Fungal Biofilms As a Valuable Target for the Discovery of Natural

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Review Fungal Bioﬁlms as a Valuable Target for the Discovery of Natural Products That Cope with the Resistance of Medically Important Fungi—Latest Findings

Estefanía Butassi 1,†, Laura Svetaz 1,†, María Cecilia Carpinella 2, Thomas Efferth 3 and Susana Zacchino 1,*

1 Pharmacognosy Area, School of Biochemical and Pharmaceutical Sciences, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina; [email protected] (E.B.); [email protected] (L.S.) 2 Fine Chemical and Natural Products Laboratory, IRNASUS CONICET-UCC, Universidad Católica de Córdoba, Córdoba 5016, Argentina; [email protected] 3 Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: The development of new antifungal agents that target biofilms is an urgent need. Natural products, mainly from the plant kingdom, represent an invaluable source of these entities. The present review provides an update (2017–May 2021) on the available information on essential oils, propolis, extracts from plants, algae, lichens and microorganisms, compounds from different natural sources and nanosystems containing natural products with the capacity to in vitro or in vivo modulate fungal biofilms. The search yielded 42 articles; seven involved essential oils, two Citation: Butassi, E.; Svetaz, L.; Carpinella, M.C.; Efferth, T.; Zacchino, Brazilian propolis, six plant extracts and one of each, extracts from lichens and algae/cyanobacteria. S. Fungal Biofilms as a Valuable Twenty articles deal with the antibiofilm effect of pure natural compounds, with 10 of them in- Target for the Discovery of Natural cluding studies of the mechanism of action and five dealing with natural compounds included in Products That Cope with the nanosystems. Thirty-seven manuscripts evaluated Candida spp. biofilms and two tested Fusarium Resistance of Medically Important and Cryptococcus spp. Only one manuscript involved Aspergillus fumigatus. From the data presented Fungi—Latest Findings. Antibiotics here, it is clear that the search of natural products with activity against fungal biofilms has been a 2021, 10, 1053. https://doi.org/ highly active area of research in recent years. However, it also reveals the necessity of deepening 10.3390/antibiotics10091053 the studies by (i) evaluating the effect of natural products on biofilms formed by the newly emerged and worrisome health-care associated fungi, C. auris, as well as on other non-albicans Candida spp., Academic Editor: María Cryptococcus sp. and filamentous fungi; (ii) elucidating the mechanisms of action of the most active Auxiliadora Dea-Ayuela natural products; (iii) increasing the in vivo testing.

Received: 26 July 2021 Accepted: 26 August 2021 Keywords: fungal biofilm; antifungal resistance; natural products; Candida spp.; Fusarium spp.; Published: 30 August 2021 Cryptococcus spp.; filamentous fungi; mechanisms of antibiofilm action

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. In recent decades, fungi has emerged as a major cause of life-threatening invasive human infections, in particular among immunocompromised patients [1–3], particularly those with human immunodeficiency virus (HIV), cancer patients receiving chemotherapy, transplant recipients, extremely aged persons and subjects in intensive care units [4,5]. Copyright: © 2021 by the authors. Fungal infections lead to mortalities estimated in 1.5 million per year, having a great impact Licensee MDPI, Basel, Switzerland. on global human health [5]. The main purpose of this review is to provide an updated This article is an open access article analysis of the natural products with a capacity of inhibiting fungal biofilms, published distributed under the terms and from 2017 to May 2021. conditions of the Creative Commons We collected papers on essential oils (EOs), propolis, extracts from plants, algae, Attribution (CC BY) license (https:// lichens and microorganisms, metabolites obtained from these sources and nanosystems. creativecommons.org/licenses/by/ 4.0/).

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1.1. Most Common Etiological Agents Causing Fungal Infections The most common fungi identiﬁed in systemic infections are the yeasts of the Candida and Cryptococcus genera, as well as the ﬁlamentous fungi of the Aspergillus genus [6–8]. Candida spp. produce a 40% mortality rate [2,9], while cryptococcosis and aspergillosis produce a mortality rate between 20% to 90% [9]. Among Candida spp., C. albicans showed to be the fourth most common cause of nosocomial bloodstream infections [10], although a recent global shift in epidemiology towards non-albicans Candida spp., such as C. glabrata, C. parapsilopsis, C. tropicalis, C. krusei and lastly, C. auris, has been detected [11,12]. Apart from the mentioned fungi, other species such as Pneumocystis jirovecii, Histoplasma capsulatum and the mucormycetes of Mucor, Absidia and Rhizopus genera, are important fungal pathogens responsible for the majority of serious fungal diseases [13–15].

1.2. Available Antifungal Agents Currently, five classes of antifungal agents, the polyenes, azoles, allylamines, echinocandins and pyrimidines are used for the treatment of fungal infections in human beings [16,17]. Some of them target ergosterol, an essential component of the fungal membrane, either by binding to it (i.e., polyenes such as amphotericin B) or by interfering with different steps of its biosynthesis (i.e., triazoles and allylamines) [16,17]. In particular, the interaction or sequestration of ergosterol by amphotericin B disturbs the membrane, which leads to an increased permeability and the leakage of the intracellular components resulting in the death of the pathogen [18–20]. Previous publications demonstrated that amphotericin B is also able to induce the generation of reactive oxygen species (ROS), as an additional mechanism to achieve the fungicidal activity [21]. On the other hand, the triazoles (i.e., fluconazole, voriconazole, itraconazole, isavuconazole and posaconazole) inhibit the enzyme lanosterol 14α-demethylase responsible for the demethylation of 14α-lanosterol [16] to form ergosterol in a process dependent on the cytochrome P450 system [22,23]. Ally- lamines, such as terbinafine, interfere with the activity of squalene epoxidase, the enzyme that catalyzes the stereospecific epoxidation of squalene to 2,3-(S)-oxidosqualene involved in ergosterol synthesis [24–26]. In addition to ergosterol, 1,3-β glucan, present in fungal cell walls, is another target for antifungal drugs due to its role in fungal growth, integrity and division as well as its participation as a virulence factor [27,28]. The echinocandins (i.e., caspofungin, micafungin and anidulafungin) exhibit a noncompetitive inhibition of the 1,3-β-D-glucan synthase, leading to a deficiency of this polymer [23,29]. The pyrimidine analogue flucytosine, selectively interferes with fungal DNA synthesis by inhibiting the thymidylate synthetase, a key enzyme acting as a crucial source of thymidine. Although the different types of discovered antifungal drugs have enabled progress in the management of fungal infections, serious problems regarding side effects, limited options of antifungals and mainly the development of drug resistance [30–33], highlight the urgent need of exploring new therapeutic approaches that overpass the unmet clinical needs [34–36].

1.3. Fungal Biofilms The most difficult-to-eradicate mycoses are not caused by planktonic cells, but by im- mobilized fungi (sessile cells) that form well-structured biofilms with the ability to adhere to different surfaces, organs or to medical devices such as catheters [37,38]. Biofilms (Figure1) are the predominant growth lifestyle of many opportunistic fungal pathogens, e.g., albicans and non-albicans Candida spp., Cryptococcus neoformans, Cryptococcus gatti, Trichosporon asahii, Rhodotorula spp., Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Coccidioides immitis, Pneumocystis spp., Fusarium spp. and many others [39]. The National Institute of Health estimated that microbial biofilms were responsible for over 80% of all infections [40]. A biofilm is defined as a community of microorganisms encapsulated in a self- produced exopolysaccharide matrix (EPM) attached to a biotic or abiotic surface [41,42], which plays a key role in its resistance [43]. The composition of the EPM produced by differ- Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 33

[39]. The National Institute of Health estimated that microbial biofilms were responsible for over 80% of all infections [40]. A biofilm is defined as a community of microorganisms encapsulated in a self-produced exopolysaccharide matrix (EPM) attached to a biotic or abiotic surface [41,42], which plays a key role in its resistance [43]. The composition of the EPM produced by different fungi has been reviewed by Mitchell et al. [44]. Particularly, it contains carbohydrates, hexosamine, phosphorus, proteins, uronic acid and environmental DNA (eDNA) which were initially identified in C. albicans EPM [45]. This matrix is composed by 55% protein, 25% carbohydrate, 15% lipid and 5% nucleic acids [46]. The most abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β- 1,6-glucans (13%) in an apparent mannan-glucan complex, while β-1,3 glucan comprised only a small portion of the total EPM carbohydrates [46]. The presence of β-1,3 glucan has been recently reported in C. glabrata biofilms [47] being a key carbohydrate component in fungal cell walls [47,48].

