South African Journal of Botany 99 (2015) 115–121

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South African Journal of Botany

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Antimicrobial activities of selected essential oils against Fusariumoxysporum isolates and their biofilms

M.C. Manganyi a,b, T. Regnier a,⁎, E.I. Olivier b

a Department of Biotechnology and Food Technology, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africab Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa

⁎ Corresponding author. Tel.: +27 12 3826202; fax: +E-mail address: regniert@tut.ac.za (T. Regnier).

http://dx.doi.org/10.1016/j.sajb.2015.03.1920254-6299/© 2015 SAAB. Published by Elsevier B.V. All rig

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 February 2015Received in revised form 5 March 2015Accepted 10 March 2015Available online xxxx

Edited by Vanessa Steenkamp

Keywords:AntifungalAntibiofilmCloveConfocal laser scanning microscopyEssential oilsFusariumThyme

Fusarium infections, such as keratitis, are becoming increasingly difficult to control due to the build-up ofresistance of fungi towards conventional antibiotics. Resistance is further enhanced by their ability to form fungalbiofilms. Poor storage conditions and inadequate cleaning of contact lenses often lead to corneal infections. Thesolution to problems associatedwith contamination and resistance to antimicrobials could lie in the discovery ofnew, affordable, efficacious antimicrobial compounds. The purpose of this study was to evaluate the antifungaland antibiofilm properties of selected essential oils. The plant-based essential oils were evaluated for in vitro an-tifungal activity against Fusarium spp. using the Toxic Medium Assay (TMA) for preliminary screening. Clove andthyme oils, as well as pure citral, eugenol and thymol at 500 μL/L, exhibited the highest antimicrobial activityagainst Fusarium isolates. Antibiofilm capacity was investigated using soft contact lenses as substrate, while con-focal laser scanning microscopy (CLSM) was used for visual observation of biofilm architecture. Although severedamage to the lenswas observedwhen the essential oils were applied at 500 μL/L, no visible alteration of the con-tact lenses was detected at 50 μL/L and therefore the antibiofilm property of thyme and clove EOswas evaluatedat this concentration. Clove and thyme oils prevented cell attachment, biofilm development and caused total in-hibition of biofilm formation on soft contact lenses. The use of diluted clove and thyme oils could therefore be anoption to prevent biofilm formation of soft contact lenses.

© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Fusaria are filamentous fungi that occur as saprophytic organisms insoil (Nelson et al., 1994) and are opportunistic pathogens of plants, an-imals and humans (Evans et al., 2004). This genus includes more than50 species with the ability to produce mycotoxins (Afsah-Hejri et al.,2013). Fusarium mycotoxins contaminate most agricultural commodi-ties and cause fusariosis in humans and animals affecting the liver,kidneys, lungs and the heart (Antonissen et al., 2014; Nordkvist andHäggblom, 2014). Persistent infection may lead to mortality (Eleniand Anaissie, 1997; Peska and Smolinski, 2005). Fusarium infections,such as keratitis, are gradually becoming an epidemic worldwide inimmunocompromised individuals (Scheel et al., 2013). It has beenestimated that microbial biofilms are responsible for 60–80% of infec-tions in developing countries and that these biofilms contribute to thedevelopment of pathogen resistance to antibiotics (Saviuc et al., 2009).

Diverse classes of microorganisms, including fungi, yeasts, bacteria,algae, protozoa and viruses, are capable of forming biofilms (Hardinget al., 2009; Lindsay and Holy, 2006). Biofilms are defined as sessilecommunities of microbial cells that are irreversibly attached to surfaces

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(substrate or cell–cell adhesion). Their formation has a significantclinical implication due to their reduced susceptibility to antimicrobialagents when compared to their planktonic counterparts (Abdi-Aliet al., 2014; Dodds et al., 1999; Stewart and Costerton, 2001). Williamet al. (1997) proved that Staphylococcus aureus biofilms on a siliconesurface were ten times more resistant to the antibiotic, vancomycin,than the corresponding planktonic cells. The genus Fusarium is consid-ered the most resistant to modern antifungal agents due to the produc-tion of biofilms (Guarro et al., 1999; Musiol et al., 2014).

