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A non-thermal plasma (NTP) is a promising tool against the development of bacterial, viral, and fungal diseases. The recently revealed development of microbial resistance to traditional drugs has increased interest in the use of NTPs. We have studied and compared the physical and microbicidal properties of two types of NTP sources based on a cometary discharge in the point-to-point electrode configuration and a corona discharge in the point-to-ring electrode configuration. The electrical and emission properties of both discharges are reported. The microbicidal effect of NTP sources was tested on three strains of the bacterium Staphylococcus aureus (including the methicillin-resistant strain), the bacterium Pseudomonas aeruginosa, the yeast Candida albicans, and the micromycete Trichophyton interdigitale. In general, the cometary discharge is a less stable source of NTP and mostly forms smaller but more rapidly emerging inhibition zones on agar plates. Due to the point-to-ring electrode configuration, the second type of discharge has higher stability and provides larger affected but often not completely inhibited zones. However, after 60 min of exposure, the NTP sources based on the cometary and point-to-ring discharges showed a similar microbicidal effect for bacteria and an individual effect for microscopic fungi.
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Candida albicans has several virulence factors at its disposal, including yeast-hyphal transition associated with biofilm formation, phospholipases, proteases and hemolytic activity, all of which contribute to its pathogenesis. We used synthetic derivative LL-III/43 of antimicrobial peptide lasioglossin LL-III to enhance effect of azoles on attenuation of C. albicans virulence factors. LL-III/43 was able to inhibit initial adhesion or biofilm formation of C. albicans strains at 50 µM. Azoles, however, were ineffective at this concentration. Using fluorescently labeled LL-III/43, we observed that peptide covered C. albicans cells, partially penetrated through their membranes and then accumulated inside cells. LL-III/43 (25 µM) in combination with clotrimazole prevented biofilm formation already at 3.1 µM clotrimazole. Neither LL-III/43 nor azoles were able to significantly inhibit phospholipases, proteases, or hemolytic activity of C. albicans. LL-III/43 (25 µM) and clotrimazole (50 µM) in combination decreased production of these virulence factors, and it completely attenuated its hemolytic activity. Scanning electron microscopy showed that LL-III/43 (50 µM) prevented C. albicans biofilm formation on Ti-6Al-4 V alloy used in orthopedic surgeries and combination of LL-III/43 (25 µM) with clotrimazole (3.1 µM) prevented biofilm formation on urinary catheters. Therefore, mixture of LL-III/43 and clotrimazole is suitable candidate for future pharmaceutical research.
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Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Azóis/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Fosfolipases/antagonistas & inibidores , Peptídeos Catiônicos Antimicrobianos/síntese química , Biofilmes/crescimento & desenvolvimento , Eritrócitos/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Fatores de VirulênciaRESUMO
Non-thermal plasma (NTP), generated at atmospheric pressure by DC cometary discharge with a metallic grid, and antibiotics (gentamicin-GTM, ceftazidime-CFZ and polymyxin B-PMB), either alone or in combination, were used to eradicate the mature biofilm of Pseudomonas aeruginosa formed on Ti-6Al-4V alloy. Our aim was to find the conditions for NTP pre-treatment capable of enhancing the action of the antibiotics and thus reducing their effective concentrations. The NTP treatment increased the efficacy of relatively low concentrations of antibiotics. Generally, the highest effect was achieved with GTM, which was able to suppress the metabolic activity of pre-formed P. aeruginosa biofilms in the concentration range of 4-9 mg/L by up to 99%. In addition, an apparent decrease of biofilm-covered area was confirmed after combined NTP treatment and GTM action by SYTO®13 staining using fluorescence microscopy. Scanning electron microscopy confirmed a complete eradication of P. aeruginosa ATCC 15442 mature biofilm from Ti-6Al-4V alloy when using 0.25 h NTP treatment and subsequent treatment by 8.5 mg/L GTM. Therefore, NTP may be used as a suitable antibiofilm agent in combination with antibiotics for the treatment of biofilm-associated infections caused by this pathogen.
