
Compared to resveratrol, pterostilbene showed potent inhibitory and antimicrobial activity against Gram-positive bacteria. Gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to classify bacteria into two broad categories according to their cell walls. The stain will either stain the cells a purple colour (for positive) or pink colour (for negative). Gram-positive bacteria have a thick cell wall made from a protein called peptidoglycan. These bacteria retain the crystal violet dye (one of the 2 main chemicals used for gram staining).
The minimum inhibitory concentration (MIC) of pterostilbene was 8~16-fold lower than that of resveratrol against drug-resistant S. aureus meaning it is more potent at lower doses. The minimum bactericidal concentration (MBC) was a significant 128-fold lower than resveratrol, meaning pterostilbene is more potent at killing bacteria than resveratrol by a significant margin.
Next they tested both compounds for their ability to suppress the growth of P. aeruginosa, a Gram-negative bacteria. Resveratrol and pterostilbene were found to be around the same in their ability to inhibit growth. In live MRSA strain ATCC 33591 neither compound affected MRSA viability at a concentration of 0.01 mM but significantly reduced growth at higher concentrations compared to the control. The inhibition zone had a diameter of 6.9 mm for resveratrol and 10.3 mm for pterostilbene when measured by the researchers. There was no significant difference in the inhibition zones between the two compounds for P. aeruginosa.
Finally, the researchers looked at protein reduction in bacteria exposed to the two compounds. Protein reduction measures the stress response in bacteria treated with antibiotics. The total protein amount in MRSA was examined after exposure to the compounds. Resveratrol and pterostilbene caused a 51% and 56% decrease in total protein compared to the control sample respectively. This makes pterostilbene marginally more effective in this respect.
Antibacterial activity against biofilm and intracellular MRSA
The control sample of MRSA had a smooth and bright appearance on the cellular surface. When the bacteria were treated with pterostilbene, the cellular surface became lost its structure and became empty, suggesting the loss of cytoplasmic contents. The researchers observed that the typical grainy appearance of intact bacterial cells was lost after exposure to pterostilbene and suggest osmotic disturbance caused damage to the MRSA cells.
As we discussed earlier, the biofilm is how bacterial communities protect themselves and how MRSA can resist antibiotics and be so virulent. Pterostilbene was effective against MRSA biofilm formation and there was a decrease of biomass and thickness (from 18 to 10 μm) upon exposure to it. The MRSA death rate was significantly increased in the biofilm upon exposure to pterostilbene showing it had penetrated the biofilm. MRSA is difficult to eliminate with traditional antibiotics because of the intracellular persistence.
Macrophages are one of the most important immune cells responsible for tissue repair and are some of the first immune cells to respond to bacterial invasion. The researchers investigated if pterostilbene could be ingested by macrophages for the intracellular destruction of MRSA.
THP-1 cells, an immortalized monocyte-like cell line were infected with MRSA and then treated with pterostilbene. The result was a dramatic reduction of live MRSA within the cell, survival of MRSA feel around 75%. The treatment also had no cytotoxic effect on the THP-1 cells. The researchers note that there was no significant difference in the level of bacterial inhibition from pterostilbene on differing amounts of MRSA burden, this means that it was effective even when there are high levels of MRSA bacteria.
Cutaneous Absorption Test
The researchers next tested the absorption of resveratrol and pterostilbene. The penetration of pterostilbene into the skin was 6-fold greater than the that of resveratrol. They also investigated the possible anti-MRSA effect of both compounds following topical application, and so calculated the therapeutic index (TI) of both. The TI of pterostilbene was higher than resveratrol by an 8~9-fold margin with the expectation of it being an effective bactericide.
Finally to determine the therapeutic efficacy of pterostilbene, mice were infected with MRSA followed by topical application of pterostilbene. In the control group of mice the abscess grew worse as a result of infection following but in the group treated with pterostilbene this increase was significantly lessened. After seven days post infection the mice were euthanized and the MRSA burden in the skin was determined.
