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Rosmarinus officinalis L. (rosemary) extract has antibiofilm effect similar to the antifungal nystatin on Candida samples

Abstract

Candida spp. are naturally opportunistic and can promote infections. These yeasts can form biofilm, after penetration and adhesion to the biotic or abiotic surfaces. Preexisting diseases, treatments with drugs and radiation therapy, medical procedures, and parafunctional habits favor the installation of a fungal infection. Increased resistance to the available antifungals has become a concern. Therefore, alternative methods to control them have been evaluated, including the use of plant substances. In this study, the antibiofilm effect of R. officinalis L. extract was analyzed on C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis. A phytochemical analysis of the extract was performed. Biofilms were formed for 48 h and exposed to the different concentrations of the extract (50, 100, and 200 mg/mL) for 5 min or 24 h. The effect of the plant extract was compared to the antifungal nystatin. Rosmarinus officinalis L. extract was constituted of phenols and flavonoids, highlighting the presence of chlorogenic acid derivatives in its composition. Biofilm reductions were observed after exposure to the plant extract for both periods. The plant extract provided a reduction similar to the antifungal. Thus, R. officinalis L. extract showed antibiofilm effect on Candida spp. comparable to the nystatin.

Key words
Antifungal effect; Biofilms; Candida spp

INTRODUCTION

Biofilms are microecosystems composed of microorganisms involved by an extracellular matrix of own production, being adhered to a surface (Ammons et al. 2014AMMONS MC, TRIPET BP, CARLSON RP, KIRKER KR, GROSS MA, STANISICH JJ & COPIÉ V. 2014. Quantitative NMR metabolite profiling of methicillin-resistant and methicillin- susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes. J Proteome Res 13: 2973-2985., Harriott & Noverr 2009HARRIOTT MM & NOVERR MC. 2009. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob Agents Chemother 53: 3914-3922.).

Candida spp. can form biofilm and germ tubes, have adhesion proteins, as well as phospholipases, proteases, and other extracellular enzymes that contribute to their diffusion through the host tissue, because these enzymes are located at the extremity of hyphae (Gauch et al. 2014GAUCH LMR, GOMES FS, ESTEVES RA, PEDROSA SS, GURGEL ESC & ARRUDA AC. 2014. Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Rev Soc Bras Med Trop 47: 389-391.). These fungi are opportunistic and can cause diseases known as candidiasis, using factors that promote their penetration, diffusion, installation, and development in the host (Soll 2002SOLL DR. 2002. Candida commensalism and virulence: the evolution of phenotypic plasticity. Acta Tropica 81: 101-110.). Some conditions such as acquired immunodeficiency syndrome (AIDS), diabetes, antibiotic therapy, corticoid therapy, chemotherapy, radiotherapy, surgeries, the presence of catheters, and probes may favor the development of candidiasis. About 80% of the hospital fungal infections are caused by Candida spp. Besides, cases of resistance to the antifungal therapy and to the host defenses have been reported (Ramage & López-Ribot 2005RAMAGE G & LÓPEZ-RIBOT JL. 2005. Techniques for antifungal susceptibility testing of Candida albicans biofilms. Methods Mol Med 118: 71-79.).

In the oral mucosa, the presence of Candida spp. is common and related to the adhesion capacity of the yeast to the epithelial cells, considered as an essential factor of virulence to the biofilm formation after microbial adhesion (Thein et al. 2006THEIN ZM, SAMARANAYAKE YH & SAMARANAYAKE LP. 2006. Effect of oral bacteria on growth and survival of Candida albicans biofilms. Arch Oral Biol 51: 672-680.). Oral biofilms are polymicrobial, composed by different species of microorganisms as well as of various Candida species, such as C. parapsilosis, C. tropicalis, C. krusei, C. glabrata and C. guilliermondii (Vidotto et al. 2003VIDOTTO V, MANTOAN B, PUGLIESE A, PONTÓN J, QUINDÓS G, AOKI S & ITO-KUWA S. 2003. Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells. Rev Iberoam Micol 20: 52-54.).

Candida albicans is the primary species responsible for causing oral candidiasis. Its ability of adhesion to the epithelial cells and the possibility of transformation from yeast to hyphae are important factors of virulence of this species (Shapiro et al. 2009SHAPIRO RS, UPPULURI P, ZAAS AK, COLLINS C, SENN H, PERFECT JR, HEITMAN J & COWEN LE. 2009. Hsp90 Orchestrates Temperature-Dependent Candida albicans Morphogenesis Via Ras1-PKA Signaling. Curr Biol 19: 621-629., Silva et al. 2011SILVA S, HENRIQUES M, HAYES A, OLIVEIRA R, AZEREDO J & WILLIAMS DW. 2011. Candida glabrata and Candida albicans co-infection of an in vitro oral epithelium. J Oral Pathol Med 40: 421-427.). This yeast can invade the gingival sulcus and grow in anaerobiosis, which may aggravate the symptoms of existing periodontal disease. Moreover, C. albicans can form a complex microbial community with Streptococcus mutans, facilitating its adhesion to the polymicrobial biofilm (Ten Cate et al. 2009TEN CATE JM, KLIS FM, PEREIRA-CENCI T, CRIELAARD W & DE GROOT PW. 2009. Molecular and cellular mechanisms that lead to Candida biofilm formation. J Dent Res 88: 105-115., Gregoire et al. 2011GREGOIRE S, XIAO J, SILVA BB, GONZALEZ I, AGIDI OS, KLEIN MI & KOO H. 2011. Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces. Appl Environ Microbiol 77: 6357-6367.). Additionally, the cariogenic potential of Candida spp. has been reported (Nikawa et al. 2003NIKAWA H, YAMASHIRO H, MAKIHIRA S, NISHIMURA M, EGUSA H, FURUKAWA M & SETIJANTO D. 2003. In vitro cariogenic potential of Candida albicans. Mycoses 46: 471-478.). The yeast can also inhibit the action of polymorphonuclear leukocytes (Maccarinelli et al. 2001MACCARINELLI G, BELOTTI R, SAVOLDI E, GERVASONI M & COCCHI D. 2001. Phagocytosis and killing of Candida albicans of polymorphonuclear cells in patients with organ transplant of periodontal disease. Minerva Stomatol 50: 345-349.) and produces enzymes that degrade immunoglobulins (Hägewald et al. 2002HÄGEWALD S, BERNIMOULIN JP, KÖTTGEN E & KAGE A. 2002. Salivary IgA subclasses and bacteriareactive IgA in patients with aggressive periodontitis. J Periodontal Res 37: 333-339.).