1.4. Stages in the Development of Biofifilms The development of a biofilm involves five stages that were clearly explained and graphed by Stoodley et al. [49], Ramage et al. [50], Mayer et al. [51] and other authors [43,52–54]. In stage 1, planktonic cells adhere to a biotic or abiotic surface, followed by a yeast-to-hyphal transition (Figure 1a,b). In the second stage, EPM is produced, resulting in a firmly adhered irreversible attachment. In stage 3 an early biofilm architecture is de-

Antibiotics 2021, 10, 1053 veloped, while in stage 4 the biofilm reaches maturation in a three-dimensional structure3 of 31 (Figure 1c). Finally, in stage 5, single planktonic cells are dispersed from the mature biofilm. This dispersion is an important step in the fungal biofilm development cycle, which can induce either bloodstream or invasive fungal infections [55] leading to an increased pathogenicityent fungi has [56] been and reviewed high risk by Mitchellof mortality et al. [57,58]. [44]. Particularly, This consortium it contains of cells carbohydrates, offers the optimumhexosamine, conditions phosphorus, for the proteins,fungi to obtain uronic nu acidtrients, and environmentaldispose waste products DNA (eDNA) and protect which fromwere the initially environment identiﬁed [58]. in OnceC. albicans the biofilmEPM [is45 formed,]. This matrix the sessile is composed cells communicate by 55% protein, with each25% other carbohydrate, through 15%quorum lipid sensing and 5% (QS) nucleic molecules acids [46 that]. The induce most abundantthe fungal polysaccharides population to cooperateincluded inα-1,2 diverse branched defenseα-1,6-mannans behaviors such (87%) as associatedvirulence and with biofilm unbranched formationβ-1,6-glucans [59–61]. The(13%) sesquiterpene in an apparent farnesol mannan-glucan and the aromatic complex, alcohol while tyrosolβ-1,3 glucan are the comprised two reported only natural a small occurringportion of QS the molecules total EPM [62,63]. carbohydrates The first [46 inhibits]. The presence filamentation of β-1,3 and glucan biofilm has formation been recently in C.reported albicans in[64]C. while glabrata tyrosolbioﬁlms stimulates [47] being the germ a key tube carbohydrate formation componentand, thus, filamentation in fungal cell [63].walls [47,48].

(b)

(a) (c)

FigureFigure 1. 1. (a(a) )The The five-stage five-stage process process involved involved in in the the development development of of biofilm: biofilm: 1. 1. adherence adherence of of yeasts yeasts to to a a surface surface followed followed byby yeast-to-hyphal yeast-to-hyphal transition; transition; 2. 2. the the ex exopolymericopolymeric matrix matrix (EPM) (EPM) is isproduced produced resu resultinglting in in a afirmly firmly adhered adhered “irreversible” “irreversible” attachment; 3. early biofilm architecture is developed; 4. the biofilm reaches maturation in a three-dimensional structure attachment; 3. early biofilm architecture is developed; 4. the biofilm reaches maturation in a three-dimensional struc- and 5. single planktonic cells are dispersed from the mature biofilm. Reproduced from Stoodley et al., 2002 [49]. Image ture and 5. single planktonic cells are dispersed from the mature biofilm. Reproduced from Stoodley et al., 2002 [49]. Image credit: D. Davies, with permission of Prof. David Davies. (b) A clearer scheme of stage 1. Yeasts adhere to host cell surfaces. Contact to host cells triggers the yeast-to-hyphal transition and directed growth via thigmotropism. Reproduced from part of Figure1a from Mayer et al. [ 51], with permission. (c) A scan electron microscopy (SEM) image of a mature (48 h) Candida albicans biofilm (formed in stage 4 of Figure1a). Bar = 10 µm. Yeasts, hyphae, and pseudohyphae can be observed. Most EPM was lost during the SEM procedures. Reproduced from Ramage et al. [50] with permission.

1.4. Stages in the Development of Biofifilms The development of a biofilm involves five stages that were clearly explained and graphed by Stoodley et al. [49], Ramage et al. [50], Mayer et al. [51] and other authors [43,52–54]. In stage 1, planktonic cells adhere to a biotic or abiotic surface, followed by a yeast-to-hyphal transition (Figure1a,b). In the second stage, EPM is produced, resulting in a firmly adhered irreversible attachment. In stage 3 an early biofilm architecture is developed, while in stage 4 the biofilm reaches maturation in a three-dimensional structure (Figure1c). Finally, in stage 5, single planktonic cells are dispersed from the mature biofilm. This dispersion is an important step in the fungal biofilm development cycle, which can induce either bloodstream or invasive fungal infections [55] leading to an increased pathogenicity [56] and high risk of mortality [57,58]. This consortium of cells offers the optimum conditions for the fungi to obtain nutrients, dispose waste products and protect from the environment [58]. Once the biofilm is formed, the sessile cells communicate with each other through quorum sensing (QS) molecules that induce the fungal population to cooperate in diverse defense behaviors such as virulence and biofilm formation [59–61]. The sesquiterpene farnesol and the aromatic alcohol tyrosol are the two reported natural Antibiotics 2021, 10, 1053 4 of 31

occurring QS molecules [62,63]. The first inhibits filamentation and biofilm formation in C. albicans [64] while tyrosol stimulates the germ tube formation and, thus, filamentation [63].

1.5. Factors and Mechanisms Associated with Biofilm Resistance One of the major characteristics of biofilms is the enhanced resistance to antimicrobial agents [65–69]. It has been reported that they possess about 5- to 4000-times less susceptibility to antifungals than equivalent populations of planktonic cells [60,69–72], which is a decisive factor driving therapeutic failures [73]. Several factors appear to be responsible for the resistance of biofilms [74–76]: (i) EPM acts as a physical barrier, preventing the entry of antifungals to biofilms, by either slowing drug diffusion or through specific sequestration mechanisms [74], with EPM components playing key roles [75]. For instance, soluble 1,3-β glucan, released from the fungal cell wall of C. albicans and A. fumigatus is able to sequester antifungal molecules, especially azole and polyene drugs, thus preventing their access to biofilm cells [68,76,77]; (ii) ergosterol levels are significantly lower at intermediate and mature phases of biofilms, compared to those in early phase biofilms and, therefore, those antifungals which target ergosterol have a poorer effect [78]. (iii) Although most sessile cells can show susceptibility to antifungal agents, a small sub-population of cells (persisters) stay alive [79], protected by the EPM and with a smaller oxidative imbalance compared to planktonic cells [80]. These persister cells can survive both the antifungal treatment and the immune system [81]. When the concentration of antifungal decreases, persister cells revive and repopulate the biofilm [81–83]. (iv) One of the major mechanisms of resistance to several classes of antifungals is the overexpression of efflux pumps [73]. These proteins imbibed in membranes are able to export the antimicrobial agents out of the cells, leading to a reduced intracellular concentration, which hinders the desired pharmacological effect. The expression of different genes associated with resistance occurs during biofilm formation and maturation. The most prominent classes of transporters are those belonging to the ATP binding cassette (ABC) and major facilitator superfamily (MFS) [84]. In Candida spp., the ABC transporters Cdr1p and Cdr2p, and the MFS transporter Mdr1p, emerged as the main contributors in biofilm-associated resistance to a range of antifungal agents, mainly to the azoles [6,85]. The expression of the transporters Cdr1p, Cdr2p and Mdr1p showed a dependency with the developmental phases of biofilms, therefore leading to different degrees of susceptibility associated with the outward transport of drugs. The overexpression of CDR1, CDR2 and MDR1 genes occurred at the early stages of biofilm formation, being the prominent mechanism for the tolerance to fluconazole, while in mature biofilms other resistance mechanisms are also involved in the diminished susceptibility to this azole drug [65,78,86]. In turn, the increase of drug resistance to antifungals, such as fluconazole and amphotericin B during biofilm maturation, seems to be associated with a significant decrease in the total ergosterol content, with changes in the expression of some ERG genes involved in its biosynthesis compared to planktonic cells [87]. Mutations or overexpression in the ERG11 gene, which encodes lanosterol 14α-demethylase [88,89], as well as in other ERG genes such ERG1 (encodes squalene epoxidase), ERG3 (encodes D5,6-desaturase), ERG7 (encodes squalene cyclase), ERG9 (encodes squalene synthase) or ERG25 (encodes C-4 methyl sterol oxidase) play important roles in the resistance of fungi to different antifungal agents [72,90–92].

