Effects of resveratrol on the growth and enzyme production of Stenotrophomonas maltophilia: a burn wound pathogen

The importance of natural products in curing various diseases is increasing day by day. The polyphenols of grape seeds (Vitis vinifera) have been recognised for their beneficial role in human health.^1,2 The grape seed has been shown to exhibit anti-oxidant, anti-inflammatory, antibacterial, anticancer, antiviral, cardioprotective, hepatoprotective, neuroprotective, anti-aging and antidiabetic properties.^3,4,5,6,7 Resveratrol (3,4ʹ,5-trihydroxy-trans-stilbene), found in grape peel and skin, is a stilbenoid, a type of natural phenol and a phytoalexin, has been found to promote antimicrobial activity against burn wound pathogens.^8,9

In India, around one million people per annum suffer from burn injuries. Burn wounds are most common in women and children. Burns are the 11th leading cause of death of children aged 1–9 years and are the fifth most common non-fatal childhood injury according to WHO reports.¹⁰ Burn wound infection is a major contribution to the clinical pathology of the condition.^{11,12,13,14,15,16}Stenotrophomonas maltophilia is an organism frequently isolated from burn wounds and has developed multi-drug resistance.^16,17,18 In addition, Stenotrophomonas maltophilia produces hyaluronidase and protease.^19,20,21 These enzymes are known to worsen the wound healing process by degrading the host tissue and infiltrating the vasculature.^21,22 This study aims to explore the antimicrobial properties of resveratrol against Stenotrophomonas maltophilia and its modifying effect in regulating the expression of these two enzymes.

Review of the literature

Burn wounds

Burn wounds remain a huge public health issue, especially in developing countries.^23,24 The survival rates depend on the extent of skin damage incurred by a patient. Burn wounds support polymicrobial communities. Delaying the commencement of appropriate wound management significantly increases the risk of wound infection. Around 80 percent of the population with burn wounds develop wound infections after 48–72 hours.^12,16 Infection causes serious consequences, such as septicaemia and organ failure, that influence morbidity and mortality.^13,16,25 This is because injury to the skin causes the release of various inflammatory mediators such as cytokines, prostaglandins, leukotrienes and vasoactive prostanoids, and can cause damage to the surrounding tissues.^26,27 These accumulated factors then enter the systemic circulation. This leads to decreased chemotaxis of the neutrophils, phagocytic and bactericidal activity, lymphokine production by macrophages, T helper cell production and increased T suppressor cell production.²⁶ In addition, damage in the skin barrier, which is rich in nutrients, causes coagulation of host and microbial proteins and this, combined with avascularity, leads to colonisation of microbes within the wounded areas.^23,26,27 The increased bacterial burden on the surface and in the wound increases the metabolic requirements of the wound and of the host's response to it; it affects tissue oxygen availability as it competes with the host cells for oxygen and nutrients.²⁷ The microbes invade the host tissue by producing enzymes such as hyaluronidase, collagenase, gelatinase, caseinase, alkaline proteases and toxins which degrade the host tissues and vasculature. These enzymes cause destruction of tissue fibres and cell membrane, leading to infiltration of the pathogens into the systemic circulation, thereby effectively evoking host inflammatory mediators such as monocytes, macrophages and leukocytes, producing damaging free oxygen radicals and worsening and delaying wound healing mechanisms.^23,27

Stages of wound healing

The stages of wound healing include:^28,29,30

• Inflammation: This lasts for about 24–48 hours and is characterised by haemostasis initially, which forms an ischaemic environment consisting of fibrin clots and abundant neutrophils and platelets. Significant vasodilation is also observed.
• Cell proliferation and repair of the matrix: This lasts for about 5–10 days after injury and a scar is observed. Old cells are removed and replaced with newer cells. Angiogenesis is also observed.
• Remodelling: This stage lasts for a year or longer. During this, most of the repair processes have ceased and apoptosis of endothelial cells, macrophages and myofibroblasts takes place. Type III collagen is actively converted to type I collagen and epithelial–mesenchymal interactions maintain skin integrity.