Candida dubliniensis may be more prevalent in patients infected with human immunodeficiency virus (HIV) (Loreto et al. 2010LORETO ES, SCHEID LA, NOGUEIRA CW, ZENI G, SANTURIO JM & ALVES SH. 2010. Candida dubliniensis: epidemiology and phenotypic methods for identification. Mycopathologia 169: 431-443.). It is an emerging pathogen that shares many phenotypic characteristics with C. albicans. Besides, C. dubliniensis can produce germ tubes and lipolytic enzymes, which are considered important virulence factors. The establishment of the infection by C. dubliniensis is triggered by the release of lipolytic enzymes used for dissemination by the host’s tissue. Adhesion accompanies this fact; synergistic interaction with other hydrolytic enzymes; non-specific activation of inflammatory reaction; and self-defense, mediated by the elimination of the competing microbiota (Park et al. 2013PARK M, DO E & JUNG WH. 2013. Lipolytic enzymes involved in the virulence of human pathogenic fungi. Mycobiology 41: 67-72., Sardi et al. 2013SARDI JC, SCORZONI L, BERNARDI T, FUSCO-ALMEIDA AM & MENDES GIANNINI MJ. 2013. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 62: 10-24., Trofa et al. 2008TROFA D, GÁCSER A & NOSANCHUK JD. 2008. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 21: 606-625.).

Candida glabrata is frequently found in cases of candidemia (Oren & Paul 2014OREN I & PAUL M. Up to date epidemiology, diagnosis and management of invasive fungal infections. 2014. Clin Microbiol Infect 20: 1-4.). This species does not present polymorphism, being only found in yeast form in the oral cavity. In polymicrobial infections, C. glabrata can penetrate the oral epithelium with the aid of C. albicans (Silva et al. 2011SILVA S, HENRIQUES M, HAYES A, OLIVEIRA R, AZEREDO J & WILLIAMS DW. 2011. Candida glabrata and Candida albicans co-infection of an in vitro oral epithelium. J Oral Pathol Med 40: 421-427.). Prolonged exposure to antifungals may increase the probability of infections by C. glabrata and C. krusei. In addition, cases of infections by these yeasts have increased in patients with debilitating conditions (Oren & Paul 2014OREN I & PAUL M. Up to date epidemiology, diagnosis and management of invasive fungal infections. 2014. Clin Microbiol Infect 20: 1-4., Sampaio & Pais 2014SAMPAIO P & PAIS C. 2014. Epidemiology of Invasive Candidiasis and Challenges for the Mycology Laboratory: Specificities of Candida glabrata. Curr Clin Microbiol Rep 1: 1-9.). Both species have shown resistance to the fluconazole and amphotericin B (Shirani et al. 2017SHIRANI M, SAMIMI A, KALANTARI H, MADANI M & KORD ZANGANEH A. 2017. Chemical composition and antifungal effect of hydroalcoholic extract of Allium tripedale (Tvautv.) against Candida species. Curr Med Mycol 3: 6-12.).

Candida tropicalis is usually associated with infections, once presents some characteristics related to its virulent potential, such as high adhesion capacity and biofilm formation (Negri et al. 2010NEGRI M, MARTINS M, HENRIQUES M, SVIDZINSKI TI, AZEREDO J & OLIVEIRA R. 2010. Examination of potential virulence factors of Candida tropicalis clinical isolates from hospitalized patients. Mycopathologia 169: 175-182.). Patients with cancer, infections, neutropenia, or treated by radiotherapy are the most affected by this species. Gamma radiation causes increased virulence of C. tropicalis, since morphological and physiological alterations may occur (Silva et al. 2017SILVA EM, MANSANO ESB, MIAZIMA ES, RODRIGUES FAV, HERNANDES L & SVIDZINSKI TIE. 2017. Radiation used for head and neck cancer increases virulence in Candida tropicalis isolated from a cancer patient. BMC Infect Dis 17: 783.).

Alternative methods to prevent and treat diseases orally transmitted are needed due to: (i) incidence of cases; (ii) increased resistance of microorganisms to the available antimicrobials; (iii) financial conditions; and (iv) harmful effects of some drugs. Phytotherapy medicines could be one of these methods once they can present biocompatibility and efficacy (Palombo 2011PALOMBO EA. 2011. Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 680354.). Studies related to the alternative therapies with plant products have consistently appeared. Substances isolated from medicinal plants, such as extracts, have stimulated the interest of the medical field, since they can be less aggressive to the organism.