1.6. Methodologies to Assess Antibiofilm Activity At present, in vitro and in vivo assays are used for quantifying biofilms [93]. Several methods for the in vitro assessing of the minimum inhibitory concentrations of biofilm sessile cells have been developed. The most commonly used assay is based on a static model using a microtiter plate [94], where the microbial biomass or metabolic activity are quantitated using dyes such as crystal violet (CV), 2,3-bis (2-methoxy-4-nitro-5- sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), or fluorescein Antibiotics 2021, 10, 1053 5 of 31

diacetate or resazurin [95]. Additionally, colony-forming units (CFU) counting has been extensively used [96]. Flow cytometry, using different fluorophores, can be used for de- termining CFU [7]. Another stain is calcofluor white (CW), which binds to 1,3 and 1,4 carbohydrate linkages and fluoresces under long-wave UV light [97]. Other biofilm devices or microscopy techniques such as light, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), among others, have been extensively used [96,98,99]. To determine the inhibition of biofilm ‘formation’, compounds are incorporated during the biofilm growth phase. Instead, to assess biofilm ‘eradication’, biofilms are grown for 24 h, after which the compounds are added and biofilms are additionally incubated 24–48 h in the presence of the compounds. The minimum biofilm inhibitory concentrations (MBIC, MBIC80 or MBIC50) are the minimum concentrations resulting in 100, 80 or 50% inhibition of biofilm formation, respectively [100]. Similarly, the minimum biofilm eradication concentration MBEC, MBEC80 or MBEC50 are the minimum concentrations resulting in 100%, 80% or 50% eradication of mature biofilms, respectively [101]. The worm Caenorhabditis elegans is highly used for the in vivo antibiofilm effectiveness of compounds, since hyphal formation is able to kill C. elegans [102]. Results are expressed as percentages of living or dead worms after incubation for a given number of days [93]. Several other models such as the venous catheter model in rats, rabbit or mice [93]; urinary catheters, subcutaneous implants, denture stomatitis, and oral and vaginal mucosae in different animals, are used. All these models were thoroughly reviewed by Nett and Andes in 2015 [103].

1.7. Natural Products as an Important Source of Antifungal Drugs Most antifungal agents in clinical use are natural or natural-derived compounds, mainly isolated from microbial strains [104]. For example, the polyenes nystatin and amphotericin B were isolated from Streptomyces spp. [105]; the spiro-diketone griseofulvin, was obtained from Penicillum griseofulvum [106]; the echinocandins caspofungin and anidulafungin are semisynthetic derivatives from the natural pneumocandin B, which was isolated from the fungus Glarea lozoyensis [107]. In turn, micafungin derived from the lipopeptide FR901379 was isolated from the fungus Coleophoma empetri [107], a plant pathogen associated with postharvest fruit rot in cranberries [108]. Under this scenario, it is clear that nature has provided a robust platform for finding novel scaffolds for the discovery of antifungal drugs. Natural antifungal products were typically discovered using the traditional antifungal assays, which measure the inhibition of growth of yeasts and filamentous fungi in their planktonic state, thus free-floating in the culture medium [109]. Recent works have reported extracts either from plants [110–112], algae [113,114], endophytic fungi [115] or marine fungi [116], as well as secondary metabolites such as phenols [111,117], flavonoids [118], naphtoquinones [119] and terpenoids [120], which showed growth inhibitory properties of fungi in their planktonic state. Both recent and previous reviews reported several pure natural products that have shown antifungal activity assessed with this type of techniques. However, none of these compounds have become leads for the development of new antifungal drugs [121]. The aim of this present review is to provide an update (2017–May 2021) on the natural products (extracts and secondary metabolites) that possess the ability to in vitro or in vivo modulate fungal biofilms not only constituted by C. albicans and some non-albicans Candida yeasts, but also by fungi from other genera. When available, the mechanism of action of these natural products has been included. To this purpose, most data published in the literature from 2017 were collected with the objective of drawing conclusions that can be useful for future research. Previously, some reviews on this subject were published [122–126] by Nazzaro et al. [123] and Singla and Dubey [124], with very few references each regarding the effect against fungal biofilms after 2017. Antibiotics 2021, 10, 1053 6 of 31

2. Reported Antibiofilm Activities of EOs, Propolis and Extracts from Plants, Algae and Cyanobacteria Different studies involving EOs with antibiofilm activity are recorded in Table1. Peixoto et al. [127] reported that Laurus nobilis L. (Lauraceae) EO at 2× minimum inhibitory concentration (MIC, 1000 µg/mL) inhibited the initial adhesion of C. albicans, while at 2× and 4× MIC, it inhibited biofilm formation and also reduced the eradication of mature biofilms with no significant difference when compared to the positive control, nystatin. In another study, Manoharan et al. [128] screened 83 EOs against C. albicans biofilm formation. Six of them obtained from Croton eluteria (L.) W.Wright (Euphorbiaceae) (cascarilla bark), Helichrysum coriaceum (DC.) Harv. (Asteraceae) (helichrysum oil), Eucalyptus globulus Labill. (Myrtaceae), Cymbopogon citratus (DC.) Stapf (Poaceae) (lemongrass), Citrus aurantiifolia (Christm.) Swingle (Rutaceae) (lime oil) and Coriandrum sativum L. (Apiaceae) (coriander) were demonstrated to inhibit more than 90% of biofilm formation when tested at 0.01%. Among them, cascarilla bark and helichrysum oil and their main components, α-longipinene and linalool, significantly reduced the yeast-to-hyphal transition, adherence and biofilm formation and greatly inhibited invasive hyphal growth in the nematode C. elegans. Serra et al. [129] tested different commercial EOs against two C. albicans strains. The results showed that only Pelargonium graveolens L’Hér. (Geraniaceae) (geranium) and Melisa officinalis L. (Lamiaceae) (melissa) EOs, eradicated mature biofilms with MBEC80 of 22.3 and 17.9 µg/mL, respectively for geranium and of 13.3 µg/mL on both strains, for melissa. Antibiotics 2021, 10, 1053 7 of 31

Table 1. More relevant results of essential oils (EOs) showing a capacity for inhibiting fungal bioﬁlms.

