Activity of Free and Liposome-Encapsulated Essential Oil from Lavandula angustifolia against Persister-Derived Biofilm of Candida auris

The high virulence of Candida auris, a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional antifungals. Its biofilm lifestyle certainly promotes the prolonged survival of C. auris after disinfection or antifungal treatments. In this work, for the first time, we detected persister cells in a biofilm of C. auris in a microwell plate model, following caspofungin treatment. Furthermore, we showed how persisters can progressively develop a new biofilm in situ, mimicking the re-colonization of a surface which may be responsible for recalcitrant infections. Plant-derived compounds, such as essential oils, may represent a valid alternative to combat fungal infections. Here, Lavandula angustifolia essential oil, as free or encapsulated in liposomes, was used to eradicate primary and persister-derived biofilms of C. auris, confirming the great potential of alternative compounds against emergent fungal pathogens. As in other Candida species, the action of essential oils against C. auris involves ROS production and affects the expression of some biofilm-related genes.


Introduction
Since its discovery in 2009 [1], the pathogenic yeast Candida auris has elicited several worldwide outbreaks; thus, it is now considered as a global threat to public health, with an estimated mortality range from 28% to 66%. C. auris is associated with hospital-acquired infections and clinical isolates generally possess a multidrug-resistant (MDR) profile, with about 30% of isolates showing reduced susceptibility to amphotericin B, and 5% being resistant to echinocandins [2].
Notwithstanding, the phenotypic features of C. auris are similar to other Candida species; its peculiar thermo-and osmotolerance, together with its intrinsic resistance to conventional antifungals and tolerance to common disinfectants, certainly constitute advantages for spreading and virulence [3,4].
The occurrence of C. auris on different medical devices and surfaces has been reported [5,6] to be probably associated with the formation of cell aggregates and biofilms.

Detection of Persisters Cells in C. auris Biofilms
Candida auris DSM 21092 was able to form strong biofilms in th microwell polystirene plate ( Figure 1). Total biomass of the sessile between 24 and 48 h, showing that a more mature biofilm was for incubation period.  Once established on the polystirene surface, the 48 h C. auris biofilm was treated with increasing caspofungin concentrations up to 200 × MIC, with an MIC value of the examined C. auris strain being 1 ± 0.2 µg mL −1 . Due to caspofungin treatment, a dose-dependent killing of the biofilm population was observed ( Figure 2).  The biphasic-killing curve revealed that a subpopulation of cells in the biofilm not susceptible, even to the highest antifungal concentration used.
According to similar curves reported for biofilms of several Candida spp. subjec an antifungal challenge, also in the case of C. auris, the phenotypic tolerant va constitute the so-called persisters, representing a very small fraction (0.05%) of the sessile population.
Live persister cells in the biofilm could be observed by Confocal Laser Sca Microscopy (CLSM), after staining with Syto9 and propidium iodide (PI) (Figu Indeed, since SYTO9 and PI differ in their ability to penetrate healthy cells, the use mixture SYTO9/PI was revealed to be useful in identifying persisters cells in C. biofilm population treated with 200 mg mL −1 caspofungin, where the very few persisters appear bright green ( Figure 3). The biphasic-killing curve revealed that a subpopulation of cells in the biofilm was not susceptible, even to the highest antifungal concentration used.
According to similar curves reported for biofilms of several Candida spp. subjected to an antifungal challenge, also in the case of C. auris, the phenotypic tolerant variants constitute the so-called persisters, representing a very small fraction (0.05%) of the entire sessile population.
Live persister cells in the biofilm could be observed by Confocal Laser Scanning Microscopy (CLSM), after staining with Syto9 and propidium iodide (PI) (Figure 3). Indeed, since SYTO9 and PI differ in their ability to penetrate healthy cells, the use of the mixture SYTO9/PI was revealed to be useful in identifying persisters cells in C. auris biofilm population treated with 200 mg mL −1 caspofungin, where the very few live persisters appear bright green ( Figure 3).
When fresh medium was dispensed into the wells of a microplate where a C. auris biofilm (primary biofilm) had been challenged with 200 µg mL −1 caspofungin, a new biofilm progressively developed starting from the residual living cells (persisters). Indeed, the number of living cells in the persister-derived biofilm increased over time, achieving the same value as that of the primary biofilm after 12 d (Figure 4).  When fresh medium was dispensed into the wells of a microplate where a C. auris biofilm (primary biofilm) had been challenged with 200 µg mL −1 caspofungin, a new biofilm progressively developed starting from the residual living cells (persisters). Indeed, the number of living cells in the persister-derived biofilm increased over time, achieving the same value as that of the primary biofilm after 12 d (Figure 4).