Rosmarinus officinalis L. was evaluated in this study due to its significant anti-Candida effect demonstrated on planktonic culture, monomicrobial biofilm, and in interactions with Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa (de Oliveira et al. 2017DE OLIVEIRA JR, DE JESUS D, FIGUEIRA LW, DE OLIVEIRA FE, PACHECO SOARES C, CAMARGO SE, JORGE AO & DE OLIVEIRA LD. 2017. Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells. Exp Biol Med (Maywood) 242: 625-634.). Popularly known as rosemary, R. officinalis (Lamiaceae) is originated from the Mediterranean region but is worldwide distributed. It has been used in the cosmetics industry, culinary, and complementary and alternative medicine, mainly for its aromatic essence and beneficial pharmaceutical effects (Machado et al. 2009MACHADO DG, BETTIO LE, CUNHA MP, CAPRA JC, DALMARCO JB, PIZZOLATTI MG & RODRIGUES AL. 2009. Antidepressant-like effect of the extract of Rosmarinus officinalis in mice: Involvement of the monoaminergic system. Prog Neuropsychopharmacol Biol Psychiatry 33: 642-650., Rašković et al. 2014RAŠKOVIĆ A, MILANOVIĆ I, PAVLOVIĆ N, ĆEBOVIĆ T, VUKMIROVIĆ S & MIKOV M. 2014. Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. BMC Complement Altern Med 14: 225.). Its main constituents, such as 1,8-cineole (52.2%), camphor (15.2%) and α-pinene (12.4%), are the responsible for the several biological activities (Silva et al. 2015SILVA AM, MACHADO ID, SANTIN JR, DE MELO IL, PEDROSA GV, GENOVESE MI, FARSKY SH & MANCINI-FILHO J. 2015. Aqueous extract of Rosmarinus officinalis L. inhibits neutrophil influx and cytokine secretion. Phytother Res 29: 125-133.), including antibacterial, antifungal, antibiofilm, antiproliferative, anti-inflammatory, antimutagenic, and antioxidant effects (Bozin et al. 2007BOZIN B, MIMICA-DUKIC N, SAMOJLIK I & JOVIN E. 2007. Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae) Essential Oils. J Agric Food Chem 55: 7879-7885., Pozzatti et al. 2010POZZATTI P, LORETO ES, NUNES MARIO DA, ROSSATO L, SANTURIO JM & ALVES SH. 2010. Activities of essential oils in the inhibition of Candida albicans and Candida dubliniensis germ tube formation. J Mycologie Médicale 20: 185-189., Chifiriuc et al. 2012CHIFIRIUC C, GRUMEZESCU V, GRUMEZESCU AM, SAVIUC C, LAZĂR V & ANDRONESCU E. 2012. Hybrid magnetite nanoparticles/Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity. Nanoscale Res Lett 7: 209., Rašković et al. 2014RAŠKOVIĆ A, MILANOVIĆ I, PAVLOVIĆ N, ĆEBOVIĆ T, VUKMIROVIĆ S & MIKOV M. 2014. Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. BMC Complement Altern Med 14: 225., Silva et al. 2015SILVA AM, MACHADO ID, SANTIN JR, DE MELO IL, PEDROSA GV, GENOVESE MI, FARSKY SH & MANCINI-FILHO J. 2015. Aqueous extract of Rosmarinus officinalis L. inhibits neutrophil influx and cytokine secretion. Phytother Res 29: 125-133., de Oliveira et al. 2017DE OLIVEIRA JR, DE JESUS D, FIGUEIRA LW, DE OLIVEIRA FE, PACHECO SOARES C, CAMARGO SE, JORGE AO & DE OLIVEIRA LD. 2017. Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells. Exp Biol Med (Maywood) 242: 625-634., Ksouri et al. 2017KSOURI S, DJEBIR S, BENTORKI AA, GOURI A, HADEF Y & BENAKHLA A. 2017. Antifungal activity of essential oils extract from Origanum floribundum Munby, Rosmarinus officinalis L. and Thymus ciliatus Desf. against Candida albicans isolated from bovine clinical mastitis. J Mycol Med 27: 245-249., Liakos et al. 2017LIAKOS JL, HOLBAN AM, CARZINO R, LAUCIELLO S & GRUMEZESCU AM. 2017. Electrospun fiber pads of cellulose acetate and essential oils with antimicrobial activity. Nanomaterials 7: 84.).

In the interaction of R. officinalis L. with fungi, the medicinal plant has caused a destabilization in the cytoplasmic membrane and cell wall of these microorganisms, providing a loss of internal contents of the cell fungal to the medium. This fact contributes to generate dysfunctional cells in the biofilm (da Silva et al. 2015DA SILVA BN, NAKASSUGI LP, FAGGION PINHEIRO OLIVEIRA J, KOHIYAMA CY, MOSSINI SA, GRESPAN R, NERILO SB, MALLMANN CA, ALVES ABREU FILHO B & MACHINSKI M JR. 2015. Antifungal activity and inhibition of fumonisin production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides (Sacc.) Nirenberg. Food Chem 166: 330-336.). Besides, inhibition of germ-tube formation by C. albicans has also been cited. This mechanism impairs its dissemination though organic tissues (Gauch et al. 2014GAUCH LMR, GOMES FS, ESTEVES RA, PEDROSA SS, GURGEL ESC & ARRUDA AC. 2014. Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Rev Soc Bras Med Trop 47: 389-391.). By means of these mechanisms, R. officinalis L. extract can provide an effective control of Candida spp. biofilms.

Thus, the antifungal effect of R. officinalis L. extract was evaluated in this study, both on planktonic cultures and biofilms of C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis, by exposure to the plant extract in different concentrations for 5 min or 24 h, comparing its effects with the antifungal nystatin.