Year Essential Oil From Fungal spp. Biofilm In Vitro Type of Studies Reference Inhibition of Cell Adhesion Inhibition of Biofilm Eradication of Mature Mechanism of Action or In or Hyphal Formation Formation Biofilms Vivo Assays C. albicans ATCC 60193; CBS At 1000 and 2000 µg/mL, the L. nobilis EO at 1000 µg/mL L. nobilis EO at 1000 and 2000 562; C. tropicalis ATCC 750; EO showed significant 2017 Laurus nobilis inhibited the initial adhesion µg/mL reduced the amount [127] CBS 94 and C. krusei ATCC inhibition of biofilm of C. albicans biofilms of mature biofilms 3413; CBS 73 formation Six EOs, C. eluteria, Helichrysum coriaceum, Croton eluteria reduced the Eucalyptus globulus, In vivo assay: C. eluteria EO C. albicans ATCC 10231, 2017 83 EOs Candida adherence Cymbopogon citratus diminished Caenorhabditis [128] 18804, 24433 and DAY185 by 75% andCoriandrum sativum elegans virulence inhibited 90% of biofilm formation Out of the 12 EOs tested, only C. albicans 135, BM2/94 and Pelargonium graveolens and 2018 12 EOs [129] NYCY 1363 Melissa officinalis eradicated mature biofilms The three EOs produced a Pogostemon heyneanus, C. albicans ATCC 90028; C. reduction in the hyphal Candida biofilms EPM was P. heyneanus and C. tamala 2018 Cinnamomum tamala and glabrata MTCC 6507 and C. formation with P. heyneanus reduced. A large reduction of [130] disrupted mature biofilms Cinnamomum camphora tropicalis MTCC 310 EO showing the maximum sugars was observed inhibition The MBEC of fennel oil for Foeniculm vulgare EO 50 2019 10 isolates of C. albicans 7/10 tested strains was 2-to [131] (fennel oil) 6-fold the MIC C. citratus EO reduced Cymbopogon citratus, biofilm formation of all C. Cuminum cyminum, C. tropicalis isolates T26, U7 2020 tropicalis tested strains. C. [132] Citrus limon and and V89 limon and C. cyminum EOs Cinnamomum verum showed minor effects C. verum EO at 8× MIC and Cymbopogon citratus and C. albicans ATCC 10231 C. citratus EO at 16× MIC, At 8× MIC, both EOs (lemongrass) biofilms, coated on precoated on PMMA, eradicated totally the 2021 [133] Cinnamomum verum EOs polymethyl methacrylate inhibited C. albicans biofilm pre-established fungal (cinnamom) (PMMA) resin formation by 73% and 68%, biofilms in 1 h respectively ATCC: American type culture collection (Manassa, VA, USA); CBS: Netherlands Collection- Central Bureau voor Schimmelcultires; EPM: exopolysaccharide matrix; EO: essential oil; MBEC: minimum biofilm eradicating concentration; MTCC: Microbial type culture collection and Gene Bank, Chandigarh, India; NCYC: national collection of yeast cultures, Swindon, Wiltshire, UK; MIC: minimum inhibitory concentration. Other culture collection acronyms can be found in the respective reference. Antibiotics 2021, 10, 1053 8 of 31

As reported by Banu et al. [130], EOs from Cinnamomum tamala (Buch.-Ham.) T. Nees and Eberm. (Lauraceae) (Indian cassia), Pogostemon heyneanus Benth (Lamiaceae) (Indian patchouli) and Cinnamomum camphora (L.) J.Presl (Lauraceae) (camphor) inhibited about 54%–65% of biofilms formed by C. albicans, C. glabrata and C. tropicalis. In addition, the three EOs reduced the Candida spp. preformed biofilms, with an inhibition range of 55%–67% at their MBICs (0.5%–5% v/v); with C. tamala being the most active plant sp., P. heyneanus EO showed the maximum inhibition of yeast-to-hyphal transition. On the other hand, the EOs from P. heyneanus and C. tamala disrupted Candida spp. mature biofilms and reduced the thick aggregation of the yeast cells. This result was confirmed by the observation of a decrease of sugars present in the EPM layer. The capacity of Foeniculum vulgare Mill. (Apiaceae) EO (fennel oil) to eradicate 10 strains of C. albicans biofilms was studied by Bassyouni et al. [131]. The MBEC50 of fennel oil for eradicating the 18-h-old biofilm was 2- to 16-fold of MIC, in 7/10 tested strains. Sahal et al. [132] investigated the antifungal and biofilm inhibitory effects of EOs by using C. tropicalis biofilms coated on different biomaterials. Treatments with 2%–8% of C. citratus EO coated on silicone rubber resulted in a 45%–76% reduction in biofilm formation of all the strains. Likewise, Cuminum cyminum L. (Apiaceae), Citrus limon (L.) Osbeck (Rutaceae) and Cinnamomum verum J.Presl (Lauraceae) EOs, were also effective in inhibiting the C. tropicalis biofilms in polystyrene plates at sub-MIC values. Therefore, the mentioned extracts, in particular C. citratus EO, could be used as an antibiofilm agent on silicone rubber prostheses and medical devices. C. tropicalis biofilms pose a serious risk for skin infections and may cause a shorter lifespan of the prosthesis. In an additional study, Choonharuangdej et al. [133] tested the efficacy of C. verum and C. citratus EOs for eradicating C. albicans biofilms established on heat-polymerized polymethyl methacrylate (PMMA) material and determined whether they were able to retard the formation of fungal biofilms and/or eradicate them. Results showed that cinnamom oil at 0.8 µL/mL (8× MIC) and lemongrass oil at 6.4 µL/mL (16× MIC), both coated on PMMA, inhibited the formation of C. albicans biofilms by 70.0% after 24 h of treatment. In contrast, at 8× MIC (0.8 and 3.2 µL/mL, respectively), both EOs eradicated 99% of the pre-established C. albicans biofilm in 1 h. Table2 shows the relevant studies on antifungal propolis with antibiofilm activity. Galletti et al. [134] evaluated the activity of green propolis collected in Paraná (Brazil) against Fusarium spp. biofilms that frequently cause disseminated infections in immunocompromised patients, with a high rate of mortality [135]. In the mentioned work, the authors used clinical isolates of F. oxysporum, F. solani and F. subglutinans and a standardized F. solani strain. The used propolis proved to be efficient to reduce both the total biomass (assessed with CV dye) and the number of viable cells (quantified with XTT) for all evaluated isolates. In addition, the CW fluorescence assay showed that biofilm structure was lost, leaving only isolated damaged cells. In a recent paper, Martorano Fernandes et al. [136] evaluated the inhibitory effects of Brazilian red propolis on C. albicans and a co-culture of C. albicans-C. glabrata biofilms. Metabolic activity determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell viability assessed with CFU counts and surface roughness (optical profilometry) were evaluated. Results showed that red propolis had high inhibitory mono sp-biofilm effects but low activity against co-cultured two spp. biofilm formation, compared to chlorhexidine. The surface roughness (Sa parameter) within the mono-sp. and the co-culture biofilms statistically differed among groups. Table3 shows the different studies on extracts from plants, lichens, algae and cyanobacteria with antibiofilm activity. Antibiotics 2021, 10, 1053 9 of 31

Table 2. More relevant results of propolis extracts showing a capacity for inhibiting fungal bioﬁlms.

Year Extracts Fungal Biofilms spp. In Vitro Type of Studies Reference Inhibition of Cell Adhesion or Hyphal Biofilm Formation Eradication of Mature Biofilms Formation Fusarium spp. isolated from Propolis from Paraná state (Brazil) onichomycoses and deposited in The total mass of biofilms and the 2017 diluted in EtOH (PE) quantified UEM; F. oxysporum FO42; F. solani number of viable cells were [134] in its total phenol content FS04 and ATCC 36031 and F. significantly reduced by PE subglutinans (FSub39) RPE at 3% showed a high and low EtOH-H2O extract of red propolis C. albicans ATCC 90028 mono sp. inhibitory capacity of inhibiting 2020 (RPE) biofilm and C. albicans-C. glabrata the formation of mono sp.- and [136] from Paraiba state, Brazil ATCC 2001 co-cultures biofilms co-cultured two spp. –biofilms, respectively ATCC: American type culture collection (Manassas, VA, USA); EtOH: ethanol; UEM: mycological collection of the laboratory of medical mycology of the Universidade Estadual de Maringá, Brazil.

Table 3. More relevant results of extracts from plants, algae and cyanobacteria showing a capacity for inhibiting fungal bioﬁlms.