Composition of L. angustifolia Oil
According to GC-MS analysis, the following major constituents, as shown in Table 1

Characterization of Liposome-Encapsulated Oil of L. angustifolia
Liposomes loaded with oil of L. angustifolia were characterized. The hydrodynamic diameter (DH) and polydispersity index (PDI) were measured using dynamic light scattering (DLS). Three independent experiments were performed for each sample and each measurement was performed at least in triplicate. Liposome solutions present a polydispersity index (PDI) < 0.3 indicating a good size distribution ( Table 2). Moreover, our data clearly indicate that the L. angustifolia oil is completely encapsulated in the experimental conditions used in this study. In particular, the ratio between the oil volume and the lipid volume is 0.1, indicating that the encapsulated oil is not able to destroy the liposome packing. When C. auris cultures were incubated in the presence of increasing concentrations of either L. angustifolia free or liposome-encapsulated oil, growth reduction was monitored during 24 h incubation. In Figure 5A,B, the effect of the examined compounds on planktonic fungal growth is reported; a significative growth reduction was already observed when cells were incubated in the presence 0.01% v/v free or encapsulated oil, but a slight effect was already visible at a concentration two-fold lower. Both 48 h primary and persister-derived biofilm of C. auris were eradicated when treated with L. angustifolia free or encapsulated oil, in a concentration dependent manner ( Figure 6). Eradication of Antibiotics 2022, 11, 26 7 of 16 biofilms was obtained at concentrations higher than those determining planktonic growth reduction, in accordance with the general lower susceptibility of biofilms to antimicrobial compounds. Furthermore, no significant difference in biofilm eradication was observed between the two oil formulations, with a value of more than 80% eradication achieved at a concentration of 0.5% v/v ( Figure 6). No eradication at all was detected by using the empty liposome (data not shown), showing that the eradication effect was due to the encapsulated oil, and not to the liposome components. during 24 h incubation. In Figure 5A,B, the effect of the examined compounds on planktonic fungal growth is reported; a significative growth reduction was already observed when cells were incubated in the presence 0.01% v/v free or encapsulated oil, but a slight effect was already visible at a concentration two-fold lower. Both 48 h primary and persister-derived biofilm of C. auris were eradicated when treated with L. angustifolia free or encapsulated oil, in a concentration dependent manner ( Figure 6). Eradication of biofilms was obtained at concentrations higher than those determining planktonic growth reduction, in accordance with the general lower susceptibility of biofilms to antimicrobial compounds. Furthermore, no significant difference in biofilm eradication was observed between the two oil formulations, with a value of more than 80% eradication achieved at a concentration of 0.5% v/v ( Figure 6). No eradication at all was detected by using the empty liposome (data not shown), showing that the eradication effect was due to the encapsulated oil, and not to the liposome components.  