The use of contact lenses has been estimated at 125–140 millionwearer's worldwide (Selan et al., 2009). It is well established that thehuman eye is a suitable environment formicrobial pathogens toflourish(Fong et al., 2004; Klotz et al., 2000). Prolonged wearing or storageleads to an increasing number of microbia colonizing the conjunctivaand lid of the eye, ultimately resulting to infection of the cornea (Fonget al., 2004; Willcox et al., 2001). Contact lens care solutions are formu-lated to kill a range of pathogens, including Gram-negative andGram-positive bacteria, and even some fungi. Manufacturers of lenscleaners focus on the control of planktonic or single-cell microbes, andorganisms responsible for microbial keratitis. However, demonstratingefficacy against biofilms is not considered as a criterion. Nonsteroidalanti-inflammatory drugs, including sodium salicylate have beenreported to inhibit extracellular bacterial biofilm production mainly by

116 M.C. Manganyi et al. / South African Journal of Botany 99 (2015) 115–121

Staphylococcus epidermidis and Pseudomonas aeruginosa (Bryers et al.,2006; Farber et al., 1995). However, their use for cleaning of lensesand their storage containers remains limited and removal of the biofilmfrom contact lenses relies mainly on how often the disinfectant is re-placed (Kilvington and Lonnen, 2009). The solution to problems associ-ated with contamination and resistance to antimicrobials lies in thediscovery of new, affordable, efficacious antimicrobial compounds.

Plant-based medicinal essential oils (EOs) for example have beenthe focus of many studies aimed at identifying natural fungicides(Gurib-Fakim, 2006; Karamaouna et al., 2013; Lahlou, 2004). Theyhave been applied asflavourants and preservatives and are componentsof many cosmetics products (Koul et al., 2008). Due to their antimicro-bial activities against bacteria (Edris, 2007), parasitic protozoa(Anthony et al., 2005; Boyom et al., 2003), fungi (Kalemba andKunicks, 2003) and viruses (Edris, 2007), EOs are currently investigatedwidely for their pharmacological applications. Clove (Syzygiumaromaticum) and thyme oil (Thymus vulgaris) and their volatiles havebeen reported to exhibit a broad spectrum of antimicrobial activities(antibiofilm properties) against planktonic micro-organisms (Al-Jiffriet al., 2011; Nzeako et al., 2006; Sandasi et al., 2010).

This study investigated the use of EOs for their antimicrobial/antibiofilm activity against Fusarium oxysporum species complex[FOSC]. Seven isolates, linked to outbreaks of keratitis in Pennsylvania,USA, were selected as test pathogens. Soft contact lenses were used assubstrates for the formation of biofilms, since they are known to be as-sociated with infections, which often result in corneal injury due tospore contamination. Since the aim of the studywas to use EOs to devel-op a cleaning solution for contact lenses, the ability of the selected EOswere evaluated to control biofilm formation on soft contact lenses,without damaging the matrix.

2. Materials and methods

2.1. Pathogens

Fusarium isolates (D-2383,M-3125, O-1885, S-1187, S-1372, S-2067,R-9963) were kindly supplied by the Department of Plant Pathology(Pennsylvania State University). The identification of these isolates byProf Geiser was based on distinctive characters, including the shapesand sizes of macro- and microconidia, the presence and absence ofchlamydospores and pigmentation and growth rates on agar media. Inaddition, the polymerase chain reaction with restriction fragmentlength polymorphism (PCR-RFLP) of intergenic spacer (IGS) region(Geiser et al., 2004) was used to verify the identification. Thefreeze-dried isolates were subcultured andmaintained on a potato dex-trose agar (PDA, Merck, Johannesburg, RSA) slant. These cultures weresubsequently revived on PDA plates at room temperature (25 °C).Cultures were allowed to grow for 7–10 days to ensure spore formation.Sabouraud dextrose broth (2%, SDB; Difco Laboratories, Detroit, MI, USA),supplemented with chloramphenicol (250 mg/L of medium, Rolab (Pty),Johannesburg, South Africa) was used to maintain the pathogens. Thesterilized supplementedmedia containing the Fusarium isolateswere cul-tured for 40 h at 37 °C in a shaking incubator. A hemocytometerwas usedto determine the concentration of the spore suspension (in triplicate).This was then standardized to 106 conidia/mL (Sun et al., 2010).