Year Extracts Fungal Biofilms spp. In Vitro Type of Studies Reference Source and Solvent Inhibition of Cell Adhesion or Biofilm Formation Eradication of Mature Biofilms Hyphal Formation PLANT EXTRACTS Eugenia leitonii, Treatment with E. leitonii seed E. brasiliensis, and E. brasiliensis seed and leaf 2017 E. myrcianthes, C. albicans ATCC 90028 [137] extracts at 10 × MIC, reduced E. involucrata leaf, pulp, seed C. albicans biofilm viability and bark EtOH-H2O extracts M. sylvestris extract at 0.19 M. sylvestris EtOH extract at mg/mL (1/4 MIC), Malva sylvestris root EtOH 0.78 and 1.56 mg/mL (MIC and 2017 C. albicans ATCC 10231 down-regulated the expression [138] extract 2× MIC) reduced biofilm of the hypha-specific gene formation HWP1 Candida biofilms at 500× MIC underwent a decrease in the C. albicans ATCC MYA2876, number of CFU/mL. Biofilm Anadenantera colubrina bark ATCC 90028 and a clinical structural alterations and 2019 [139] EtOH-H2O extract isolate; C. parapsilopsis ATCC cellular destruction were 22019 and C. krusei ATCC 6258 observed, being C. parapsilosis and C. krusei the most affected biofilms. CFL, FAL and FAB produced a The extracts inhibited biofilm Clematis flammula fresh leaves very low germ tube formation formation with MBIC50 = 250 (CFL) and Fraxinus angustifolia of 7.57, 2.29 and 1.17%, µg/mL for FAL and 500 2020 C. albicans ATCC 10231 [140] fresh leaves (FAL) and bark respectively in comparison to a µg/mL for FAB, while CFL (FAB) EtOH extracts growth of 50.89% for the showed a MBIC50 > 1000 control group µg/mL Antibiotics 2021, 10, 1053 10 of 31

Table 3. Cont.

Year Extracts Fungal Biofilms spp. In Vitro Type of Studies Reference Source and Solvent Inhibition of Cell Adhesion or Biofilm Formation Eradication of Mature Biofilms Hyphal Formation Hs extract eradicated C. albicans Hs extract inhibits the Fungal cells incubated with Hs biofilms at 3.12 mg/mL. In vivo C. albicans isolated from yeast-to-hyphal transition and extract at 2.5 mg/mL ( 1 MIC), Hibiscus sabdariffa flower (Hs) 2 assay with Caenorhabtidis 2020 vulvo-vaginal candidiasis biofilm adherence (50% at 1.5 [141] DMSO extract inhibited the biofilm elegans showed that Hs mg/mL and 80% at 6.25 maturation and, thus, the decreased the CFU of C. mg/mL) biofilm formation albicans i O. aristatus extract at 2 mg/mL Mild inhibition of C. albicans Orthoshipon aristatus purple 2020 C. albicans ATCC 10231 reduced the adhesion of C. growth at the biofilm [142] leaf n-hexane extract albicans cells development stage at 2 mg/mL LICHENS Seven extracts showed Seven extracts displayed antibiofilm capacity. Among Thirty eight lichen acetone anti-maturation effect. Among them, E.prunastri, Cladonia extracts of nine different them, Evernia prunastri and 2017 C. albicans ATCC 3153 uncialis (R. fastigiata and [143] families (mainly Parmeliaceae Ramalina fastigiata were the Xanthoparmelia conspersa and Cladoniaceae) most promising lichens (IC < 50 (showed IC values <10 4 µg/mL) 50 µg/mL MICROALGAE AND CYANOBACTERIA C. albicans and C. parapsilopsis biofilm formation was inhibited by 308 extracts. C. albicans biofilms were particularly sensitive to extracts from 675 hexane, ethyl acetate and Cryptophyta, Euglenophyta, methanol extracts obtained C. albicans and C. parapsilopsis 2019 and Glaucophyta (three [144] from 225 microalgae and (voucher N◦s not stated) completely unrelated lineages), cyanobacteria with MBIC50 = 8 µg/mL. Instead, Rhodophyta spp. showed activity against C. parapsilopsis with MBIC50 = 64 µg/mL

ATCC: American type culture collection; CFU: colony forming units; EtOH: ethanol; IC50: concentration that inhibits 50% growth; MBIC: minimum bioﬁlm inhibitory concentration; MIC: minimum inhibitory concentration. Antibiotics 2021, 10, 1053 11 of 31

Hydroethanol extracts of leaves, pulps, seeds and barks of several Eugenia spp. (Myr- taceae) such as E. leitonii Legrand, E. brasiliensis Lam., E. myrcianthes Nied. and E. involucrata DC. were tested for their capacity to eradicate mature C. albicans biofilms [137]. Among them, extracts from E. leitonii seeds and E. brasiliensis seeds and leaves reduced C. albicans biofilm viability by 54%, 54% and 55% at 156.2, 156.2 and 312.5 µg/mL (10× MIC), respectively, with better activity than that of nystatin which showed a 42% reduction. At these concentrations, all extracts caused damage to biofilm architecture and integrity, which could be observed by SEM. Alizadeh et al. [138] determined by CV assay that the ethanol extract of Malva sylvestris L. (Malvaceae) root at 0.78 and 1.56 mg/mL (MIC and 2× MIC, respectively) reduced C. albicans biofilm growth. By light microscopy, the authors observed that the extract was able to decrease biofilm thickness and cellular density. Silva et al. [139] determined that the hydroethanol extract of Anadenanthera colubrina Vell. Bre- nan (Fabaceae) barks, from the Caatinga biome (Brazil), showed the capacity to eradicate mature biofilms formed by four C. albicans strains, and one of each C. parapsilopsis and C. krusei, causing, in the last two, a 100% decrease of biofilms at 500× MIC. The Algerian Clematis flammula L. (Ranunculaceae) ethanol leaves (CFL) and Fraxinus angustifolia Vahl. (Oleaceae) leaves (FAL) and bark (FAB) extracts at 500 µg/mL showed 36.8%, 62.4% and 54.8% inhibition of C. albicans biofilm formation, respectively, which was probably related to the disruption of the cell surface hydrophobicity (CSH) and to the inhibition of germ tube and hyphae formation [140]. After four h incubation, 66.32% of hyphal form was seen in the control group, while 3.96%, 2.11% and 1.65% of hyphae were formed in the presence of CFL, FAL and FAB, respectively [140]. As reported by Dwivedi et al. [141], the Hibiscus sabdariffa L. (Malvaceae) flower DMSO extract inhibited the yeast-to-hypha transition with hyphae showing morphological changes, and also adherence of C. albicans cells (80% at 6.25 mg/mL). It also inhibited biofilm formation as well as disrupted the pre-formed C. albicans biofilm by 50% when tested at 3.12 mg/mL. The hexane extract of purple leaves from Orthosiphon aristatus (Blume) Miq. (Lamiaceae) was tested on different biofilm stages. [142]. Treatments of C. albicans with the extract at 2 mg/mL, showed a 69.2% decrease in cell viability (assessed with MTT) at the adhesion stage, while fluconazole at 6 µg/mL caused a 54.7% reduction. It is important to highlight that a 50% reduction in cell density compared to the negative control was observed in the presence of 1.3 mg/mL of the extract, as determined by the CV assay. At the development stage, a 57.1% and 57.3% inhibition on C. albicans growth, in the presence of the extract and fluconazole respectively, was observed. A low inhibition of approximately 20% was observed on mature biofilms after treatment with both the extract and fluconazole [142]. The ability to inhibit the formation (named in this paper ‘anti-maturation’) or to eradicate preformed 24 h-old C. albicans biofilms (named ‘antibiofilm’) were evaluated for 38 lichen acetone extracts, by XTT assay [143]. Among them, eleven extracts showed antibiofilm activity, with seven displaying both anti-maturation and antibiofilm properties. Of them, extracts from Evernia prunastri (L.) Ach (Parmeliaceae) and Ramalina fastigiata (Pers.) Ach. (Ramalinaceae) were the most promising ones, with half inhibitory concentration (IC50) values <4 µg/mL for anti-maturation. E. prunastri, Cladonia uncialis (L.) Weber ex F.H.Wigg (Cladoniaceae), R. fastigiata and Xanthoparmelia conspersa (Ehrh. ex Ach.) (Parmeliaceae) extracts showed IC50 values <10 µg/mL for antibiofilm eradication. Cepas et al. [144] tested 675 hexane, ethyl acetate and methanol extracts of 225 microalgae and cyanobacteria against C. albicans and C. parapsilopsis biofilms, which were inhibited by 308 extracts. Among the 11 phy- lum, the lowest activity was reported for Euglenophyta, with MBIC50 and MBIC90 of 8 and 16 µg/mL, respectively; Cryptophyta showed MBIC50 and MBIC90 values of 8 and 128 µg/mL while Glaucophyta presented MBIC50 and MBIC90 values of 8 and 256 µg/mL, respectively, against C. albicans. Instead, Rhodophyta spp. showed MBIC50 and MBIC90 values of 64 and 512 µg/mL, respectively, against C. parapsilopsis. Antibiotics 2021, 10, 1053 12 of 31