It is known that the exposure of EOs on Candida cells provokes an incre intracellular Reactive Oxygen Species (ROS), which in turn may determine a c damage and eventually cell death, which in the case of sessile cells leads to It is known that the exposure of EOs on Candida cells provokes an increase in intracellular Reactive Oxygen Species (ROS), which in turn may determine a cellular damage and eventually cell death, which in the case of sessile cells leads to their detachment. ROS increase was detected also in C. auris cells in the persister-derived biofilm, when treated with free or encapsulated oil for eradication, and the ROS increase was concentration-dependent ( Figure 7). derived biofilm: free oil (dark grey); liposome-encapsulate oil (light grey). Bars with di (a-e) are significantly different, bars with the same letters (a-e) are not significa (Tukay's test, p < 0.05).
It is known that the exposure of EOs on Candida cells provokes an intracellular Reactive Oxygen Species (ROS), which in turn may determin damage and eventually cell death, which in the case of sessile cells lea detachment. ROS increase was detected also in C. auris cells in the persi biofilm, when treated with free or encapsulated oil for eradication, and the R was concentration-dependent ( Figure 7).  To elucidate the potential mechanisms by which L. angustifolia essential oil alone or encapsulated in a liposome (concentration of 0.05%) eradicated C. auris persister cells biofilms, genes expression analysis of ALS5, ERG11, CDR1 and HOG1 was performed using qRT-PCR ( Figure 8, Table S2).
Data demonstrated that ERG11 (p < 0.0001) was significantly downregulated in biofilms treated with liposome confirming that one of the mechanisms of antifungal activity could be by binding to the membrane ergosterol also in a secondary biofilm, while upregulated in persister biofilm treated with oil alone. L. angustifolia essential oil upregulated this gene with a 2.6-fold increase (Figure 8, Supplementary Materials: Table S2). In addition, the expression of ALS5 was also inhibited after treatment with liposome of 2-fold, on the contrary significantly upregulated in biofilm treated with only essential oil with a 3.2-fold increase in expression levels (see also Table S2). HOG1 gene was up-regulated (4.8-fold) and down-regulated (2.6-fold) by L. angustifolia liposome-encapsulated oil and L. angustifolia oil, respectively (p < 0.0001; Figure 8). Finally, CDR1 was up-regulated by both treatments (2.5-fold and 2.8-fold increase in expression levels, respectively), as seen also in Table S2. To elucidate the potential mechanisms by which L. angustifolia essential oil alone or encapsulated in a liposome (concentration of 0.05%) eradicated C. auris persister cells biofilms, genes expression analysis of ALS5, ERG11, CDR1 and HOG1 was performed using qRT-PCR ( Figure 8, Table S2).  Table S2 for the values). A Brown-Forsythe test Sisak's test (**** p < 0.0001; *** p < 0.001).
Data demonstrated that ERG11 (p < 0.0001) was significantly downregulated in biofilms treated with liposome confirming that one of the mechanisms of antifungal activity could be by binding to the membrane ergosterol also in a secondary biofilm, while upregulated in persister biofilm treated with oil alone. L. angustifolia essential oil upregulated this gene with a 2.6-fold increase (Figure 8, Supplementary Materials: Table  S2). In addition, the expression of ALS5 was also inhibited after treatment with liposome of 2-fold, on the contrary significantly upregulated in biofilm treated with only essential oil with a 3.2-fold increase in expression levels (see also Table S2). HOG1 gene was upregulated (4.8-fold) and down-regulated (2.6-fold) by L. angustifolia liposomeencapsulated oil and L. angustifolia oil, respectively (p < 0.0001; Figure 8). Finally, CDR1 was up-regulated by both treatments (2.5-fold and 2.8-fold increase in expression levels, respectively), as seen also in Table S2.