2.2. Essential oils

Essential oil of Lippia scaberrima and two batches of Lippia rehmanniiEOwere obtained from Prof Combrinck (Department of PharmaceuticalSciences, Tshwane University of Technology, Pretoria, RSA). Menthaspicata, Helichrysum splendidum, Cymbopogon citratus, T. vulgaris,S. aromaticum, Cinnamon zeylanicum and Cinnamomum camphora EOswere obtained fromHolistic Emporium (Johannesburg, RSA). Pure com-pounds (R-(−)-carvone, S-(+)-carvone, eugenol, limonene, neral,

citral, limonene and thymol) used in this experiment were purchasedfrom Sigma-Aldrich (Pty) Ltd. (Johannesburg, RSA).

2.3. Gas chromatography analyses of essential oils

Each sample was diluted to 20% (v/v) with dichloromethane andanalyzed using an Agilent 6890N gas chromatograph, coupled to aflame ionization detector (FID) and a 5973 Mass Spectrometer (AgilentTechnologies, RSA), as described by Kamatou et al. (2012). The identifi-cation of the compounds was carried out by comparing mass spectraand retention indices of authentic reference standards, as well as byusing the National Institute of Standards and Technology (NIST), MassFinder and Flavor data libraries.

2.4. In vitro antifungal activities of essential oils

Essential oils were screened in a toxic medium assay at concentra-tions of 100, 250, 500, 1000, 2000, 2500 and 3000 μL/L. A specificamount of EO was premixed with 200 μL of the surfactant Tween-80(Polysorbate 80, Sigma-Aldrich, Johannesburg, RSA), prior to supple-mentation of sterilized PDA to yield the exact required concentration.Negative controls consisted of PDA plates containing surfactant, butwithout any supplementation, while ICA-Thiabendazole® 500SC(ICA International Chemicals (Pty) Ltd., Stellenbosch, RSA) served asthepositive control. Ten replicateswere prepared in 90mmPetri dishes,whereafter a 5 mm agar plug of fungal mycelia was placed asepticallyonto the center of each PDA plate. After 9 days incubation at 25 °C, themycelial growth (mm) was measured with a 150 mm digital caliper(Absolute Digimatic-Mitutoyo Corp. Japan). Data were expressed aspercentage inhibition of mycelial growth relative to the control, accord-ing to the method described by Plaza et al. (2004). Average values andstandard deviations for replicate measurements were determinedusing Microsoft Office Excel 2010 Version 14.0. However, no testswere done to determine whether differences were significant or not,since the only criterion used to determine efficacy was total pathogencontrol.

2.5. Physical effects of oils on soft contact lenses

In this study, three concentrations (250, 100, 50 μL/L) of clove andthyme oil, previously mixed with 200 μL/L of a hydrophilic nonionicsurfactant (Tween®-80, Sigma-Aldrich, Johannesburg, RSA), wereamended to contact lens solution (Cooper Vision, UK). Approximately2 mL of each mixture (volume necessary to cover the lens) was trans-ferred directly onto the container containing the sterile contact lens.Damages on the lenses were visually assessed 1 h, 2 h, 3 h and 24 hafter immersion. In addition, long term effects were established bysoaking the lenses over a three month period.

2.6. Biofilm formation and inhibition

Soft contact lenses (Cooper Vision, UK) were used as an adhesionsurface to cultivate biofilms (Sun et al., 2010). The substrate was usedto determine whether the Fusarium isolates have the ability to producea biofilm on the synthetic surface. Contact lens containers (SauflonPharmaceuticals Ltd, UK) were used to hold the lenses. For attachment,2 to 4 mL of themycelium suspension in 2% SDB was transferred onto alens, which was then incubated in a shaking incubator for 90 min at37 °C (attachment phase). After incubation, lenses were gently rinsedwith phosphate buffered saline (PBS, Merck, Johannesburg, RSA) to re-move all non-adherent cells. Sterile Sabouraud Dextrose Broth (2%)was used to enhance biofilm growth at 37 °C for 48 h on a rocking plat-form. The formation of hyphae on the soft lens was confirmed by usingthe tetrazolium XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 carboxanilide] assay as described by Sun et al. (2010).