3. Reported Antibiofilm Activities of Pure Natural Compounds Pure natural compounds with antibiofilm activity are summarized in Table4 and detailed below. Liu et al. [145] demonstrated that the formyl-phloroglucinol meroditerpenoid, eucarobustol E (EE), isolated from Eucalyptus robusta Sm. (Myrtaceae), suppressed 73% of C. albicans biofilm formation at 32 µg/mL, destroyed nearly all mature biofilms (92%) at 128 µg/mL, blocked the yeast-to-hyphal transition and reduced the CSH at 16 µg/mL. EE downregulated the expression of genes involved in hyphal growth (EFG1, CPH1, TEC1, EED1, UME6, and HGC1), cell surface proteins (ALS3, HWP1, and SAP5) and in ergosterol biosynthesis (ERG6, ERG13, ERG252, ERG11, ERG10, and ERG7). This activity resulted in the reduction of ergosterol, which alters cell membrane functions, leading to cell death. In turn, EE upregulated the farnesol-encoding gene DPP3, which negatively regulated biofilm formation. According to the authors, EE differs from clinical antifungal agents in their antibiofilm mechanisms and so it is certainly worth considering for further development as an antifungal drug. Shi et al. [146] reported that berberine (BBR) inhibited biofilm formation of 13 strains of C. tropicalis and one strain of each C. albicans, C. parapsilosis and C. glabrata with MBICs ranging from 64 to 256 µg/mL. The mRNA expression of ERG11 and of the efflux proteins CDR1 and MDR1 were 1.43–2.10-fold upregulated by BBR at 16 µg/mL. Behbehani et al. [147] found that the lignan magnolol (2-(2-hydroxy-5-prop- 2-enylphenyl)-4-prop-2-enylphenol), isolated from Magnolia officinalis Rehder and E.H. Wilson (Magnoliaceae), showed strong antibiofilm formation activity against C. albicans, C. dubliniensis and C. glabrata, with 69.5%, 46.7% and 35.6% at 32 µg/mL, respectively, as determined by MTT assay. Six EO’s components such as thymol, carvacrol, cinnamaldehyde, citral, menthol and eugenol were tested by Kumari et al. [148]. These compounds proved to be effective on biofilm formation and on preformed C. neoformans and C. laurentii biofilms in the following order: thymol > carvacrol > citral > eugenol = cinnamaldehyde > menthol with MBIC80 ranging from 32 to 128 µg/mL and MBEC80 ranging from 64 to 256 µg/mL, determined by XTT assay. SEM and CLSM showed the absence of EPM, reduction in cellular density and alteration of the surface morphology of biofilm cells. Cryptococcosis is a systemic infection [149] very difficult to treat due to the ability of these fungi to form biofilms resistant to standard antifungal treatments. Kumari et al. [150] deepened the study of the C. neofomans antibiofilm activity of thymol (16 µg/mL), carvacrol (32 µg/mL) and citral (64 µg/mL) using field emission scanning electron microscopy (FE-SEM), atomic force microscopy (ATM) and Fourier transform infrared spectroscopy. The three terpenes appear to act through the interaction with ergosterol or the inhibition of its biosynthesis, and the disruption of the biofilm cell surface, with pore formation and efflux of the K+/intracellular content. Morphological changes and qualitative/quantitative alterations in the EPM and in cellular components of C. neoformans biofilm cells were also observed. The terpenes-treated cells showed 35%−45% reduction in total carbohydrates, with varia- tion in the type of glycosyl residues. Li et al., showed that the eudesmane sesquiterpene ent-isoalantolactone (ent-iLL) showed inhibition of the yeast-to-hyphal conversion of a mu- tant of C. albicans in assays performed in liquid and solid media at 8 µg/mL and 4 µg/mL, respectively [151]. In addition, ent-iLL at 16 µg/mL reduced the presence of ergosterol in the membrane, through inhibiting the activity of Erg11 and Erg6 [151]. The polyphenol curcumin (Cur) was evaluated for their antibiofilm properties against C. albicans by Alalwan et al. [152]. The MBIC80, was 200 µg/mL, as determined by XTT assay. Furthermore, Cur at 50 µg/mL was able to decrease C. albicans adhesion to a PMMA denture base material, an effect that could be enhanced by pre-treatment of the yeasts with the polyphenol. Regard- ing its molecular effects, Cur down-regulated the adhesin ALS3, with minimal effect on its related ALS1. On the contrary, the clustered aggregative and flocculation genes AAF1, EAP1, and ALS5 transcripts were up-regulated. Three gingerols (6-, 8- and 10-gingerols) and three shogaols (6-, 8-, and 10-shogaols) isolated from Zingiber officinale Roscoe (Zingib- eraceae) showed antibiofilm and anti-virulence activities against a fluconazole-resistant C. albicans strain. Results showed that only 6-shogaol at 10 µg/mL and 6- and 8-gingerols at 50 µg/mL, significantly reduced the C. albicans biofilm formation, suggesting that the Antibiotics 2021, 10, 1053 13 of 31