Discussion
Nowadays, failure in the treatment of Candida spp. infection has highlighted the relevant role exerted by the so-called persister cells in biofilms.  Table S2 for the values). A Brown-Forsythe test Sisak's test (**** p < 0.0001; *** p < 0.001).

Discussion
Nowadays, failure in the treatment of Candida spp. infection has highlighted the relevant role exerted by the so-called persister cells in biofilms.
In this work, we suggest that persister cells are present also in biofilms of the emerging pathogen C. auris. Indeed, C. auris with multiple drug-resistances against prominent antifungals has occurred independently in different countries, so that a clinical alert identifying C. auris as an emerging "superbug" has been released [27,28]. The incoming of C. auris also in Italy is recent, and its spread has probably been facilitated during the SARS-CoV-2 pandemic, due to the increase in antimicrobial resistance for the unconditioned use of broad-spectrum antimicrobials [29].
Here, using an in vitro model, we showed that when a mature biofilm of C. auris was treated with caspofungin, a small fraction of cells tolerant to high concentrations of the antifungal was detected. C. auris persisters, which survived the caspofungin challenge, were able to re-colonize the support when incubated in the presence of fresh medium. This proliferation mimics the re-colonization of a surface which may follow a strong antifungal treatment, as in the case of the lock therapy [30], which utilizes prolonged instillation of a solution containing high concentrations of antimicrobial agent within an infected intravascular catheter, in an attempt to sterilize it and control bloodstream infections. Therefore, due to the survival of the tolerant persister subpopulation, in the long-term, re-population of the catheter interior may occur, thus leading to chronic infections.
Essential oils fractionally distilled from plants have drawn increased attention because of their multiple pharmacological properties such as antibacterial, antifungal, and antiviral activities. To date very few scientific papers have investigated the effect of EOs [18] and their components such as monoterpene phenols against planktonic C. auris [31], and none reported the effect of EOs on C. auris biofilms. In particular, L. angustifolia oil on C. auris has not been investigated so far.
In the L. angustifolia oil examined, the main components are linalyl acetate and linalool, according to the composition of this oil as reported by other authors [32]. The same authors attributed to these two components, and especially to the monoterpene linalool, the antifungal activity against C. albicans, though the antimicrobial effects of the oil may be the result of the synergistic action of its major and minor components [33].
Our results clearly show that low concentrations of free and liposome-encapsulated L. angustifolia oil are sufficient to inhibit the growth of C. auris. Further, and more interestingly, the same compounds were effective to eradicate biofilms formed by C. auris in vitro, either the biofilm developed starting form planktonic cells (primary biofilm) and that developed from persister cells. The effective concentrations remained unchanged, indirectly showing that persisters were transient phenotypic variants instead of resistant cells, giving rise to a new biofilm which responds to the antifungal treatment similarly to the primary biofilm.
L. angustifolia encapsulation into liposomes can be used to overcome some critical issues. The use of liposomes also provides the necessary protection against essential oil oxidation, and thus enhances the oil solubility.
Researchers have investigated the complex action modes of EOs from various perspectives. The main mechanism is based on their lipophilic character, which facilitates the access of the hydrophobic compounds to the cytoplasmic membrane, causing membrane damage, loss of intracellular substances, and finally microbial death [34]. The effect of oil or its active components on Candida spp. is certainly mediated by ROS generation [35]. Additionally, for C. auris persister cells in the biofilm, following exposure to free or encapsulated oil, we detected an increase in intracellular ROS depending on used concentrations, showing that the antifungal effect of the two compounds is mediated by ROS accumulation. It is likely that also for C. auris, ROS production is associated with cell damage and to the activation of the apoptotic pathway.
The impact on genes encoding for efflux pumps (CDR1), ergosterol biosynthesis (ERG11), hyphae-specific gene (ALS5) and stress related (HOG1) in the cells of the persister derived biofilm confirmed the treatment efficacy.
Indeed, in our study, the gene related to ergosterol biosynthesis, ERG11, was downregulated during eradication with liposome encapsulated oil, and this effect could be attributed to its cell membrane-targeting activity, while we could observe a significant increase in expression of the ERG11 gene when the selected L. angustifolia oil were applied to persister-derived biofilm.
Although it had been previously shown that linalool, one of the major components of Lavandula oil, inhibited hyphal growth and biofilm formation of C. albicans by downregulating, between others, the expressions of ALS3 [36], in the present study, we observed that only the gene expression of hyphae-specific ALS5 in biofilm-persister cells treated with L. angustifolia liposome-encapsulated were downregulated. It appears that in persister-cells derived biofilm treated with essential oil only, other hyphal growth and biofilm formation genes had been involved to diminish its virulence.
HOG1 was upregulated, when encapsulated oil was used, and since HOG1 is involved in the oxidative stress response, this result strongly supports the implication of the HOG1 pathway in the C. auris response to L. angustifolia encapsulated oil. Our results are interesting, since part of the side effects caused by antifungal compounds are due to their ability to trigger oxidative stress. When intracellular ROS is elevated, C. auris will initiate an effective oxidative stress response regulated by the transcription factor HOG1, as shown for other Candida spp. Regarding the expression of CDR1, one of the genes encoding for the efflux pump, we observed that both the application of free lavender and the encapsulated one upregulated this gene. We hypothesized that other genes correlated with resistance should be investigated.