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Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Ligas , Pressão Atmosférica , Ceftazidima/farmacologia , Gentamicinas/farmacologia , Microscopia Eletrônica de Varredura , Gases em Plasma , Polimixina B/farmacologia , Pseudomonas aeruginosa/metabolismo , Titânio/químicaRESUMO
Candida albicans is the main causative agent of vulvovaginal candidiasis (VVC), a common mycosis in women, relapses of which are difficult to manage due to biofilm formation. This study aimed at developing novel non-toxic compounds active against Candida spp. biofilms. We synthesised analogues of natural antifungal peptides LL-III (LL-III/43) and HAL-2 (peptide VIII) originally isolated from bee venoms and elucidated their structures by nuclear magnetic resonance spectroscopy. The haemolytic, cytotoxic, antifungal and anti-biofilm activities of LL-III/43 and peptide VIII were then tested. LL-III/43 and VIII showed moderate cytotoxicity to HUVEC-2 cells and had comparable inhibitory activity against C. albicans and non-albicans spp. The lowest minimum inhibitory concentration (MIC90) of LL-III/43 was observed towards Candida tropicalis (0.8 µM). That was 8-fold lower than that of antimycotic amphotericin B. Both peptides can be used to inhibit Candida spp. bio film f ormation. Biofilm inhibitory concentrations (BIC50) ranged from 0.9 to 58.6 µM and biofilm eradication concentrations (BEC50) for almost all tested Candida spp. strains ranged from 12.8 to 200 µM. Als o pro ven were the peptides' abilities to reduce the area colonised by biofilms , inhibit hyphae formation and permeabilise cell membranes in biofil ms . LL-III/43 and VIII are promising candidates for further development as therapeutics against VVC.
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Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/química , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Anfotericina B/farmacologia , Antifúngicos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Candidíase Vulvovaginal/microbiologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Hifas/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
In recent decades, the non-thermal plasma, i.e. partially or completely ionized gas produced by electric discharges at ambient temperature, has become of interest for its microbiocidal properties with potential of use in the food industry or medicine. Recently, this interest focuses not only on the planktonic forms of microorganisms but also on their biofilms. The works in this interdisciplinary field are summarized in this review. The wide range of biofilm-plasma interactions is divided into studies of general plasma action on bacteria, on biofilm and on its oral and dental application; a short overview of plasma instrumentation is also included. In addition, not only biofilm combating but also an important area of biofilm prevention is discussed. Various DC discharges of the point-to-plane type. Author's photograph, published in Khun et al. (Plasma Sources Sci Technol 27:065002, 2018).
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Biofilmes/efeitos da radiação , Gases em Plasma/farmacologia , Bactérias/crescimento & desenvolvimento , Bactérias/efeitos da radiação , Biofilmes/crescimento & desenvolvimento , Materiais Dentários/farmacologia , Desinfecção/instrumentação , Desinfecção/métodos , Indústria Alimentícia , Fungos/efeitos da radiação , CicatrizaçãoRESUMO
The paper reports a low-cost handheld source of a cold air plasma intended for biomedical applications that can be made by anyone (detailed technical information and a step-by-step guide for creating the NTP source are provided). The plasma source employs a 1.4 W corona discharge in the needle-to-cone electrode configuration and is an extremely simple device, consisting basically of two electrodes and a cheap power supply. To achieve the best bactericidal effect, the plasma source has been optimized on Escherichia coli. The bactericidal ability of the plasma source was further tested on a wide range of microorganisms: Staphylococcus aureus as a representative of gram-positive bacteria, Pseudomonas aeruginosa as gram-negative bacteria, Candida albicans as yeasts, Trichophyton interdigitale as microfungi, and Deinococcus radiodurans as a representative of extremophilic bacteria resistant to many DNA-damaging agents, including ultraviolet and ionizing radiation. The testing showed that the plasma source inactivates all the microorganisms tested in several minutes (up to 105-107 CFU depending on a microorganism), proving its effectiveness against a wide spectrum of pathogens, in particular microfungi, yeasts, gram-positive and gram-negative bacteria. Studies of long-lived reactive species such as ozone, nitrogen oxides, hydrogen peroxide, nitrite, and nitrate revealed a strong correlation between ozone and the bactericidal effect, indicating that the bactericidal effect should generally be attributed to reactive oxygen species. This is the first comprehensive study of the bactericidal effect of a corona discharge in air and the formation of long-lived reactive species by the discharge, depending on both the interelectrode distance and the discharge current.