increase in the length of the side chain decreased the activity. CLSM showed that biofilms treated with 6-gingerol and 6-shogaol were reduced in density and in thickness. In addition, both compounds inhibited hyphal growth in embedded colonies and free-living planktonic cells, and prevented cell aggregation, which was confirmed by SEM. Both entities significantly altered the expressions of some hypha-specific (HWP1 and ECE1), biofilm- related (HWP1 and RTA3) and multidrug transporter (CDR1 and CDR2) related genes [153]. Yan et al. [154] reported that the 1,4-naphtoquinone derivative shikonin (SK), at 32 µg/mL, inhibited almost totally C. albicans biofilm formation and eradicated the preformed mature biofilms, as evidenced with XTT assay and confirmed by CLSM. SK inhibited hyphae formation, showing a complete inhibition at 0.5 µg/mL in Lee´s medium and reduced CSH by 70.3% at 2 µg/mL. Several hypha and adhesion specific genes such as of ECE1, HWP1, EFG1, CPH1, RAS1, ALS1, ALS3 and CSH1 were downregulated while TUP1, NRG1 and BCR1 were upregulated by SK. In addition, SK induced the production of farnesol at 8 µg/mL, which enhanced its antibiofilm activity. Saibabu et al. [155] showed that different C. albicans strains treated with 62.5 µg/mL of vanillin showed few or no adherence to buccal epithelial cells. By MTT assay, it was observed the adherence of Candida cells to polystyrene surface was reduced by 52%, the biofilm formation was decreased by 49%, and the mature biofilm eradication decreased by 52%. At 125 µg/mL, vanillin protected C. elegans against C. albicans infection and enhanced its survival [155]. Kischkel et al. [156] evaluated farnesol on preformed F. solani complex biofilms particularly formed by Fusarium keratoplasticum, which is the most prevalent fungi related to biofilm formation in hospital water systems and in internal pipelines. Farnesol showed activity against F. keratoplasticum preformed biofilms, and was effective also during its formation at different times (at adhesion and at 24, 48 and 72 h) with a complete inhibition at 700 µM, as assessed by counting the CFU number and by CV and XTT assays. A recent report from Wang et al. [157] described the isolation of 14 new terpenoids from the liverwort Heteroscyphus coalitus (Hook.) Schiffner (Geocalycaceae), including eight ent-clerodane diterpenoids, four labdane diterpenoids, heteroscyphsic acids A-I, heteroscyphins A-E, a harziane type diterpenoid and one guaiane sesquiterpene together with a known ent-junceic acid. At 4–32 µg/mL most of these compounds inhibit hyphal and biofilm formation of an efflux pump deficient strain of C. albicans, but not of the wild type of strain. The most effective molecule was heteroscyphin D, which suppressed the ability of C. albicans to adhere to A549 cells and to form biofilms with a complete inhibiton at 8 µg/mL, determined by XTT assay. In addition, this compound was able to modulate the transcription of related genes in this fungus, as described in Table4. Das et al. [158] showed that artemisinin (Ar), the sesquiterpene lactone isolated from Artemisia annua L. (Asteraceae), and scopoletin (Sc), the 7-hydroxy-6-methoxy coumarin present in several spp., were tested on mature biofilms of different albicans and non-albicans Candida strains. The results demonstrated that Ar was more active than Sc in disrupting the preformed EPM-covered biofilm structure and in killing the sessile cell population at their respective MBEC10. In the same year, Lemos et al. [159] demonstrated that Sc, at its MIC (50 µg/mL), was able to reduce the preformed biofilms of a resistant C. tropicalis strain in 68.2%, and to inhibit the biofilm formation. Kipanga et al. [160] showed that the drimane sesquiterpene dialdehydes warburganal and polygodial, obtained from Warburgia ugandensis Sprague (Canellaceae), inhibited C. albicans biofilm formation with MBIC50 of 4.5 and 10.8 µg/mL, respectively, and with MBIC50 of 49.1 and 50.6 µg/mL, respectively, against C. glabrata. Regarding biofilm eradication, warburganal and polygodial showed MBEC50 of 16.4 and 16.0 µg/ml, respectively, against C. albicans but did not inhibit C. glabrata biofilm eradication. The higher potency of warburganal over polygodial for inhibiting biofilm formation and eradication could be attributed to the hydroxyl group present at position C-9, that differentiates both sesquiterpenes. The triterpenoid saponins gypenosides, isolated from Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae), showed MBIC80 > 128 µg/mL against two fluconazole-resistant strains of C. albicans, as determined by XTT. No significant reduction in the density and in the length of the hyphae were observed [161]. Antibiotics 2021, 10, 1053 14 of 31

Zhao et al. [162] showed that turbinmicin, a highly functionalized polycyclic compound isolated from the marine microbiome, completely disrupted extracellular vesicle (EV) delivery, during biofilm growth at 16 µg/mL, and this impaired the subsequent assembly of the biofilm EPM. C. albicans biofilm EVs have a pivotal role in EPM production and biofilm drug resistance [163]. This property was observed by a combination of flow cytometry, image confirmation, and fluorescence sensitivity on C. albicans, C. tropicalis, C. glabrata, C. auris and A. fumigatus. By SEM, it was determined that turbinmicin at 2.5 µg/mL eliminated EPM, thus rendering the drug-resistant biofilm communities susceptible to the antifungal effects of TBM itself, as well as to clinical antifungal agents. Zainal et al. [164] demonstrated that allicin, the organosulfur compound obtained from Allium sativum L. (Amaryllidaceae), was able to eradicate 50% of C. albicans biofilm formed on self-polymerized acrylic resin when administered at a sub-MIC concentration of 4 µg/mL. Feldman et al. [165] reported that cannabidiol (CBD) obtained from Cannabis sativa L. (Cannabaceae) exerted an inhibitory effect on biofilm formation with a MBIC90 of 100 µg/mL. At 25 µg/mL, the metabolically active cells (assessed by MTT) in 24, 48 and 72 h-biofilms decreased by 48%, 64% and 87%, respectively. At 1.56 and 3.12 µg/mL, mature biofilms decreased by 28% and 44%, respectively. Furthermore, CBD reduced the thickness of fungal biofilm and EPM production accompanied by a downregulation of genes involved in EPM synthesis. At 25 µg/mL, CBD inhibited 90% of the ATP synthesis and enhanced mitochondrial membrane potential and 1 ROS levels. At 4 MBIC90, CBD produced upregulation of yeast-associated genes and downregulation of hyphae-specific genes. As reported by Ivanov et al. [166], camphor inhibited more than 50% of the biofilm biomass in C. albicans strains at 62–250 µg/mL, while in C. tropicalis, the inhibition was achieved at 175 µg/mL. On the other hand, eucalyptol showed the same effect in the tested C. albicans strains at higher concentrations (>3000 µg/mL) and in C. tropicalis, C. parapsilosis and C. krusei at >1000 µg/mL [166]. Camphor, applied at 125 µg/mL, reduced ROS generation in one strain of C. albicans, although eucalyptol failed to exert this effect. The compounds did not interfere with ERG11 expression. Neosartorya fifischeri antifungal protein 2 (NFAP2), a novel member of small cysteine-rich and cationic antifungal proteins from filamentous ascomycetes (crAFPs), showed low activity against C. auris biofilms [167]. When NFAP2 was tested in combination with clinical antifungals, an enhancement of the activity was observed. The results of combinations were not included in this review. AntibioticsAntibiotics 2021, 10 ,20 x 21FOR, 10 PEER, x FOR REVIEW PEER REVIEW 16 of 3316 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 16 of 33 Antibiotics 2021, 10, x FOR PEER REVIEW 16 of 33