MATERIALS AND METHODS

Plant extract

Rosmarinus officinalisL. extract was commercially purchased at 200 mg/mL in propylene glycol (Seiva Brazilis, São Paulo, Brazil). Plant extract was obtained from leaves and flowers of the vegetable, being chemically composed of acid saponin, borneol acetate, camphene, camphor, cineol, free borneol, glucosidic compounds, little tannin, oleanolic acid and pinene, according to the manufacturer.

Phytochemical analysis

Determination of soluble solids

The plant extract (5 mL) was dried at 80°C until total elimination of liquids. Then, the solid compounds were cooled in a desiccator and the amount of soluble solids from the extract was calculated.

Determination of total phenol

A stock solution of extract diluted in ethanol and distilled water was prepared (1:100). After, 0.2 mL of this solution was added in 5 mL of distilled water. To this new solution, 0.8 mL of Folin-Ciocalteau reagent (Merck, Germany) was added. Posteriorly, 1.2 mL of 20% sodium carbonate-tartrate buffer was added between 1 and 8 min. The solution was kept in a water bath at 20°C for 2 h. Absorbance of this solution was read in a spectrophotometer (760 nm) and the amount of total phenol was determined by the straight equation of gallic acid (Bankova & Marcucci 2000BANKOVA V & MARCUCCI MC. 2000. Standardization of propolis: present status and perspectives. Bee World 81: 182-188.). The procedure was performed in triplicate.

Determination of total flavonoid

A stock solution of extract in methanol was prepared (1:100) and 0.2 mL was added in 5 mL of methanol. To this solution, 0.2 mL of aluminum chloride was added and the volume was completed to 10 mL with methanol. This new solution was kept in a water bath at 20°C for 30 min. Absorbance of this solution was read in a spectrophotometer (425 nm) and the concentration of total flavonoid was determined by the straight equation of quercetin (Bankova & Marcucci 2000BANKOVA V & MARCUCCI MC. 2000. Standardization of propolis: present status and perspectives. Bee World 81: 182-188.). The procedure was performed in triplicate.

Antioxidant activity

Elimination of free radicals by the extract was verified using 2,2-diphenyl-1-picrylhydrazyl (DPPH - Sigma-Aldrich, St. Louis, USA), according to Brand-Williams et al. (1995)BRAND-WILLIAMS W, CUVELIER ME & BERSET C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 28: 25-30., with some modifications. Then, 0.5 mL of DPPH (0.2 mmol/L of methanol) was added in 0.5 mL of extract at different concentrations suspended in methanol. The samples were maintained at room temperature for 30 min, with absence of light. The absorbance of this solution was read in a spectrophotometer (517 nm). The optical density (OD) values were converted to μg/mL and the concentration that eliminated 50% of free radicals (EC50) was determined by non-linear regression (Veiga et al. 2017VEIGA RS, DE MENDONÇA S, MENDES PB, PAULINO N, MIMICA MJ, LAGAREIRO NETTO AA, LIRA IS, LÓPEZ BG, NEGRÃO V & MARCUCCI MC. 2017. Artepillin C and phenolic compounds responsible for antimicrobial and antioxidant activity of green propolis and Baccharis dracunculifolia DC. J Appl Microbiol 122: 911-920.). The procedure was performed in triplicate.

Chromatographic analysis

High-performance Liquid Chromatography (HPLC) was used to characterize and quantify the content of markers in the plant extract. Thus, a chromatograph with a photodiode detector (HPLC-DAD) and an automatic injector (D-7000 Merck-Hitachi) was used. The mobile phase of the chromatography was composed of water-formic acid solution (Merck) diluted in the ratio of 95:5 (solvent A) and methanol HPLC grade (Merck) (solvent B). The flow was 1 mL/min and the linear gradient started with 0% B and ended with 70% B in a running time of 50 min at 280 and 340 nm (Veiga et al. 2017VEIGA RS, DE MENDONÇA S, MENDES PB, PAULINO N, MIMICA MJ, LAGAREIRO NETTO AA, LIRA IS, LÓPEZ BG, NEGRÃO V & MARCUCCI MC. 2017. Artepillin C and phenolic compounds responsible for antimicrobial and antioxidant activity of green propolis and Baccharis dracunculifolia DC. J Appl Microbiol 122: 911-920.).

Yeast strains

Reference strains (ATCC - American Type Culture Collection) of C. albicans (ATCC 18804), C. dubliniensis (ATCC MYA646), C. glabrata (ATCC 9030), C. krusei (ATCC 6258), and C. tropicalis (ATCC 13803), from the Laboratory of Microbiology and Immunology (ICT/UNESP), were used in this study. Yeasts were stored at -80°C in Yeast Extract Peptone Dextrose broth (YPD - Himedia, Mumbai, India) with 16% glycerol.