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Gases em Plasma , Gases em Plasma/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Candida albicans/efeitos dos fármacosRESUMO
Antibiotic resistance (ATBR) is increasing every year as the overuse of antibiotics (ATBs) and the lack of newly emerging antimicrobial agents lead to an efficient pathogen escape from ATBs action. This trend is alarming and the World Health Organization warned in 2021 that ATBR could become the leading cause of death worldwide by 2050. The development of novel ATBs is not fast enough considering the situation, and alternative strategies are therefore urgently required. One such alternative may be the use of non-thermal plasma (NTP), a well-established antimicrobial agent actively used in a growing number of medical fields. Despite its efficiency, NTP alone is not always sufficient to completely eliminate pathogens. However, NTP combined with ATBs is more potent and evidence has been emerging over the last few years proving this is a robust and highly effective strategy to fight resistant pathogens. This minireview summarizes experimental research addressing the potential of the NTP-ATBs combination, particularly for inhibiting planktonic and biofilm growth and treating infections in mouse models caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The published studies highlight this combination as a promising solution to emerging ATBR, and further research is therefore highly desirable.
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Antibacterianos , Biofilmes , Gases em Plasma , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Gases em Plasma/farmacologia , Animais , Humanos , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Camundongos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Farmacorresistência Bacteriana , Resistência Microbiana a Medicamentos , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Modelos Animais de Doenças , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Due to the emerging resistance of microorganisms and viruses to conventional treatments, the importance of self-disinfecting materials is highly increasing. Such materials could be silver or its nanoparticles (AgNPs), both of which have been studied for their antimicrobial effect. In this study, we compared the biological effects of AgNP coatings with and without a plasma-polymerized hexamethyldisiloxane (ppHMDSO) protective film to smooth silver or copper coatings under three ambient conditions that mimic their potential medical use (dry or wet environments and an environment simulating the human body). The coatings were deposited on 3D printed polylactic acid substrates by DC magnetron sputtering, and their surface morphology was visualized using scanning electron microscopy. Cytotoxicity of the samples was evaluated using human lung epithelial cells A549. Furthermore, antibacterial activity was determined against the Gram-negative pathogenic bacterium Pseudomonas aeruginosa PAO1 and antiviral activity was assessed using human rhinovirus species A/type 2. The obtained results showed that overcoating of AgNPs with ppHMDSO creates the material with antibacterial and antiviral activity and at the same time without a cytotoxic effect for the surrounding tissue cells. These findings suggest that the production of 3D printed substrates coated with a layer of AgNPs-ppHMDSO could have potential applications in the medical field as functional materials.
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The emergence of antibiotic resistance in opportunistic pathogens represents a huge problem, the solution for which may be a treatment with a combination of multiple antimicrobial agents. Sodium salt of cobalt bis-dicarbollide (COSAN.Na) is one of the very stable, low-toxic, amphiphilic boron-rich sandwich complex heteroboranes. This compound has a wide range of potential applications in the biological sciences due to its antitumor, anti-HIV-1, antimicrobial and antibiofilm activity. Our study confirmed the ability of COSAN.Na (in the concentration range 0.2-2.48 µg/mL) to enhance tetracycline, erythromycin, and vancomycin action towards Staphylococcus epidermidis planktonic growth with an additive or synergistic effect (e.g., the combination of 1.24 µg/mL COSAN.Na and 6.5 µg/mL TET). The effective inhibitory concentration of antibiotics was reduced up to tenfold most efficiently in the case of tetracycline (from 65 to 6.5 µg/mL). In addition, strong effect of COSAN.Na on disruption of the cell envelopes was determined using propidium iodide uptake measurement and further confirmed by transmission electron microscopy. The combination of amphiphilic COSAN.Na with antibiotics can therefore be considered a promising way to overcome antibiotic resistance in Gram-positive cocci.
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The increasing risk of antibiotic failure in the treatment of Pseudomonas aeruginosa infections is largely related to the production of a wide range of virulence factors. The use of non-thermal plasma (NTP) is a promising alternative to antimicrobial treatment. Nevertheless, there is still a lack of knowledge about the effects of NTP on the virulence factors production. We evaluated the ability of four NTP-affected P. aeruginosa strains to re-form biofilm and produce Las-B elastase, proteases, lipases, haemolysins, gelatinase or pyocyanin. Highly strains-dependent inhibitory activity of NTP against extracellular virulence factors production was observed. Las-B elastase activity was reduced up to 82% after 15-min NTP treatment, protease activity and pyocyanin production by biofilm cells was completely inhibited after 60 min, in contrast to lipases and gelatinase production, which remained unchanged. However, for all strains tested, a notable reduction in biofilm re-development ability was depicted using spinning disc confocal microscopy. In addition, NTP exposure of mature biofilms caused disruption of biofilm cells and their dispersion into the environment, as shown by transmission electron microscopy. This appears to be a key step that could help overcome the high resistance of P. aeruginosa and its eventual elimination, for example in combination with antibiotics still highly effective against planktonic cells.