Stenotrophomonas maltophilia

Stenotrophomonas maltophilia, a burn wound pathogen previously known as Pseudomonas maltophilia and Xanthomonas maltophilia is a Gram-negative, aerobic, motile bacterium, and the majority of strains are multi-drug resistant to the currently available broad-spectrum antibiotics.^19,31,32,33Stenotrophomonas maltophilia also produces a wide range of extracellular enzymes, including DNase, RNase, fibrinolysin, lipases, hyaluronidase, protease and elastase.^{15,16,17,20,32} Production of enzymes and motility are some of the factors contributing to the pathogenicity of the organism.²² Patients with burn wounds infected with Aspergillus, Scedosporium and Mucor species were most commonly co-infected with Stenotrophomonas maltophilia, contributing about 21.1% of the hospital-acquired burn wound infection according to Centre for Disease Control and Prevention, as reported in a single-centred case study for a period of four years.^34,35 Overall, the mortality rate among hospitalised patients with burn wounds was about 30.7%, the major contribution being bacteraemia due to Stenotrophomonas maltophilia infection. Hospital-acquired Stenotrophomonas maltophilia infections contribute about 37.5% to the mortality according to a study in Europe in 2017.³⁴ Treatment of Stenotrophomonas maltophilia infections becomes difficult as the organism is resistant to beta-lactam antibiotics, aminoglycosides and cephalosporins. Currently, Stenotrophomonas maltophilia infections are treated using trimethoprim-sulfamethoxazole if the organism is susceptible to it. Resistance is conferred due to the impermeability of the membranes towards antimicrobials. The resistance to beta-lactam antibiotics is due to the production of beta-lactamase—a zinc-containing penicillinase or a cephalosporinase. Some strains are susceptible to a combination of ticarcillin and a beta-lactamase inhibitor, clavulanic acid.³⁶

Resveratrol

Resveratrol (3,4ʹ,5-trihydroxy-trans-stilbene) is a natural phytoalexin stilbene present in grapes, wine and other natural products. It is a polyphenol biphenyl and its multiple hydroxyl groups contribute to its bioactivities. It is a compound well known for its antimicrobial, anti-inflammatory, anti-oxidant and immunomodulatory properties relevant for healing burn wounds. It modulates lipid synthesis and metabolism, confers neural and cardiovascular protection, and inhibits platelet aggregation and thrombosis, resulting in protection from atherosclerosis. It can also protect against cancer, diabetes, obesity and ageing. It also induces the expression of the Fas–Fas ligand system, mitogen-activated protein kinase (MAPK) pathway and the expression of p53.^{5,6,7,37,38,39,40}

In India, medicines of herbal origin are popular and widely used for many diseases. Hence, this article aims to find out the antibacterial activity of resveratrol on Stenotrophomonas maltophilia and on the regulation of two enzymes: hyaluronidase and protease produced by the organism.

Methods

Stenotrophomonas maltophilia (ATCC 17666) was grown in nutrient broth (HiMedia, India) and the concentration of cells was standardised to 0.5 McFarland standard using a spectrophotometer as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Resveratrol (Sigma Aldrich, India) was dissolved in 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich, India) and 1mg/ml stock solution was prepared. Mueller–Hinton agar (HiMedia, India) and tetrazolium chloride (TTC) (Sigma Aldrich, India) were used for well diffusion assay. Casein agar, skimmed milk agar and Mueller–Hinton agar containing 0.04% hyaluronic acid (HiMedia, India) were used for qualitative assays to check production of protease and hyaluronidase enzymes, respectively. The dye used for microdilution was resazurin (Sigma Aldrich, India). The quantity of hyaluronidase was identified with hyaluronic acid (HiMedia, India). Protease estimation was performed using bovine serum albumin (BSA) (Sigma Aldrich, India) as standard. All the other chemicals used were of analytical grade. All experiments were performed in triplicate.

Anti-microbial assays

Well diffusion assay: To 100mL of Mueller–Hinton agar, 750µL of tetrazolium chloride (TTC) and 1500µL of fresh Stenotrophomonas maltophilia culture was added. The agar was poured and the wells were punched into the agar. The concentrations of resveratrol chosen for this study were 50µg, 100µg, 150µg, 200µg, 500µg, 1mg, 2mg, 3mg and 5mg/ml. Ciprofloxacin (2mg/ml) was used as a positive control for this study. The plates were left for overnight incubation at 37°C and triplicates were maintained.⁴¹

Resazurin dye binding method: This microdilution protocol included resazurin (1mg/ml) as the indicator. Resazurin works on the REDOX principle. The blue compound turns red when there is growth of bacteria as the growth of the organism causes the reduction of the dye from resazurin to resorufin. Hence, it is used to analyse bacterial growth.⁴²

Resveratrol had a stock concentration of 1mg/ml. To all the wells, 100µL of uninoculated broth was added and 100µL of 250µg/ml resveratrol was serially diluted in the ratio of 1:2. A volume of 10µL of the inoculum diluted in the ratio of 1:100 was added to all the wells and the plate was incubated overnight. After overnight incubation, 30µL of 0.01% resazurin was added to all wells and results were documented after 1 hour of incubation. Positive control consisted of only the inoculum and uninoculated broth and negative control consisted only of the uninoculated broth. The drug was not added to either the positive or negative control. The minimum inhibitory concentration (MIC) was determined by identifying the well loaded with the least concentration of resveratrol that did not show a visible colour change due to the reduction of the indicator.⁴²

The microdilution protocol was carried out with the standard drugs silver sulfadiazine (SS), cotrimoxazole (CT) and gentamicin (G) and the MICs were recorded.