Antifungal effect on planktonic cultures

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of R. officinalis L. extract were determined by broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI 2002CLSI. 2002. Reference method for broth dilution in tests for determining the sensitivity to antifungal therapy of yeast. Approved standard, NCCLS document M27-A2. 2nd ed., USA: CLSI, 2002., 2012CLSI. 2012. Reference method for broth dilution antifungal susceptibility testing of yeasts. Fourth Informational Supplement M27-S4, USA: CLSI, 2012.). Initially, strains were grown on Sabouraud Dextrose agar (SD - Himedia) at 37°C for 24 h. Fungal suspensions were prepared in sterile saline solution (0.9% NaCl), whose turbidity was adjusted in a spectrophotometer (Micronal, São Paulo, Brazil) with the parameters of 530 nm and optical density (OD) of 0.284 ± 0.02, to obtain 106 colony-forming units per milliliter (CFU/mL). After dilution of this solution, a concentration between 5 x 102 and 2.5 x 103 CFU/mL was used. Ten serial dilutions of the plant extract were performed in microplates (TPP, Trasadingen, Switzerland). For this purpose, RPMI 1640 broth with glutamine, red phenol indicator, and bicarbonate-free (Himedia), buffered at pH 7 ± 0.1 by 3-(N-morpholino) propanesulfonic acid (MOPS - Sigma-Aldrich, St. Louis, USA) was added in each well (100 μL/well). Microdilution was initiated from the first well, adding 100 μL of plant extract to the RPMI 1640. The ten dilutions of the plant extract were obtained by successive transfers of 100 μL/well. After, standardized inoculum was added in each well (100 μL/well). Thus, evaluated concentrations were from 50 to 0.09 mg/mL. Wells for growth control (medium plus inoculum) and medium alone were added. After incubation (37°C/24 h), MIC was determined in the first well with absence of microbial turbidity, next to the well with apparent microbial growth. For determination of the MFC, MIC and concentrations above it were added on SD agar. After incubation (37°C/48 h), MFC was determined in plate with absence of colony growth, containing the lowest concentration of the plant extract. Propylene glycol was also checked on the yeasts.

Biofilm formation

Yeasts were grown on SD and after in Yeast Nitrogen Base broth (YNB - Sigma-Aldrich) at 37°C for 24 h each growth. Fungal suspension was centrifuged at 2000 rpm/10 min (MPW-350, Warsaw, Poland), supernatant was replaced with saline (0.9% NaCl) and another centrifugation was performed, this procedure was performed twice. Turbidity of the fungal suspension was adjusted in a spectrophotometer (530 nm; 0.381 ± 0.02) to obtain an inoculum of 107 CFU/mL. This standardized suspension was added in microplate wells (200 μL/well) and incubation (37°C/90 min) under agitation (75 rpm - Quimis, Diadema, Brazil) was performed. After pre-adherence of the fungal cells, supernatant was replaced with YNB broth and biofilm was formed for 48 h. Culture medium was exchanged after 24 h of incubation (de Oliveira et al. 2017DE OLIVEIRA JR, DE JESUS D, FIGUEIRA LW, DE OLIVEIRA FE, PACHECO SOARES C, CAMARGO SE, JORGE AO & DE OLIVEIRA LD. 2017. Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells. Exp Biol Med (Maywood) 242: 625-634.).

Antibiofilm effect

Wells were washed with saline solution (200 μL/well) to remove cells not adhered to the biofilm. Then, biofilms were exposed to the effective concentrations of R. officinalis L. extract (200 μL/well), previously determined on planktonic cultures. Nystatin (100,000 IU/mL) and saline solution (0.9% NaCl) were used as controls. Ten replicates were performed in each experimental group. Biofilms were exposure to the products for 5 min or 24 h. Posteriorly, wells were washed with saline solution (200 μL/well) to remove affected cells. Biofilms were disaggregated by ultrasonic homogenizer (Sonopuls HD 2200 - Bandelin Eletronic, Berlin, Germany) for 30 s and 25% power. The suspension generated was diluted and 20 μL of each dilution was added on SD agar in triplicate. After drying the drops (~ 5 min), the plate was inverted and incubated (37°C/24 h). Colonies from each drop were counted and mean values ​​were presented in CFU/mL.

Statistical analysis

Data were presented in mean values (± standard deviation) and checked by one-way analysis of variance (ANOVA) and Tukey’s test, for comparison between treated groups and control groups (CFU/mL), or T-test, for comparison between exposure times in each treated group (reduction percentage). It was considered significant statistical difference when P ≤ 0.05. Ten replicates were performed in each experimental group. GraphPad Prism 5.0 program was used for this purpose.

RESULTS

Phytochemical analysis

Rosmarinus officinalis L. extract showed in its composition: (i) 2.28 ± 0.00% of solids soluble in ethanol; (ii) 3.37 ± 0.00 μg/mL of total phenol; (iii) 2.08 ± 0.37 μg/mL of total flavonoid; and (iv) eliminated free radicals with EC50 of 84.30 ± 2.44 μg/mL. By chromatographic analysis, it was observed some chlorogenic acid derivatives at the retention time of 12.61 min (Figure 1).

Figure 1
High-performance Liquid Chromatography (HPLC) analysis. Chromatogram of R. officinalis L. extract highlighted the presence of chlorogenic acid derivatives in its composition at the retention time of 12.61 min.

Antifungal effect on planktonic cultures

Rosmarinus officinalis L. extract showed MIC of 50 mg/mL on C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis. Therefore, MIC (50 mg/mL), 2 x MIC (100 mg/mL) and 4 x MIC (200 mg/mL) were evaluated on biofilms. Propylene glycol did not show antifungal effect, since microbial growth was observed at all evaluated concentrations. Thus, absence of interference of this vehicle on the yeasts was confirmed.

Antibiofilm effect after 5 min exposure

Rosmarinus officinalis L. extract provided significant reductions of C. albicans biofilm (CFU/mL), in relation to the control group (4 x 104 ± 2.03 x 104) after exposure to 50 (2.11 x 104 ± 1.2 x 104) and 100 mg/mL (7.3 x 102 ± 6.12 x 102). At 200 mg/mL, a total elimination of the biofilm was observed. These concentrations of the extract had antifungal effect similar to the nystatin (1.67 x 103 ± 0) (Figure 2a). Thereby, concentrations of 100 and 200 mg/mL showed higher reduction, when compared to the concentration of 50 mg/mL (Figure 2b). These results were similar to the reductions presented by the nystatin (Table I).