Materials and Methods
In Figure 9 the flow chart of the experimental design is reported. are interesting, since part of the side effects caused by antifungal compounds are due to their ability to trigger oxidative stress. When intracellular ROS is elevated, C. auris will initiate an effective oxidative stress response regulated by the transcription factor HOG1, as shown for other Candida spp. Regarding the expression of CDR1, one of the genes encoding for the efflux pump, we observed that both the application of free lavender and the encapsulated one upregulated this gene. We hypothesized that other genes correlated with resistance should be investigated.

Materials and Methods
In Figure 9 the flow chart of the experimental design is reported.
. Figure 9. Flow chart of study design. Figure 9. Flow chart of study design.

Strain and Culture Condition
Candida auris strain DSM 21092 (DSMZ German Collection) was maintained in glycerol at −80 • C. The strain was cultured in shake flasks containing Tryptic Soy Broth (TSB) (OXOID, Basingstoke, UK) with 1% w/v glucose, starting from an overnight pre-culture prepared in the same medium. Shake flasks were incubated at 37 • C, 200 rpm.

GC-MS Analysis of L. angustifolia Oil
Composition of L. angustifolia oil (Natura Zen Srl, Italy) (Lot n. 0281-4392105-2023/2; expiry date 12/2023) was determined by GC-MS analysis performed with an Agilent 6850 GC (Milan, Italy), equipped with an HP-5MS capillary column (5% phenyl methyl poly siloxane stationary phase), coupled to an Agilent 5973 Inert MS detector operated in the full scan mode (m/z 18-550) at a frequency of 3.9 Hz and with the EI ion source and quadrupole mass filter temperatures kept, respectively, at 230 • C and 200 • C. Helium was used as carrier gas at a flow rate of 1 mL·min −1 . The injector temperature was 180 • C and the temperature ramp raised the column temperature from 50 • C to 240 • C: 50 • C for 5 min; 5 • C·min −1 until reaching 180 • C; and 10 • C·min −1 until reaching 240 • C. The solvent delay was 3 min. Compounds were identified by comparing their EI mass spectra at 70 eV with spectra of known substances present in the NIST 14 mass spectral library (NIST 14, https://www.nist.gov/srd/nist-standard-reference-database-1a (accessed on 3 November 2021)). Moreover, the identification was supported by Kovats retention index (RI) calculated for each analyte by the Kovats equation, using the standard n-alkane mixture in the range C7-C40 (Sigma-Aldrich, Saint Louis, MO, USA).
The relative percentage of each compound is defined by the area of selected peak divided for the sum of the peak area.
Small unilamellar vesicles (SUV) consisting of PC/Chol (70/30 mol/mol) were prepared. The liposomes were prepared at a total lipid concentration of 0.1 mM, mixed aggregates of PC, Chol and L. angustifolia oil were dissolved in chloroform and subsequently the solvent was evaporated under a stream of nitrogen gas. Appropriate amounts of L. angustifolia, in particular 20 µL were added to 200 µL of liposomes, so that oil in the liposome formulation had a concentration of 10% v/v. Following, lipid films were hydrated with water for 1 h. The lipid suspension was freeze-thawed 6 times, Liposomes were obtained sonicating the solution for 40 min. Unloaded drugs were removed by the Sephadex G50 column to purify the final formulation and evaluate the efficiency of encapsulation. Dynamic light scattering (DLS) measurements were made using Zetasizer Nano-ZS (Malvern In-struments, Worcestershire, UK), to check the Zeta potential and the size ( Table 2). The analysis was performed with He-Ne laser 4 mW operating at 633 nm at scattering angle fixed at 173 • and at 25 • C. The results were determined three times for each sample and each measurement was performed at least in triplicate.