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Gases em Plasma , Infecções por Pseudomonas , Antibacterianos/farmacologia , Biofilmes , Endopeptidases/farmacologia , Gelatinases/farmacologia , Proteínas Hemolisinas/farmacologia , Humanos , Elastase Pancreática , Peptídeo Hidrolases , Plâncton , Gases em Plasma/farmacologia , Pseudomonas aeruginosa , Piocianina , Percepção de Quorum , Fatores de VirulênciaRESUMO
The current pandemic resulted in a rapidly increasing demand for personal protective equipment (PPE) initially leading to severe shortages of these items. Hence, during an unexpected and fast virus spread, the possibility of reusing highly efficient protective equipment could provide a viable solution for keeping both healthcare professionals and the general public equipped and protected. This requires an efficient decontamination technique that preserves functionality of the sensitive materials used for PPE production. Non-thermal plasma (NTP) is a decontamination technique with documented efficiency against select bacterial and fungal pathogens combined with low damage to exposed materials. We have investigated NTP for decontamination of high-efficiency P3 R filters from viral respiratory pathogens in comparison to other commonly used techniques. We show that NTP treatment completely inactivates SARS-CoV-2 and three other common human respiratory viruses including Influenza A, Rhinovirus and Adenovirus, revealing an efficiency comparable to 90°C dry heat or UVC light. Unlike some of the tested techniques (e.g., autoclaving), NTP neither influenced the filtering efficiency nor the microstructure of the filter. We demonstrate that NTP is a powerful and economic technology for efficient decontamination of protective filters and other sensitive materials from different respiratory pathogens.
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This study evaluated the effect of low-molecular weight chitosan on Staphylococcus epidermidis, a common colonizer of joint implants and other prosthetic devices. We have also attempted to elucidate its mechanism of action. Chitosan was found to be effective against both the planktonic and biofilm cells (MIC80 35-40 mg/L; MBIC80 40-150 mg/L), in contrast to the antibiotics erythromycin and tetracycline with no antibiofilm activity (MBIC80 not found). In combination, chitosan had an additive effect with antibiotics on suspension growth of S. epidermidis (FICi 0.7-1.0), and the combinatory action caused a complete inhibition of biofilm metabolic activity in some cases. In addition, chitosan caused rapid cellular damage and enhanced antihaemolytic activity of tetracycline in combination towards S. epidermidis biofilm cells. Chitosan efficiently inhibited S. epidermidis growth acting via cell membrane damage, yet the extent of antimicrobial and antibiofilm activities was quite strain-specific. It was proved to be a very efficient antimicrobial agent worth further examination as a potent candidate in pharmaceutical research. Apart from antimicrobial activity, it also acted as antivirulence enhancing agent which is a very promising strategy for alternative infectious diseases treatment.
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Anti-Infecciosos , Quitosana , Antibacterianos/farmacologia , Biofilmes , Membrana Celular , Quitosana/farmacologia , Testes de Sensibilidade Microbiana , Peso Molecular , Staphylococcus epidermidisRESUMO
The acronym ESKAPE refers to a group of bacteria consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. They are important in human medicine as pathogens that show increasing resistance to commonly used antibiotics; thus, the search for new effective bactericidal agents is still topical. One of the possible alternatives is the use of non-thermal plasma (NTP), a partially ionized gas with the energy stored particularly in the free electrons, which has antimicrobial and anti-biofilm effects. Its mechanism of action includes the formation of pores in the bacterial membranes; therefore, resistance toward it is not developed. This paper focuses on the current overview of literature describing the use of NTP as a new promising tool against ESKAPE bacteria, both in planktonic and biofilm forms. Thus, it points to the fact that NTP treatment can be used for the decontamination of different types of liquids, medical materials, and devices or even surfaces used in various industries. In summary, the use of diverse experimental setups leads to very different efficiencies in inactivation. However, Gram-positive bacteria appear less susceptible compared to Gram-negative ones, in general.