Figure 2
Mean values (± standard deviation) of CFU/mL and reduction percentage (%) of the biofilms of C. albicans (a,b), C. dubliniensis (c,d), C. glabrata (e,f), C. krusei (g,h), and C. tropicalis (i,j) obtained after 5 min exposure to the R. officinalis L. extract (50, 100, and 200 mg/mL) or to the nystatin (100,000 IU/mL), compared to the control group (0 mg/mL – 0.9% NaCl). Different letters indicate significant statistical difference. (n = 10; One-way ANOVA and Tukey’s test; P ≤ 0.05).
Table I
Reduction percentage of the fungal biofilms after exposure to the R. officinalis L. extract or nystatin.

Candida dubliniensis biofilm was significantly affected by the plant extract at 100 (5.38 x 106 ± 1.56 x 106) and 200 mg/mL (2.25 x 106 ± 2.33 x 106), compared to the control group (9.38 x 106 ± 1.69 x 106). However, at 50 mg/mL (6.56 x 106 ± 2.08 x 106), the reduction did not statistically differ from the control group. On the other hand, the concentrations of the plant extract were similar to the nystatin (4.36 x 106 ± 1.53 x 106) (Figure 2c). Thereby, the highest rate of biofilm reduction was found after exposure to 200 mg/mL. Comparable values were observed at 50 and 100 mg/mL (Figure 2d). The groups treated with plant extract presented similar reduction percentages concerning the groups treated with nystatin, as shown in Table I.

The plant extract was effective on C. glabrata biofilm after exposure to 50 (1.36 x 103 ± 0.31 x 103), 100 (6.33 x 102 ± 3.98 x 102) and 200 mg/mL (6.67 x 102 ± 2.36 x 102), in relation to the control group (2.5 x 103 ± 0.97 x 103). Antifungal effect presented by these concentrations was similar to the effect demonstrated by the nystatin (1.67 x 103 ± 0) (Figure 2e). Therefore, similarities between the reduction indices were observed in the groups exposed to the extract at 50, 100, and 200 mg/mL, as well as to the nystatin (Figure 2f; Table I).

Reductions of the C. krusei biofilm were observed after exposure to the plant extract at 50 (1.5 x 103 ± 0.24 x 103), 100 (1.56 x 103 ± 0.1 x 103), and 200 mg/mL (1.67 x 103 ± 0), compared to the control group (2.78 x 103 ± 0.48 x 103). Treated groups were similar to each other and nystatin (1.67 x 103 ± 0) (Figure 2g). Thus, similar reduction indices were observed in the groups exposed to 50, 100, and 200 mg/mL, and to the antifungal nystatin (Figure 2h; Table I).

Candida tropicalis biofilm was impaired after treatment with the plant extract. In the group treated with 200 mg/mL there was a total elimination of the biofilm, as well as in the group exposed to the nystatin. In addition, reductions of this biofilm were observed in the groups treated with extract at 50 (2.37 x 103 ± 0.9 x 103) and 100 mg/mL (6.67 x 102 ± 4.71 x 102), in relation to the control group (1.87 x 104 ± 0.84 x 104). Groups treated with extract at 100 and 200 mg/mL or nystatin demonstrated similar antibiofilm effect (Figure 2i). Therefore, reduction rates of the biofilm were similar between the groups treated with extract at 100 and 200 mg/mL and nystatin, different from the treatment with extract at 50 mg/mL (Figure 2j; Table I).

Antibiofilm effect after 24 h exposure

Rosmarinus officinalis L. extract significantly reduced the C. albicans biofilm after treatment with concentrations of 50 (7.65 x 106 ± 1.52 x 106), 100 (3.87 x 104 ± 0.46 x 104), and 200 mg/mL (1.87 x 104 ± 0.56 x 104), in relation to the control group (1.94 x 107 ± 0.55 x 107). Concentrations of 100 and 200 mg/mL showed effect similar to the nystatin (1.71 x 105 ± 0.92 x 105) (Figure 3a). Thus, the lowest reduction index of the biofilm was observed after exposure to the plant extract at 50 mg/mL. However, concentrations of 100 and 200 mg/mL presented indices similar to the nystatin (Figure 3b; Table I).

Figure 3
Mean values (± standard deviation) of CFU/mL and reduction percentage (%) of the biofilms of C. albicans (a,b), C. dubliniensis (c,d), C. glabrata (e,f), C. krusei (g,h), and C. tropicalis (i,j) obtained after 24 h exposure to the R. officinalis L. extract (50, 100, and 200 mg/mL) or to the nystatin (100,000 IU/mL), compared to the control group (0 mg/mL – 0.9% NaCl). Different letters indicate significant statistical difference. (n = 10; One-way ANOVA and Tukey’s test; P ≤ 0.05).

Candida dubliniensis biofilm was decreased after exposure to the plant extract at 50 (7.43 x 106 ± 1.51 x 106) and 100 (8.33 x 105 ± 0), and the total elimination of this biofilm was observed at 200 mg/mL, compared to the control group (2.98 x 107 ± 0.73 x 107). Concentrations of 100 and 200 mg/mL demonstrated effect similar to the nystatin (1.11 x 104 ± 0.82 x 104) (Figure 3c). Thereby, the highest reduction percentages were observed in the treatments with 100 and 200 mg/mL, which were similar to the nystatin. The lowest index was obtained at 50 mg/mL (Figure 3d; Table I).