Determination of Caspofungin Minimum Inhibitory Concentration
The minimum inhibitory concentrations (MIC) of caspofungin (Sigma-Aldrich Co, St. Louis, MO, USA) against C. auris were determined with a broth microdilution method, as described by CLSI-M27-A3 [37] with some modifications. Concentrations of caspofungin from 0.1 to 5 µg mL −1 were added to wells of a 96-well microplate containing the microorganism in TSB with 1% w/v glucose. The plate was incubated at 37 • C for 24 h. MIC value was determined as the lowest concentration inhibiting fungal growth at 590 nm using a microplate reader (Synergy H4; BioTek Instruments, Agilent Technologies, Winooski VT 05404 USA).

Effect of L. aungustifolia Oil and Liposome-Encapsulated Oil on Planktonic C. auris Cells
The effect of both L. angustifolia oil and liposome-encapsulated oil on planktonic cells of C. auris was evaluated incubating fungal cultures in shake flasks containing TSB medium with 1% w/v glucose, in the presence of different oil or liposome-encapsulated oil concentrations, ranging from 0.005 to 0.5% v/v. Stock solutions of the oil were prepared diluting it in 0.1% v/v Tween 80; stock solutions of the liposome-encapsulated oil were prepared in distilled water. The cultures were inoculated with proper aliquots of an overnight pre-culture prepared in the same conditions. The flasks were incubated at 37 • C for 24 h at 200 rpm. Cell growth was monitored spectrophotometrically at OD 590 and growth reduction expressed as percentage of the control.

Biofilm Formation and Quantification
C. auris cells used to start biofilm formation were obtained collecting cells in midexponential phase from shake flask cultures in TSB with glucose 1% and re-suspending them in RPMI 1640 (w/o glutamine) (Lonza, Switzerland) to 1 × 10 6 CFU mL −1 . Aliquotes of 100 µL of cell suspensions were loaded into a polystyrene 96-well microplate and incubated at 37 • C for 24 or 48 h. Wells were then washed three times with 200 µL PBS to remove non-adherent cells.
Quantification of the biofilm biomass was performed by crystal violet staining, as previously described [33]. To quantify the biofilm formation, the optical density cut off value (ODc) was calculated as the three standard deviations above the mean OD of the negative. Final OD value of the tested strain was interpreted as negative (OD ≤ ODc), weak (ODc ≤ OD ≤ 2ODc), moderate (2ODc < OD ≤ 4ODc), or strong (4ODc < OD) biofilm former [38].

Detection of Persisters Cells
The quantification of persister cells in C. auris biofilm was performed as previously described [11], with some modifications. Briefly, the 48 h C. auris biofilm, grown as previously described, was challenged with caspofungin buffered to pH 7 with 0.165 M morpholinopropanesulfonic acid (MOPS, Sigma-Aldrich Co, St. Louis, MO, USA) at concentrations ranging from 5 to 200 µg mL −1 for 24 h at 37 • C in RPMI medium. Caspofungin stock solution at concentration of 500 mg mL −1 was prepared in DMSO 1.3% v/v. Preliminarly, it was shown that DMSO was not toxic against C. auris up to concentrations of 5% v/v (data not shown).
Biofilms were abundantly washed with PBS, then disrupted by scraping and vortexed vigorously for 30 s before serial dilution and plating on YPD (1% yeast extract, 2% bactopeptone, 2% glucose) agar medium. Plates were incubated at 37 • C for 48 h. The number of surviving cells (persisters) was expressed as CFU per well.

Development of Persister-Derived Biofilm of C. auris
The small number of cells not susceptible to caspofungin treatment (persisters) in the primary 48 h biofilm were the inoculum of a new biofilm (persister-derived biofilm), which developed in situ in the 96-well microplate.
Therefore, to obtain the persister-derived biofilm, following the caspofungin treatment of the primary biofilm, the wells were abundantly washed with PBS, and incubation was allowed to continue for 12 d during which 200 µL YPD medium were added to each well every 24 h. Biofilm mass was estimated every 48 h by CFU assay, as described above.

