The Effect of Juniperus virginiana Essential Oil on Candida albicans Biofilm Formation

albicans is one of the most important natural microbiotas in human. It is responsible for creating various types of candidiasis as opportunistic yeast. Biofilm formation is one of the pathogenic factors of Candida albicans that triggers infection from any surface on which it is formed. In this study, the effect of Juniperus virginiana essential oil (JVEO) on growth, germ tube production and biofilm formation against albicans ATCC 10231 was investigated. JVEO effectively inhibited formation with a minimum inhibitory concentration (MBIC50) of <1250µg/ml. Moreover, it suppressed hyphal growth and germ tube and biofilm formation in that strain. Transcriptomic analysis carried out by qRT-PCR revealed that the expression of HWP1, ALS3 adhesion related genes and CPH1, CYR1, UME6, HGC1 and EED1 hypha formation and maintenance related genes were all suppressed by JVEO. Furthermore, in the cAMP-dependent protein kinase pathway, RAS1 was down regulated but EFG1 was not affected. These results suggest that JVEO may be a potential therapeutic treatment and control of Candida biofilm formation.


Introduction
Biofilms are special microbial structures that play significant roles in triggering infectious diseases and in different deviceassociated infections [1,2]. Candida species are considered to be the most important agents of fungal biofilm which cause infection.
The biofilm formation on abiotic and biotic surfaces is shown to be the most important property of these species. Candida species are commensal organisms that are found in healthy individuals (25-75%), with any tissue damage and inflammation [3]. These species include 50-70% of the commensal organisms which may cause a wide range of superficial to life-threatening infections [4,5]. The presence of biofilm can aggravate the infection due to the establishment of a structure with high tolerance to antifungal agents comparing to planktonic cells and act as a resource for the cells to disseminate to different parts of the body, including an imperative need to investigate new antifungal compounds against C. albicans biofilms. Plants are rich sources of agents with biological effects as have been used for many years in traditional medicine. The natural extract or essential oil obtained from various plants contains many organic compounds with therapeutic usage [9,10]. Terpenoids, terpenes and aromatic substances are three main components of essential oils. From very ancient times these oily liquids have been shown to have strong activity against fungi and bacteria as reported in a number of studies [11,12]. It has been demonstrated that essential oils have significant role in the pharmaceutical industries; moreover, these can be measured as a principal alternative in controlling microorganisms including fungi [13]. Juniperus virginiana is a juniper which is distributed and native in the eastern of USA. J. virginiana is identified with aromatic smell and aptitude to prevent flying insects [14].
Ethanolic and superficial fluid extraction of J. virginiana has been shown to possess inhibitory effect against wood decay fungi [15]. Many studies have been done to demonstrate potential strategies for eradicating and controlling biofilm production in C.
albicans [16]. Furthermore, various studies have been carried out to discover the compounds or products with natural base and antibiofilm or antifungal activity against C. albicans. For instance, Raut et al. [17] have identified eight terpenoids with inhibitory effect on mature biofilm of C. albicans. It has been reported that filamentation, intercellular adherence, and biofilm development were inhibited by phenazines produced by P. aeruginosa [18]. Wong et al. [19] have discovered a small molecule with activity against Candida spp both in vitro and in vivo. Adhesion is the initial step of biofilm formation that is followed by cell proliferation, hypha formation, extracellular matrix production, eventually, cell dispreading [20]. Numerous studies have been conducted on the effect of natural materials on cell adhesion, hypha formation and biofilm formation in C. albicans [21,22]. In the present study, we aimed to investigate the activity of

Materials and Methods
C. albicans ATCC10231 was obtained from pathogenic fungi culture collection of the Pasteur Institute of Iran, cultured on Sabouraud dextrose agar (1% w/v peptone, 4% w/v dextrose, and 1.8% w/v agar) medium and incubated at 28°C for 48h. To prepare cell suspension, the strain was cultured in YPD (1% yeast extract, 2% peptone, and 2% dextrose) liquid medium in orbital  for 24h [21]. After removing the medium, biofilm was lightly washed by sterile PBS buffer and the cells were scrapped from the bottom of the flask. The yeast cells were homogenized by glass beads. Total RNA was extracted using GITC (Guanidium isothiocyanate) reagent and treated with RNase-free DNase [27]. The cDNA was prepared 72°C for 15 sec [21]. The reactions were repeated in triplicate.
The gene expression was normalized by 18S rRNA gene, and the folding changes were determined using the relative threshold method (2−∆∆CT). All data were analyzed by One way ANOVA using GRAPHPAD PRISM 6 (GraphPad Prism Software Inc, San Diego, CA, USA). The differences with P < 0.05 were considered to be significant.  It has been demonstrated that 65%-80% of Candida infections are related to the biofilm formation [29]. A special structure of biofilm is suitable as source of recurrence and represented tolerance to antifungal agents; so, the development of antifungal strategies to avoid biofilm formation or biofilm elimination has become a challenge [30]. Our results showed that essential oil at different concentration inhibited the growth of C. albicans ATCC10231.

Results and Discussion
The results revealed that the fungal growth was inhibited dose with the MIC of 625<MBIC50<1250 µg/ml (Figure 3). The effect of EO on biofilm formation was improved as the concentration of EO was increased; therefore, no biofilm formation was observed in maximum concentration of JVEO (2500 µg/ml). Many studies revealed that natural compounds such as phytochemicals are able to inhibit biofilm formation in C. albicans [31][32][33][34][35]. They reported that the mechanism of action of these compounds were inhibition of the hypha formation and disruption of adhesion; as two essential steps of biofilm formation mechanism. Some studies have been carried out on the antimicrobial and antifungal effects of JVEO previously [12,15]. Here we investigated that JVEO inhibits growth, germ    It has been demonstrated that hypha formation contributes as one of the major virulence of C. albicans [36] and any disruption in this process leads to produce abnormal biofilm formation that is simply removed from the surface [37]. The first step of biofilm formation in C. albicans, is adhesion. Agglutinin-like sequence (ALS) is one of the families of adhesion molecules which include eight members (ALS1-ALS9). This family is expressed by ALS genes that encode cell wall glycosyl-phosphatidylinositol proteins which are required for the attachment of the yeasts to the surfaces [16,31]. Within this family, ALS3, due to its important role in biofilm production, has been shown to be of great importance among other genes in the family. Furthermore, ALS3, is represented to be highly expressed in oral epithelial cell infection [38,39]. HWP1, another adhesion protein, is a manoprotein which exists in the cell wall of albicans including: BCR1, TEC1 and EFG1 [41]. The genes consist of SAPs, LIP and PLBs also have essential role in colonization and disseminated infection by effect on the host cell membrane [42].
The data showed that the expression of CPH1, EED1 and CYR1 genes was suppressed but not as much as previous genes. The expression of EFG1 didn't change much. HST7 showed increased relative expression in two concentrations (312.5 and 1250 µg/ml) ( Figure 5). In general, qRTPCR indicated that treatment with JVEO can strongly affect significant genes in relation to hypha and biofilm formation in C. albicans. Terpinen-4-ol is an isomer of terpineol that is found in the essential oil of aromatic plants such as tea tree oil.
It revealed biological properties such as antimicrobial, antioxidant and anti-inflammatory activity [43,46]. Here we studied on JVEO the highest percentage of which is reported to be Terpinen-4-ol previously [47]. Based on this, it can be concluded that the antibiofilm effect of the extract is due to the presence of this compound.
Given the widespread prevalence of fungal infections, finding effective ways to more adequately treat patients is essential today.
This is achieved by studying the various pathogenic properties of these species, including biofilm formation.