Table 1Inhibitory effects (%) of ten selected essential oils and their major components againstF. oxysporum isolate S-1187.

Concentration in μL/L

Inhibitor 100 250 500 1000 2000 2500 3000Helichrysum splendidum (A) – – – – – – 0Helichrysum splendidum (B) – – – – – – 58Cymbopogon citratus – – – – 46 100 100Thymus vulgaris – 61 100 100 100 100 100Lippia rehmannii – – 72 100 100 100 100Lippia scaberrima – – – – – – 87Mentha spicata – – – – 79 100 100Syzigium aromaticum – 83 100 100 100 100 100Cinnamon zeylanicum – – 92 100 100 100 100Cinnamomum camphora – – – – – – 49R-(−)-carvone – – – – – – 89S-(+)-carvone – – – – – – 93Eugenol – 85 100 100 100 100 100Citral – 88 100 100 100 100 100Neral – – – – 100 100 100Limonene – – – – – – 47Thymol – 87 100 100 100 100 100

H. splendidum A = Holistic Emporium, Johannesburg RSA, B = Burgess and Finch, CapeTown RS.

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Once the biofilm formation on contact lenses was confirmed for allthe isolates, the same experiment was conducted to evaluate theantibiofilm property of each EO (thyme and clove) at a concentrationof 50 μL/L, using only the isolate S-1187. This isolate was selectedbased on its ability to form a dense biofilm on contact lenseswhen com-pare to the other isolates tested. In this case, after attachment, lenseswere gently rinsedwith PBS to remove all non-adherent cells. Lenses in-oculatedwith the isolate, but without any treatment, served as controls.Essential oil solutions were transferred onto the surfaces of biofilm-infiltrated contact lenses and were incubated at 37 °C for 48 h on arocking platform (150–180 rpm). To assess the antibiofilm property ofthe oil, the architecture of biofilms formed on soft contact lenseswas in-vestigated using confocal scanning laser microscopy (CSLM). CalcofluorWhite (Sigma-Aldrich, USA) was used as a staining dye. Fluorescencewas observed at a UV-excitation wavelength of 405 nm and emissionwavelengths between 420–480 nm. The stain illuminates hyphae witha blue fluorescence by binding to cellulose and chitin residues of cellwall polysaccharides. The fluorescence dye (10–20 μL) was transferredonto each lens in a dark environment. After 3–5min, the lenswas gentlyrinsed with distilled, deionized water. Lenses were flattened by cuttingat four edges. Stained biofilms were observed under the confocal scan-ning laser microscope (Imamura et al., 2008).

3. Results and discussion

3.1. Characterization of essential oils

Since the compositions of EOs from a particular botanical or brandare known to be highly variable (Badi et al., 2004; Debabrata et al.,2014; Lohani et al., 2012), chemical analysis of the oil is critical to ensurereproducible antifungal action. Gas chromatographic analysis of the oilsused in this study indicated that the oils contained one or more of thefollowing compounds in high concentrations: carvone, citral (a mixtureof the isomers neral and geranial), 1,8-cineole, geranial and thymol.These constituents of EOs are known to have good antimicrobial proper-ties towards various organisms (Zomorodian et al., 2013). As expected,the essential oil of C. camphora ct. cineole selected for the study wasfound to comprise 50% 1,8-cineole and 14% limonene. Two knowncitral-rich essential oils, lemongrass and L. rehmannii were also used.The lemongrass EO was richer in citral (39% geranial, 31% neral), thanthe oil of L. rehmannii, which contained only 34.5% geranial and 20.2%neral. As previously reported by Linde et al. (2010), the origin ofL. rehmannii plants used for the extraction of the oil significantly affectsthe ratio of geranial/neral of the oil. Oils of H. splendidum, purchasedfrom two suppliers (A = Holistic Emporium, Johannesburg RSA, B =Burgess and Finch, Cape Town RSA) contained 2.90% and 22.7% α-pinene, respectively. Significant variations in the volatile compositionsand yields have been reported for oils obtained from different popula-tions of H. splendidum (Mashigo et al., 2015). In this study, spearmintoil yielded a higher concentration of carvone (77.9%) thanL. scaberrima (43.8%). Of the eugenol-rich oils tested, clove EO wasfound to contain 82.3% eugenol, β-caryophyllene (11.0%), and α-humulene (2.19%) corresponding to the results of Viuda-Martos et al.(2007), who reported 85% eugenol and 11% β-caryophyllene. Eugenol(12.8%) andβ-caryophyllene (8.23%)were themain constituents of cin-namon essential oil. In this study, the essential oil of red thyme pur-chased from Holistic Emporium was characterized by a high contentof thymol (45.2%), terpinen-4-ol (12.3%), γ-terpinene (8.81%) and cis-sabinene hydrate (8.05%), which is in accordance with the content re-ported by Viuda-Martos et al. (2007) and later by Grigore et al.(2010). It is essential that an EO applied as a disinfectant for biofilm con-trol should have a reasonably constant chemical composition that com-plies with a predetermined specification range. Most of the oils used inthis study can be easily purchased in large quantities and are suppliedwith a certificate of analysis. These oils are characterized by a singleprincipal active component. The selection was aimed at oils of which