Rosmarinus officinalis L. extract showed antibiofilm action on C. glabrata after exposure to concentrations of 50 (6.4 x 105 ± 4.93 x 105), 100 (8.33 x 105 ± 0), and 200 mg/mL (1.67 x 102 ± 0), demonstrating an effect similar to the nystatin (1.58 x 104 ± 0.12 x 104). In the control group, 6.97 x 106 ± 3.97 x 106 CFU/mL were obtained (Figure 3e). Thereby, the reduction percentages were similar between the groups treated with extract at 50, 100, and 200 mg/mL, and treated with nystatin (Figure 3f; Table I).

Candida krusei biofilm was affected by the plant extract at 50 (8.33 x 103 ± 3.85 x 103), 100 (3.33 x 102 ± 1.67 x 102), and 200 mg/ml (2.08 x 102 ± 0.83 x 102), compared to the control group (1.78 x 105 ± 0.51 x 105). The plant extract had antibiofilm effect similar to the nystatin (7.78 x 102 ± 0.96 x 102) (Figure 3g). Thus, the reduction rates observed in the groups exposed to the extract at 50, 100, and 200 mg/mL did not differ from the indices presented by the group treated with nystatin (Figure 3h; Table I).

The plant extract effectively contributed to eliminate the C. tropicalis biofilm after exposure to the concentrations of 50 (1 x 105 ± 0), 100 (1.67 x 102 ± 0), and 200 mg/mL (1.67 x 104 ± 0), in relation to the control group (4.56 x 105 ± 1.62 x 105). The antibiofilm effect showed by the concentrations of 100 and 200 mg/mL was similar to that showed by the nystatin, which completely eliminated this biofilm (Figure 3i). Thus, the reduction percentages of the biofilm were similar between the groups exposed to the extract at 100 and 200 mg/mL, and to the nystatin. The lowest reduction rate was observed at the concentration of 50 mg/mL (Figure 3j; Table I).

5 min versus 24 h

A comparison between the reduction percentages obtained from the experimental groups after exposure to R. officinalis L. extract for 5 min or 24 h was shown in Figure 4. A higher reduction percentage of the C. albicans biofilm was observed after 24 h exposure to the extract (50 mg/mL) and to the nystatin, compared to the exposure for 5 min. Similar reduction percentages were observed after exposure to the plant extract at 100 and 200 mg/mL (Figure 4a).

Figure 4
Comparison of mean values (± standard deviation) of reduction percentage of the biofilms of C. albicans (a), C. dubliniensis (b), C. glabrata (c), C. krusei (d), and C. tropicalis (e) between the exposure times of 5 min and 24 h to the R. officinalis L. extract (50, 100, and 200 mg/mL) or to the nystatin (100,000 IU/mL). Asterisks indicate significant statistical difference between the exposure times in each experimental group. (n = 10; T-test; P ≤ 0.05).

Exposure for 24 h to the extract at 50 and 100 mg/mL or to the nystatin promoted higher reduction percentages of the C. dubliniensis biofilm in relation to the exposure for 5 min. However, similar percentages were observed after treatment with extract at 200 mg/mL (Figure 4b).

Candida glabrata biofilm had the highest reduction percentages after 24 h exposure to the plant extract at 50 and 200 mg/mL, as well as to the nystatin, compared to the 5 min exposure. Similarities were found in the treatment with extract at 100 mg/mL (Figure 4c).

All the experimental groups showed a higher reduction percentage of the C. krusei biofilm after 24 h exposure, than in the exposure for 5 min (Figure 4d).

Concentrations of 50 and 200 mg/mL showed higher reduction percentage of the C. tropicalis biofilm after 5 min exposure. The highest reduction percentage was found in the group exposed to the extract at 100 mg/mL for 24 h. In addition, exposure to the nystatin provided a reduction percentage similar between both exposure times (Figure 4e).

DISCUSSION

In this study, the MIC of R. officinalis L. extract was 50 mg/mL on planktonic cultures of C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis. Therefore, MIC (50 mg/mL), 2 x MIC (100 mg/mL) and 4 x MIC (200 mg/mL) were evaluated on the fungal biofilms, once higher concentrations than the MIC is necessary to affect biofilms, compared to planktonic cultures (Lewis 2001LEWIS K. 2001. Riddle of Biofilm Resistance. Antimicrob Agents Chemother 45: 999-1007.).

On the C. albicans biofilm, concentrations of 50, 100, and 200 mg/mL of R. officinalis L. extract showed effect similar to the nystatin, after only 5 min exposure (Figure 2a). Similar reduction rates to the nystatin were observed after exposure for 24 h at 100 and 200 mg/mL (Figure 3a). Studies have reported that the R. officinalis L. essential oil have been able to act on clinical isolates of C. albicans, affecting the formation of germ tubes (Pozzatti et al. 2010POZZATTI P, LORETO ES, NUNES MARIO DA, ROSSATO L, SANTURIO JM & ALVES SH. 2010. Activities of essential oils in the inhibition of Candida albicans and Candida dubliniensis germ tube formation. J Mycologie Médicale 20: 185-189., Gauch et al. 2014GAUCH LMR, GOMES FS, ESTEVES RA, PEDROSA SS, GURGEL ESC & ARRUDA AC. 2014. Effects of Rosmarinus officinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Rev Soc Bras Med Trop 47: 389-391., Ksouri et al. 2017KSOURI S, DJEBIR S, BENTORKI AA, GOURI A, HADEF Y & BENAKHLA A. 2017. Antifungal activity of essential oils extract from Origanum floribundum Munby, Rosmarinus officinalis L. and Thymus ciliatus Desf. against Candida albicans isolated from bovine clinical mastitis. J Mycol Med 27: 245-249.). Additionally, the antibiofilm effect of this essential oil against C. albicans has been demonstrated. This plant product was incorporated into cellulose acetate nanofibers once it was an excellent carrier with the absence of biological activity. The union of these products resulted in a uniform structure, whose highest concentrations of the essential oil were more effective against C. albicans (Liakos et al. 2017LIAKOS JL, HOLBAN AM, CARZINO R, LAUCIELLO S & GRUMEZESCU AM. 2017. Electrospun fiber pads of cellulose acetate and essential oils with antimicrobial activity. Nanomaterials 7: 84.). Ethanolic and aqueous extracts from leaves of R. officinalis L. and Echinophora platyloba were evaluated and compared. R. officinalis L. ethanolic extract demonstrated effective antifungal activity on C. albicans, showing superior effect to the E. platyloba ethanolic extract. On the other hand, the aqueous extracts did not have significant impact against C. albicans (Sepehri et al. 2016SEPEHRI Z, JAVADIAN F, KHAMMARI D & HASSANSHAHIAN M. 2016. Antifungal effects of the aqueous and ethanolic leaf extracts of Echinophora platyloba and Rosmarinus officinalis. Curr Med Mycol 2: 30-35.).