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The influence of non-thermal plasma (NTP) treatment on the prevention of antibiotic resistance of microbial biofilms was studied. Staphylococcus epidermidis and Escherichia coli bacteria and a yeast Candida albicans, grown on the surface of Ti-6Al-4V alloy used in the manufacture of prosthetic implants, were employed. Their biofilms were exposed to NTP produced by DC cometary discharge and subsequently treated with antibiotics commonly used for the treatment of infections caused by them: erythromycin (ERY), polymyxin B (PMB), or amphotericin B (AMB), respectively. All biofilms displayed significant reduction of their metabolic activity after NTP exposure, the most sensitive was S. epidermidis. The subsequent action of antibiotics caused significant decrease in the metabolic activity of S. epidermidis and E. coli, but not C. albicans, although the area covered by biofilm decreased in all cases. The combined effect of NTP with antibiotics was thus proved to be a promising strategy in bacterial pathogen treatment.
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Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Gases em Plasma/farmacologia , Ligas , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Especificidade da Espécie , Staphylococcus epidermidis/efeitos dos fármacos , TitânioRESUMO
A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.
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PURPOSE: Antibiotic-loaded polymethylmethacrylate-based bone cement has been implemented in orthopaedics to cope with implant-related infections associated with the formation of bacterial biofilms. In the context of emerging bacterial resistance to current antibiotics, we examined the efficacy of short antimicrobial peptide-loaded bone cement in inhibiting bacterial adhesion and consequent biofilm formation on its surface. METHODOLOGY: The ability of α-helical antimicrobial peptides composed of 12 amino acid residues to prevent bacterial biofilm [methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli] formation on the surface of model implants made from polymethylmethacrylate-based bone cement was evaluated by colony-forming unit (c.f.u.) counting of bacteria released by sonication from the biofilms formed on their surfaces. The biofilms on model implant surfaces were also visualized by light microscopy after staining with tetrazolium dye (MTT) and by scanning electron microscopy. RESULTS: When incorporated in the implants, these peptides caused a mean reduction in the number of bacterial cells attached to implants' surfaces (by five orders of magnitude), and 88 % of these implants showed no bacterial adhesion after being exposed to growth media containing various bacteria. CONCLUSION: The results showed that the antibiofilm activity of these peptides was comparable to that of the antibiotics, but the peptides exhibited broader specificity than the antibiotics. Given the rapid development of antibiotic resistance, antimicrobial peptides show promise as a substitute for antibiotics for loading into bone cements.
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Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cimentos Ósseos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Polimetil Metacrilato , Próteses e Implantes/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus epidermidis/crescimento & desenvolvimentoRESUMO
Microorganisms that cause chronic infections exist predominantly as surface-attached stable communities known as biofilms. Microbial cells in biofilms are highly resistant to conventional antibiotics and other forms of antimicrobial treatment; therefore, modern medicine tries to develop new drugs that exhibit anti-biofilm activity. We investigated the influence of a plant polyphenolic compound resveratrol (representative of the stilbene family) on the opportunistic pathogen Trichosporon cutaneum. Besides the influence on the planktonic cells of T. cutaneum, the ability to inhibit biofilm formation and to eradicate mature biofilm was studied. We have tested resveratrol as pure compound, as well as resveratrol in complex plant extract-the commercially available dietary supplement Regrapex-R-forte, which contains the extract of Vitis vinifera grape and extract of Polygonum cuspidatum root. Regrapex-R-forte is rich in stilbenes and other biologically active substances. Light microscopy imaging, confocal microscopy, and crystal violet staining were used to quantify and visualize the biofilm. The metabolic activity of biofilm-forming cells was studied by the tetrazolium salt assay. Amphotericin B had higher activity against planktonic cells; however, resveratrol and Regrapex-R-forte showed anti-biofilm effects, both in inhibition of biofilm formation and in the eradication of mature biofilm. The minimum biofilm eradicating concentration (MBEC80) for Regrapex-R-forte was found to be 2222 mg/L (in which resveratrol concentration is 200 mg/L). These methods demonstrated that Regrapex-R-forte can be employed as an anti-biofilm agent, as it has similar effect as amphotericin B (MBEC80 = 700 mg/L), which is routinely used in clinical practice.