the antifungal activity could possibly be ascribed to the occurrence ofthe major compound.

3.2. In vitro antifungal activities of essential oils

A previous investigation by our research group concerning thein vitro effects of L. rehmannii, lemongrass EOs and citral against arange of fungal decay pathogens, including F. oxysporum frommaize, in-dicated that the oils have the ability to totally inhibit fungal growth atconcentrations above 3000 μL/L (Linde et al., 2010). The study also re-vealed that lemongrass oil totally inhibited the growth of F. oxysporumat a concentration of 50 μL/L and L. rehmannii and citral at 20 μL/L. Inour study, the concentration of 3000 μL/L wasfirstly selected for screen-ing, to allow themost effective EOs to be identified. Of the 10 EOs tested,only spearmint, cinnamon, clove, lemongrass, L. rehmannii and thyme,were able to totally control the mycelial growth of the Fusarium isolateS-1187 (Table 1). An assessment of the antifungal properties of themajor constituents of an EO in relation to that of the oil itself shedslight on the possible contribution of active principles. Of all the essentialoils and pure compounds tested, thyme and clove EOs and eugenol,citral and thymol proved to have the best antifungal activities, with allcausing total inhibition of fungal growth at 500 μL/L. The antimicrobialactivity of the essential oils of thyme and clove could be associatedwith their major constituents, thymol and eugenol, respectively. Thesecomponents have been reported to display antifungal properties (Burt,2004; Kamatou et al., 2012; Krist et al., 2007). Therefore, a furtherscreening using the essential oils of clove and thyme at 500 μL/L and1000 μL/L was done against all seven Fusaria isolates (Table 2). Total in-hibition of all isolates was obtained when the EOs were used at a con-centration of 1000 μL/L. The isolate R-9963 was the most resistantstrain, with both EOs exhibiting 83–100% inhibitions. Multiple mecha-nisms could be responsible for such resistance, where the microorgan-ism alters the target site of the antimicrobial agent. Generally, thehigher resistance amongst different isolates of the same species is as-cribed to complex mechanisms, such as the significant alteration oflipid layers that renders cell membranes almost impermeable to lipo-philic compounds (Ghannoum and Rice, 1999; Nazzaro et al., 2013).However, othermechanisms, including resistance against the inhibitionof lanosterol demethylase, a key enzyme in ergosterol biosynthesis,DNA and RNA synthesis or 1,3-ß-glucan synthase have also been report-ed (Loeffler and Stevens, 2003).

A large number of research papers published throughout the lastdecade attest to the antifungal properties of clove and thyme EOs

Table 2Inhibition activity (%) of clove and thyme essential oils at concentrations of 500 and1000 μL/L towards seven Fusarium isolates.

Oil Isolates Inhibition(%)at 500 μL/L

Inhibitor(%)at 1000 μL/L

Clove D-2383 100 100M-3125 93 ± 2 100O-1885 100 100R-9963 81 ± 3 100S-1187 100 100S-1372 100 100S-2067 90 ± 3 100

Thyme D-2383 91 ± 4 100M-3125 90 ± 3 100O-1885 89 ± 3 100R-9963 82 ± 4 100S-1187 100 100S-1372 88 ± 2 100S-2067 89 ± 3 100

Table 3Effects of clove, L. rehmannii (B) and thyme essential oils on contact lenses after 1, 2, 3 and24 h exposure.