Eradication of Biofilm with Free Liposome-Encapsulated L. angustifolia Oil
The eradication activity of L. angustifolia oil, fand liposome-encapsulated oil, was evaluated exposing C. auris primary and persister-derived biofilms for 24 h to different concentrations of each of the two compounds ranging from 0.05 to 1% v/v, in RPMI medium and quantified the residual biomass by crystal violet assay. The percentage of eradication was calculated as biofilm reduction % = (OD 570 control − OD 570 sample/OD 570 control) × 100, where OD 570 control and OD 570 sample corresponded to the untreated and treated biofilm, respectively.  The effect of L. angustifolia free and liposome-encapsulated oil on the expression of ERG 11, ALS 5, HOG 1 and CDR1 (Table S1) [39,40] in persister-derived biofilms of C. auris was analysed using qRT-PCR. Briefly, persister-derived biofilms were treated with 0.05% v/v oil or liposome-encapsulated oil for 24 h at 37 • C. Then, the biofilms were scraped from the polystyrene surface, and collected by centrifugation (3000× g, 5 min). The pellets were used to extract total RNA using the Direct-zolTM RNA Miniprep Plus Kit (ZYMO RESEARCH). The purity and concentration of the extracted RNA were verified using Nanodrop spectrophotometer 2000 (Thermo Scientific Inc., Waltham, MA USA). A total of 1000 ng of extracted RNA was retrotranscribed with an iScript™ cDNA Synthesis kit (Bio-Rad, Milan, Italy) and it was used as a template in a reaction containing primer (0.3 mM) and 1× SensiFASTTM SYBR Green master mix (Meridiana Bioline, Aurogene, Rome, Italy). PCR amplifications were performed in a AriaMx Real-Time PCR instrument (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions. Fluorescence was measured using Agilent Aria 1.7 software (Agilent Technologies, Inc.). The expression of gene was analyzed and normalized against ACT1 gene using REST software (Relative Expression Software Tool, Weihenstephan, Germany, version 1.9.12) based on the Pfaffl method [41,42]. Relative expression ratios greater than ±1.5 were considered significant. A Brown-Forsythe test was applied, followed by Tukey's post hoc test.

Confocal Laser Scanning Microscopy
The occurrence of persisters in C. auris biofilm was also evaluated by Confocal Laser Scanning Microscopy (CLSM). The biofilm was allowed to form for 48 h on NuncTM Lab-Tek ® 8-well Chamber Slides (n • 177445; 209 Thermo Scientific, Ottawa, ON, Canada) and treated with 200 µg mL −1 caspofungin. Then, it was thoroughly washed with PBS and stained with 5 µM SYTO9 (ThermoFisher Scientific MA, USA) that penetrates both viable and nonviable cells, followed by 20 µM propidium iodide, a non-vital nuclear stain commonly used for identifying dead cells, for 15 min at room temperature, in the dark. Observation was performed with Zeiss LSM700 microscope at 20× magnification.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism Software (version 8.02 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com (accessed on 6 November 2021)). Data were reported as the mean ± standard deviation obtained from three independent experiments. Data of development of a new biofilm from persisters and of effect of L. angustifolia free and liposome-encapsulated oil on planktonic cells were analysed by one-way ANOVA followed by Holm-Sidak's test (p < 0.05); data of eradication were analysed by two-way ANOVA followed by Tukay's test for the difference between the groups.

Conclusions
Our results show that persister cells tolerant to caspofungin are present in a biofilm of C. auris. Persisters can progressively re-colonize the support, giving rise to a new persister-derived biofilm.
L. angustifolia oil shows antifungal and antibiofilm properties in the two formulations tested towards C. auris, being effective also against persister-derived biofilm.
Overall, the results indicate the possibility of extending the use of EOs, confirming the great potential represented by the use of alternative compounds to combat emergent fungal pathogen infections.