Candida dubliniensis biofilm suffered significant reductions when treated with R. officinalis L. extract. at 200 mg/mL (Figure 2c), this concentration similarly acted to the nystatin after 5 min exposure. However, at 100 and 200 mg/mL, an action similar to the antifungal was also observed after 24 h exposure (Figure 3c). The effect of R. officinalis L. extract against C. dubliniensis biofilms have not been reported, but susceptibility of this yeast to that plant extract has been demonstrated on planktonic cultures (Höfling et al. 2010HÖFLING JF, ANIBAL PC, OBANDO-PEREDA GA, PEIXOTO IAT, FURLETTI VF, FOGLIO MA & GONÇALVES RB. 2010. Antimicrobial potential of some plant extracts against Candida species. Braz J Biol 70: 1065-1068.).

The effect of R. officinalis L. essential oil has been evaluated, as well as other aromatic plants, such as Origanum vulgare, Cinnamomum zeylanicum, Lippia graveolens, Thymus vulgaris, and Salvia officinalis on planktonic cultures of fluconazole-susceptible or -resistant C. glabrata. Only O. vulgare and L. graveolens inhibited fluconazole-susceptible C. glabrata samples, and C. zeylanicum was capable of acting on fluconazole-resistant C. glabrata isolates (Soares et al. 2015SOARES IH, LORETO ÉS, ROSSATO L, MARIO DN, VENTURINI TP, BALDISSERA F, SANTURIO JM & ALVES SH. 2015. In vitro activity of essential oils extracted from condiments against fluconazole-resistant and -sensitive Candida glabrata. J Mycol Med 25: 213-217.). In our study, inhibitory effect on planktonic culture (MIC = 50 mg/mL) and significant antibiofilm activity in both exposure times (5 min and 24 h) were found on C. glabrata, with an action similar to the nystatin (Figures 2e and 3e).

Candida krusei biofilm showed a significant reduction after exposure to the R. officinalis L. extract for 5 min and 24 h, being similar to the nystatin (Figures 2g and 3g). Action of R. officinalis L. extract on C. krusei biofilm has not yet been reported. However, an inhibitory effect was reported on planktonic cultures, using 3 µg/mL, for a dichloromethane extract, and 1 µg/mL, for a methanol extract (Höfling et al. 2010HÖFLING JF, ANIBAL PC, OBANDO-PEREDA GA, PEIXOTO IAT, FURLETTI VF, FOGLIO MA & GONÇALVES RB. 2010. Antimicrobial potential of some plant extracts against Candida species. Braz J Biol 70: 1065-1068.).

Candida tropicalis biofilm was significantly reduced after 5 min and 24 h exposure to the R. officinalis L. (Figures 2i and 3i), presenting concentrations with effects similar to the nystatin. Antifungal effect of R. officinalis L extract has also been reported on planktonic culture of C. tropicalis by MIC of 15 µg/mL, using a dichloromethane extract, and 3 µg/mL, for a methanol extract (Höfling et al. 2010HÖFLING JF, ANIBAL PC, OBANDO-PEREDA GA, PEIXOTO IAT, FURLETTI VF, FOGLIO MA & GONÇALVES RB. 2010. Antimicrobial potential of some plant extracts against Candida species. Braz J Biol 70: 1065-1068.). Besides, the ability to inhibit the adhesion and formation of polymicrobial biofilms composed by C. albicans and C. tropicalis has been demonstrated by the R. officinalis L. essential oil conjugated with nanoparticles (Chifiriuc et al. 2012CHIFIRIUC C, GRUMEZESCU V, GRUMEZESCU AM, SAVIUC C, LAZĂR V & ANDRONESCU E. 2012. Hybrid magnetite nanoparticles/Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity. Nanoscale Res Lett 7: 209.).

CONCLUSIONS

Rosmarinus officinalis L. extract had a significant antibiofilm effect at the evaluated concentrations, after 5 min and 24 h exposure. However, the extract at 100 and 200 mg/mL demonstrated the highest reduction rates for biofilms of C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis, showing action similar to the nystatin.

ACKNOWLEDGMENTS

To Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the Scientific Initiation Grant (process 15/25338-0) and Research Grant (process 15/08776-3).

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Publication Dates

  • Publication in this collection
    30 Apr 2021
  • Date of issue
    2021

History

  • Received
    26 Mar 2019
  • Accepted
    25 Aug 2019
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