Hyaluronidase expression regulation assay

Hyaluronic acid hydrolysis assay: ⁴³ Hyaluronidase activity of Stenotrophomonas maltophilia was tested by the well diffusion method. To 50mL of Mueller–Hinton agar, 1mL of 0.04% hyaluronic acid was added. Wells were punched and to these the crude extract of the cell suspension, treated and untreated with resveratrol, were added. The plates were incubated at 37°C overnight. A zone of clearance around the well was considered a positive reaction.⁴⁴

Turbidity assay: The test tubes were marked as B (blank), EBT (enzyme blank untreated sample), EBRT (enzyme blank treated sample), SB (substrate blank), T1–T6 (untreated samples) and RT1–RT6 (treated samples). 1mL of the substrate was added (0.25mL of 0.04% hyaluronic acid, 0.5mL of distilled water, 0.25mL of acidified BSA fraction V (1% w/v in 0.5M sodium acetate buffer pH 3.1)). To this, 0.5mL of the supernatant was added and was mixed and incubated at 37°C for 30 minutes. The reaction was stopped by cooling the test tubes in an ice bath. To this mixture, 0.1mL of 2N acetic acid was added. The readings were taken at 600nm spectrophotometrically. Acetic acid was added to precipitate the remaining hyaluronic acid thereby making the solution turbid.⁴⁴

Protease expression regulation assay

Casein hydrolysis assay: Protease activity of Stenotrophomonas maltophilia was tested by inoculating the organisms in nutrient agar containing 0.3% skimmed milk (obtained commercially) and casein agar plates.²¹ The plates were incubated at 37°C for one day. A transparent halo around the inoculated culture was considered a positive reaction. Bacillus spp was used as a positive control.⁴⁵

Determination of specific activity of protease using tyrosine standard:⁴⁶

Protein estimation was performed using Lowry's reagent with BSA as standard. The test tubes were marked B (Blank), S1–S5 for standards with concentrations of 150µg, 300µg, 450µg, 600µg and 750µg/ml of BSA (1mg/mL), respectively, T1–T6 for untreated samples and RT1–RT6 for resveratrol-treated samples. A 20µL aliquot of the sample was added to each of T1–T6 and RT1–RT6 and the volume in all the test tubes was made up to 1mL using distilled water. Tyrosine standard was performed and the amount of tyrosine liberated in the sample was calculated from the standard graph prepared using tyrosine.⁴⁶ One unit of enzyme activity is defined as the amount of enzyme required to release 1µM of tyrosine equivalence under standard conditions.

Results

Antimicrobial assays

Well diffusion assay: There was no evident clearance zone observed around the wells with resveratrol at concentrations from 50–500µg/ml. Resveratrol at concentrations of 1mg, 2mg, 3mg and 5mg/ml was found to be active against Stenotrophomonas maltophilia. Ciprofloxacin showed antimicrobial activity from a concentration of 50µg/ml. The zone of inhibition by ciprofloxacin observed at concentrations of 50µg, 100µg, 150µg and 200µg/ml against Stenotrophomonas maltophilia was measured as 1.2, 1.7, 1.8 and 2.2cm diameter, respectively. No zone of inhibition was observed when tested against 500µg/ml of resveratrol; a 0.7cm zone of inhibition was observed at a 1mg concentration of resveratrol. An increasing zone of inhibition with diameters 0.8cm and 1.1cm, respectively, was observed at 2 and 3mg/ml concentrations of resveratrol. The maximum diameter of 1.6cm was observed when tested with a 5mg/ml concentration of resveratrol.

Resazurin dye binding method: The MIC of resveratrol against Stenotrophomonas maltophilia was found to be 125±1.5µg/ml. Comparative studies were performed with silver sulfadiazine, cotrimoxazole, and gentamicin and it was found that these drugs had MICs of 31.25±1.75µg, 15.625±1.3µg, and 15.625±2.5µg/ml, respectively. The values represent mean with standard deviation of six replicates.

Hyaluronidase expression regulation assay

Hyaluronic acid hydrolysis assay: In the qualitative analysis, Stenotrophomonas maltophilia showed a zone of clearance in the hyaluronic acid containing media indicating the production of hyaluronidase.