Essential oils (μL/L)

1st hour 2nd hour

Clove 50 No change No change100 No change No change250 Small holes Small holes500 Opaque Opaque

3rd hour 24 h50 No change No change100 Small holes Small holes250 Opaque Opaque500 Opaque Fully opaque

1st hour 2nd hourLippia rehmannii 50 No change No change100 No change No change250 Burn with small holes Burn with small holes500 Burn with small holes Burn with small holes

3rd hour 24 h50 No change No change100 No change Partly opaque with holes

small large holes250 Small holes Opaque with holes500 Opaque with holes

1st hour 2nd hourThyme 50 No change No change100 No change No change250 Small holes Small holes500 Fully opaque Opaque and small holes

3rd hour 24 h50 No change No change100 Small holes Partly opaque with holes250 Partly opaque with holes Fully opaque500 Fully opaque Fully opaque

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(Burt, 2004; Pina-Vaz et al., 2004). It is well known that thephenylpropene, eugenol (4-allyl-2-methoxyphenol), citral and isopro-pyl cresol aremulti-purposemolecules with broad applications in phar-macy, food technologies and agriculture (Burt, 2004; Kamatou et al.,2012). Therefore, their modes of action are well documented (Burt,2004). For example, the lipophilic nature of thymol and eugenol permitthe diffusion of these molecules between the fatty acyl chains of cellmembranes. This has a profound effect on both permeability and fluid-ity of cell membranes, resulting in the impairment of ergosterol(Hyldgaard et al., 2012), in addition to disturbing cell growth and enve-lope morphogenesis (Sikkema et al., 1995).

3.3. Physical effects of clove and thyme essential oils application on lenses

As the final aim of this study was to obtain a potential sanitizer forcontact lenses, proof of concept data on the safety performance ofsuch a product is crucial. The first step towards the acquisition of suchdata is to assess the potential effect of the oil on the integrity of the con-tact lenses over time. It is well established that essential oils can affectthe structural integrity of some polymers, leading to breakdown, disso-lution and release of toxic compounds (Al Kharousi et al., 2014). In thecase of contact lenses, an application of the clove and thyme essentialoils at a concentration of 250 μL/L resulted in severe damage (holesand opacity) to the lenses (Table 3). However, no effects were observedwhen a concentration of 50 μL/L was applied. Although no scientific pa-pers report on the cytotoxicity of the essential oils on the eye, manybooks on aromatherapy and on essential oils recommend flushing theeye with a buffer after inadvertent administration of oil into the eye(Tisserand and Young, 2014). However, at a very low concentration,such as 50 μL/L, limited to no irritation was found by ten lens wearers(Patent, US 5387394 A, 1992). Nonetheless, it is recommended torinse the lenses with a fresh buffer solution.

3.4. Biofilm formation

Microbial biofilm formation by Fusarium on contact lenses has beenpreviously reported by Imamura et al. (2008). As mentioned by Davies(2003), a large proportion of humanmicrobial infections are associatedwith biofilm formation. This ability creates a sheltered environmentthat facilitatesmicrobial interactions and enhances resistance of biofilmcommunities to antimicrobial agents. Although there are many reportsavailable regarding the antimicrobial properties of essential oils, fewof these focus on the antibiofilm activities of these plant-derived ex-tracts. The antibiofilm activities of clove and thyme EOs against sevenFusarium isolates were therefore investigated using a contact lens ma-trix as support. Biofilms grown on soft contact lenses were examinedby CSLM using Calcofluor-white, a UV-excitable dye that binds cellulose