Pterostilbene was effective at inhibiting MRSA growth. They then examined the skin structure using staining. The control mice showed severe damage to the epidermis, degeneration of the dermis and the infiltration of inflammatory cells. The MRSA burden was mixed with immune cells in dermis and subcutis which indicates a deep level of inflammation. In the skin of mice treated with pterostilbene the skin structure was essentially normal with immune cell infiltration restricted beyond the areas treated with pterostilbene.
Cytotoxicity of Resveratrol and Pterostilbene
One of the concerns of using antibacterial drugs is their potential cytotoxic effect on the host’s own cells. So any antibacterial therapy must not be cytotoxic and thus safe to use without risk of harm. Resveratrol and pterostilbene at 0.01 mM were found to be non-toxic when keratinocytes (a type of skin cell) were exposed to them. At a significantly higher dose of 0.05 mM cell viability was observed for pterostilbene at 86% and resveratrol was 75%. The researchers were able to maintain cell viability greater than 80% even up to 0.125 mM.
The researchers also tested neutrophils for viability by testing LDH release associated with membrane damage. Using an LDH assay they determined that both resveratrol and pterostilbene at concentrations of 0.01~0.125 mM can maintain cell viability 80% or greater. There was no significant LDH leakage from cells in the control or compound treated groups.There was no significant difference of LDH leakage between the control and the compound-treated groups.
Skin irritation test
Finally the researchers tested pterostilbene to see if it was an irritant on nude mice. After topical application of pterostilbene a slight redness of the skin was observed, caused by increased blood flow in superficial capillaries. They then checked irritation further using a microscope and observed that the skin showed a comparative structure to the skin of untreated control mice. This suggests that topically the application of pterostilbene is not a major irritant and any irritation is trivial.
Conclusion
Pterostilbene is superior to resveratrol as an antibacterial agent, furthermore it is safe and well tolerated and holds great potential for treating MRSA and other bacterial infections.
References
(1) Hoyert, D. L., & Xu, J. (2012). Deaths: preliminary data for 2011. Natl Vital Stat Rep, 61(6), 1-51.
(2) Nelson, R. E., Slayton, R. B., Stevens, V. W., Jones, M. M., Khader, K., Rubin, M. A., … & Samore, M. H. (2017). Attributable Mortality of Healthcare-Associated Infections Due to Multidrug-Resistant Gram-Negative Bacteria and Methicillin-Resistant Staphylococcus Aureus. Infection Control & Hospital Epidemiology, 1-9.
(3) Water, J. J., Smart, S., Franzyk, H., Foged, C., & Nielsen, H. M. (2015). Nanoparticle-mediated delivery of the antimicrobial peptide plectasin against Staphylococcus aureus in infected epithelial cells. European Journal of Pharmaceutics and Biopharmaceutics, 92, 65-73.
(4) Chung, P. Y., & Toh, Y. S. (2014). Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus aureus. Pathogens and disease, 70(3), 231-239.
(5) Park, S., Cha, S. H., Cho, I., Park, S., Park, Y., Cho, S., & Park, Y. (2016). Antibacterial nanocarriers of resveratrol with gold and silver nanoparticles. Materials Science and Engineering: C, 58, 1160-1169.
(6) Pterostilbene is a methoxylated form of resveratrol that primarily exists in blueberries
(7) Ishak, S. F., Ghazali, A. R., Zin, N. M., & Basri, D. F. (2016). Pterostilbene enhanced anti-methicillin resistant Staphylococcus aureus (MRSA) activity of oxacillin. Am. J. Infect. Dis, 1, 1-10.
(8) Li, D. D., Zhao, L. X., Mylonakis, E., Hu, G. H., Zou, Y., Huang, T. K., … & Jiang, Y. Y. (2014). In vitro and in vivo activities of pterostilbene against Candida albicans biofilms. Antimicrobial agents and chemotherapy, 58(4), 2344-2355.
(9) Yang, S. C., Tseng, C. H., Wang, P. W., Lu, P. L., Weng, Y. H., Yen, F. L., & Fang, J. Y. (2017). Pterostilbene, a methoxylated resveratrol derivative, efficiently eradicates planktonic, biofilm, and intracellular MRSA by topical application. Frontiers in microbiology, 8.