Turbidity assay: In the quantitative analysis, hyaluronidase production was found to have halved in the samples treated with resveratrol compared to the untreated samples.

Protease expression regulation assay

Casein hydrolysis assay: In the qualitative analysis, Stenotrophomonas maltophilia showed a zone of clearance indicating the production of protease.

Determination of specific activity of protease: The total protein content of the crude extract from the bacterial population under study was estimated. The protein content present in the samples treated with resveratrol was less than the protein content present in the samples not treated with resveratrol (150µg versus 360µg). The specific activity of protease was found to have fallen to a quarter in the samples treated with resveratrol compared to the untreated samples. The specific activity of protease in the untreated samples was found to be 300U/mg and the specific activity of protease in the treated samples was found to be 75U/mg.

Discussion

Burn wound infections are one of the most common and traumatic forms of injury that lead to morbidity. The goal for every physician is to prevent the onset of infections but this is an enormous task. Stenotrophomonas maltophilia, an increasingly recognised nosocomial burn wound pathogen, is multi-drug resistant to the currently available broad-spectrum antibiotics.^20,31,32,33Stenotrophomonas maltophilia was chosen due to its detrimental effects and increased resistance towards already-existing drugs, such as ticarcillin and a beta-lactamase inhibitor, clavulanic acid.³⁶ Increased prevalence of multi-drug resistance is conferred by the ability of the organism to produce beta lactamases that directly inhibit the activity of even third-generation antibiotics, such as cephalosporin and its subtypes. This leads to ineffective therapy and there is a growing health concern with respect to treating wound infections.⁴⁷ There is a need to find alternative medications, able to cure Stenotrophomonas maltophilia infections. Resveratrol may be a drug able to treat Stenotrophomonas maltophilia infections. Moreover, enzymes, such as protease and hyaluronidase, are required during the initial stages of inflammation for sloughing off damaged tissues. This aids wound repair, but excessive production during the later stages of wound healing causes damage to surrounding tissues, leading to infiltration of the microorganisms into the host tissues and vasculature and giving rise to systemic infections.²²

Resveratrol is found in several natural products and it is well-known for eliciting various activities that affect the biological system.^{5,6,7,37,38,39,40} In addition to the antimicrobial and enzyme regulatory properties of resveratrol, it also shows anti-oxidant and anti-inflammatory properties.⁴⁸ Hence, this study has a greater scope clinically as resveratrol may be an ideal drug to treat burn wound infections. Standards, such as the MIC, are crucial in identifying whether the microbe is susceptible (S), intermediate (I), or resistant (R). These values are then used for pharmacokinetic or pharmacodynamic analysis, drug approval trials and post-marketing studies.⁴⁷ The findings of our research suggest that 125µg/ml is the minimum concentration of resveratrol required to inhibit the growth of Stenotrophomonas maltophilia and that there is a halving in the amount of hyaluronidase and a reduction to a quarter in the amount of protease in resveratrol-treated samples. Other studies have reported antimicrobial activity of resveratrol against Stenotrophomonas maltophilia.^37,49 ß-nitrostyrene, a modified compound of E-resveratrol, shows a varied MIC ranging between 6.25–100µg/ml.^50,51 The enzymes produced by the bacteria are said to contribute to the loss of host tissue, causing impairment in host defence mechanisms. Resveratrol is understood to disrupt membrane potential and inhibit DNA synthesis in the bacterium thereby killing it. The genes involved in cell division and ATP-binding cassette (ABC) transporters were found to be down-regulated in resveratrol-treated microbes.⁵² Another possible mechanism is the inhibition of the filamentous temperature-sensitive protein (FtsZ) which is a protein responsible for cell division. This inhibition may result in halting the cellular division and growth of the bacterium.⁵³

Limitations

The current study did not explore gene expression related to enzyme production in order to elucidate the antibacterial effect of resveratrol further.

Conclusion

Based on the resazurin dye binding assay and the enzyme activity assay, using resveratrol in Stenotrophomonas maltophilia-infected burn wounds may reduce the microbial load and enhance healing through mechanisms preventing tissue damage due to bacterial enzymes. Molecular studies, such as quantitative polymerase chain reaction (qPCR), to analyse the expression of genes involved in bacterial enzyme production would help us better understand and confirm the regulatory effects of resveratrol in enzyme production.

Reflective questions

• Are Stenotrophomonas maltophilia infections common? Have you heard about this organism before?
• What is the implication of multidrug resistance on drug development? Can resistance be overcome in the future?
• Would the usage of resveratrol reduce financial burden by reducing costs as compared to normal burn wound drugs?