and chitin and has long been used to identify bacterial and fungalbiofilms (Cruz et al., 2013; Farber et al., 1995; Harding et al., 2009).The ability of bacteria to attach to contact lenses and subsequentlyform biofilms has long been established (Zegans et al., 2002).Szczotka-Flynn et al. (2009) also confirmed biofilm formation on siliconhydrogel contact lenses by P. aeruginosa, Serratia marcescens andS. aureus clinical strains, commonly associated with contact lens kerati-tis and infection. All seven Fusarium isolates investigated in the currentstudy were found to form dense (Isolate S-1187) to limited biofilmswith distinct architecture on soft contact lenses. As shown in Fig. 1,Fusarium hyphae were able to develop on the lenses (Fig. 1A) andeven slightly penetrated the polymeric structure (Fig. 1B). Similar ob-servations have been reported by Imamura et al. (2008), Ramani andChaturvedi (2011) and Mukherjee et al. (2012) for Fusarium solaniand F. oxysporum on contact lenses. In our study, all Fusarium isolatesalso formed spores on lenses. Spore formation forms part of a commonsurvival mechanism used by microorganisms and is usually linked to alack of nutrients on the surface (Neiman, 2005). It can be concludedthat all of the selected Fusarium isolates could be a potential source ofcontamination of soft contact lenses. Isolate S-1187 was selected as asuitable pathogen for the in vivo determination of the antibiofilm prop-erties of thyme and clove EOs.

3.5. Antibiofilm assay

As reported by Imamura et al. (2008), Szczotka-Flynn et al. (2009)andWu et al. (2011), most commercially available contact lens care so-lutions demonstrate variable antimicrobial activities against bacterialcells grown under planktonic or biofilm conditions. To our knowledge,this is the first report on the use of confocal scanning laser microscopyto evaluate changes in the density of Fusarial biofilms following

Fig. 1. Biofilm formation of Fusarium spp (S-1187) on soft contact lenses 48 h after inoculation. A: Development of mycelium hyphae on the surface. B: Penetration of the hyphae into thematrix.

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exposure to thyme and clove oils. As depicted in Fig. 2 (B and D), un-treated biofilm of F. oxysporum S-1187 exhibited a dense network ofcells with extracellular polymeric matrix after 48 h. Although the con-centration of the EOs (50 μL/L) used in this trial was much lower thanthe minimum concentration of the oil found to be effective in vitro,both EOs significantly inhibited the biofilm formation of isolate S-1187 (Fig. 2A and2C) at this concentration. In addition, nometabolic ac-tivity was observed and none of the biofilm matrices were found to beviable, indicating excellent fungicidal activities of the oils tested. Anti-microbial potencies of EOs have been widely investigated through alarge number of studies aimed at confirming their potential use to over-come themicrobial drug resistance problem (Burt, 2004). Regarding dis-infectant products, numerous plant-derived EOs, such as those fromcitrus, clove, cypress, Lippia spp, oregano, rosemary and thyme, have

Fig. 2. Control of the biofilm formation of Fusarium spp (S-1187) on soft contact lenses by appliapplication. B and D — Inoculated and untreated lens, C — Thyme essential oil application.

been introduced as they have shown potential as antibiofilm agents(Bai and Vittal, 2014; Burt, 2004; Chorianopoulos et al., 2008; Leonardet al., 2010; Selim et al., 2014; Sandasi et al., 2010). Similarly, goodantibiofilm activity against both fungal and bacterial biofilms by ter-penes such as thymol (Upadhyay et al., 2014) and eugenol (Perez-Conesa et al., 2006) has been reported.

4. Conclusions

This study revealed and reinforced the significance of plant extractsin the development of new, affordable, safe and effective with dual ac-tion (antibiofilm and antimicrobial activity) antimicrobial agents. Thesuccess of clove and thyme EOs in inhibiting cell attachment by severalF. oxysporum isolates, as shown in this study, is a potential tool for

cation of clove and thyme essential oils at a concentration of 50 μL/L. A: Clove essential oil

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reducing microbial colonization on contact lenses. This potency is mostprobably a result of the high eugenol and thymol contents, respectively,of the two oils. However, as these tests have all been done in vitro, thenext logical step is further clinical in vivo investigations to confirm if in-fection can be inhibited by the EOs, demonstrating the safe use of theseoils for the prevention of Fusarial keratitis.

Acknowledgments

The authors would like to express their gratitude to TshwaneUniversity of Technology (L714) for funding, to Professor DavidGeiser from Pennsylvania State University for kindly supplying theF. oxysporum isolates, and finally to Professor Combrinck andDr Sandasifor their assistance with the chemistry and antibiofilm assays.

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