Table 4.Table More 4. relevant More relevant results resultsof natural of natural compounds compounds showing showing a capacity a capacity for inhibiting for inhibiting fungal biofilms.fungal biofilms. Antibiotics 2021, 10, 1053 15 of 31 In VitroIn Type Vitro of TypeStudies of Studies Type of TypeCompound/ of Compound/ Table 4. More relevant results of natural compounds showing a capacity for inhibiting fungal biofilms. Year Year StructureStructure and Name and Name Table 4.Strains More Strainsrelevant results of naturalInhibition compoundsInhibition of Cell showing Adhesionof Cell aAdhesion capacity for inhibitingEradication fungalEradication biofilms. of Ma- ofStudies Ma- Studiesof Mechanisms of Mechanisms Reference Reference Natural NaturalSource Source In Vitro Type of StudiesBiofilm BiofilmFormation Formation Type of Compound/ Table 4. More relevant results of natural compounds showingor HyphalInor a Vitro capacity HyphalFormation Type Formation for of inhibitingStudies fungal biofilms.ture Biofilmsture Biofilms of Actionof Action YearType of Compound/ Structure and Name Strains InInhibition Vitro Type of Cell of Studies Adhesion Eradication of Ma- Studies of Mechanisms Reference Year TypeNatural of Compound/Source Structure and Name Strains Inhibition of Cell Adhesion Biofilm Formation Eradication of Ma- Studies of Mechanisms Reference Year Type ofYear Compound/NaturalNatural Source Source Structure and Name and Name StrainsStrains In Vitro Type ofInhibitionor Studies Hyphal Formationof Cell Adhesion Biofilm Formation Eradicationture Biofilms of AtMa- 8 andStudiesofAt 16 ActionReference 8 µg/ml and of 16 MechanismsEE µg/ml re- EE re- Reference Natural Source Inhibition of Cellor AdhesionHyphal Formation BiofilmEradication Formation of Mature tureStudies Biofilms of Mechanisms of of Action Formyl-Formylphloroglu--phloroglu- Biofilm FormationEE inhibitedEE inhibited 60 and 60 and C. albicansC. SC5314albicans andSC5314 ATCC and 24433; ATCCor Hyphal EE 24433; inhibited Formationor EE Hyphal inhibited C. albicans Formation C. yeastalbicans- yeast- Biofilms tureAction Biofilms duced byofduced Action9.2% byand 9.2% 65.3% and 65.3% cinol meroterpenoidcinol meroterpenoid 73% biofilm73% formationbiofilm formation 10 Fluconazole10 Fluconazole-resistant-resistant C. albicans C. albicansto-hyphal to -transitionhyphal transition in both in both EE eradicatedEE eradicated mature thematu ergosterolreAt the 8 ergosteroland production 16 µg/ml production EE re- 2017 Source2017 Formyl Source- phloroglu- at 16 andEEat 32 inhibited16 µg/mL, and 32 60µg/mL, and At 8 and 16 µg/ml EE[145] re- [145] Formyl-phloroglu- and 8 FluconazoleC.and albicans 8 Fluconazole SC5314-susceptible and-susceptible ATCC C. al- liquid 24433; C. al- and EEliquid inhibited solid and hypha solid C. albicans-in- hypha yeast-in- -EE inhibited 60 biofilmand atbiofilm 128 µg/mL at 128 andµg/mL increasedduced and increased by by 9.2% 3.95- and by 3.95 65.3%- EucalyptuscinolEucalyptus spp. meroterpenoid and spp. and C. albicansC. albicans SC5314SC5314 and and ATCC ATCC 24433; EE inhibited C. albicansrespectively yeast-73%respectively biofilm and 100% formation and 100% At 8 and 16 µg/ml EE duced by 9.2% and 65.3% 10 Fluconazole24433; 10 -resistant C.EE albicans inhibited C. to albicans-hyphal transitionEE inhibited 60in and both 73% EE eradicatedreduced by 9.2% andmatu 65.3%re the ergosterol production Formyl-phloroglucinolcinol meroterpenoid bicans 10bicans Fluconazole -resistant C. albicansducing tomediaducing-hyphal media transition in both 73% biofilm formation EE eradicated matuand 5.43re theand-fold ergosterol 5.43 the- foldfarnesol productionthe farnesol Psidium2017 Source Psidiumspp. spp. Fluconazole-resistant C. yeast-to-hyphal transition in biofilm formationat at >32 16 and µg/mLatat 16 >32EE and eradicated µg/mL 32 µg/mL, mature biofilm the ergosterol production [145] 2017 Source 2017 Source 10 Fluconazole-resistant C. albicans to-hyphal transition in both at 16 and 32 µg/mL, EE eradicated mature the [ergosterol145] production [145] and 8albicans Fluconazoleand 8 -susceptibleboth liquid C. al- andliquid solid and solid32 µg/mL, hypha respectively-in- and at 128 µg/mL biofilmand increased at 128 by µg/mL 3.95-production, and andproduction, increased respectively respectively by 3.95- Eucalyptus2017spp. SourceEucalyptus and Psidium spp. spp. and and 8 Fluconazole-susceptible C. al- liquid and solid hypha-in- atrespectively 16 and 32 µg/mL,and 100% biofilm at 128 µg/mL and increased by 3.95- [145] Eucalyptus spp. eucarobustoland eucarobustol E (EE) E (EE) andbicans 8Fluconazole-susceptible Fluconazole-susceptibleC. hypha-inducing C. al- liquidducing media and media solid100% athypha >32 µg/mL-in- respectively and 100% biofilm5.43-fold at the 128 farnesol µg/mL and increased5.43-fold the by farnesol3.95- EucalyptusPsidium spp. spp. and bicansalbicans ducing media respectivelyat >32 µg/mL and 100% production, respectively and 5.43-fold the farnesol Psidium spp. bicans ducing media at >32 µg/mL and 5.43-fold the farnesol Psidium spp. at >32 µg/mL production, respectively eucarobustol E (EE) C. albicansC. SC5314;albicans SC5314; production, respectively eucarobustol E (EE) E (EE) C. parapsilosisC. parapsilosis ATCC 22019; ATCC C. 22019; gla- C. gla- IsoquinolineIsoquinoline alkaloid alkaloid BBR inhibitedBBR inhibited Candida Candidabio- bio- brata ATCCC.brata albicans 15126; ATCC SC5314; C. 15126; tropicalis C. tropicalis 2017 Source2017 Source C. albicans SC5314; film formationfilm formation with MBIC with MBIC [146] [146] ATCC 750C.ATCC parapsilosis andC. albicans 750other SC5314;and ATCCC tropicalisother 22019; C tropicalis C. gla- Not informedIsoquinolineNot informed alkaloid C. parapsilosis ATCC 22019; C. values ofBBRvalues 64 –inhibited256 of µg/mL 64–256 Candida µg/mL bio- Isoquinoline alkaloidIsoquinoline alkaloid C.brata parapsilosis ATCC 15126; ATCC C. 22019; tropicalisBBR C. inhibited gla- CandidaBBR inhibited Candida bio- Isoquinoline alkaloid strains brata(2203,strains glabrataATCC 2317, (2203,ATCC 15126;2006, 15126;2317, 2718,C. C. 2006, tropicalis 333, 2718, 333, BBR inhibited Candida bio- 2017 Source 2017 Source biofilm formationfilm with formation MBIC with MBIC [146] [146] 2017 Source brata tropicalisATCCATCC 15126; 750 and C. other tropicalis film formation with MBIC [146] Not informed2017 Source 087, 20026)ATCC087, 20026) 750 and other C tropicalisvalues of 64–256filmµg/mL formation with MBIC [146] Not informed ATCCC tropicalis 750 andstrains other (2203, 2317, C tropicalis values of 64–256 µg/mL Not informed berberineberberine (BBR) (BBR) strains2006, (2203, 2718, 333, 2317, 087, 20026) 2006, 2718, 333, values of 64–256 µg/mL strains (2203, 2317, 2006, 2718, 333, 087, 20026) 087, 20026) berberine (BBR) (BBR) berberine (BBR) MagnololMagnolol inhibited inhibited 35.6%–69.5%35.6% pre-–69.5% pre- 3-3’-Neolignan3-3’-Neolignan C. dubliniensisC. dubliniensis CDC 27897; CDC C. 27897; albi- C. albi- Magnolol inhibited Magnolol inhibitedformedMagnolol biofilmsformed biofilms of inhibited of C. dubliniensis CDC 27897; C. 20173-3’-Neolignan Source2017 Source cans CDCcans 27907 CDC and 27907 ATCC and 24433; ATCC 24433; 35.6%–69.5% preformed Magnolol inhibited [147] [147] albicans CDC 27907 and 35.6%the three–69.5% fungi pre- at 32 2017 Source biofilms of the threethe fungi three at 35.6% fungi–69.5% at 32 pre- [147] Magnolia3Magnolia- 3’offi-Neolignancinalis o ffi cinalis C. glabrataC.C. dubliniensis glabrataCDCATCC 24433;28621 CDCC. CDC glabrata 28621 CDC27897; C. albi- Magnolia officinalis3-3’-Neolignan C. dubliniensis CDC 27897; C. albi- 32 µg/mL 35.6%formed–69.5% biofilms pre- of 2017 3Source-3’-Neolignan magnolol.magnolol. C.cans dubliniensis 28621CDC 27907 CDC and 27897; ATCC C. 24433; albi- µg/mL formed µg/mL biofilms of [147] 2017 Source cans CDC 27907 and ATCC 24433; formedthe three biofilms fungi at of 32 [147] 2017 SourceMagnolia officinalis(2- (2 -hydroxy(2-(2-hydroxy-5-prop--25--enylphenyl)prop-2-enylphenyl) -4- -4cans-C. glabrata CDC 27907 CDC 28621and ATCC 24433; the three fungi at 32 [147] Magnolia officinalis C. glabrata CDC 28621 theµg/mL three fungi at 32 Magnolia officinalis magnolol.prop-2-enylphenol) C. glabrata CDC 28621 µg/mL prop-2-(2-(2-hydroxy-5-prop-2-enylphenyl)-4-prop-2-enylphenol)magnolol.enylphenol) µg/mL magnolol.(2-(2-hydroxy -5-prop-2-enylphenyl)-4- (2-(2-hydroxy-5-prop-2-enylphenyl)-4- MBEC80 MBECof thymol80 of thymol 5 terpenes5 terpenes and one and one (2prop-(2--hydroxy2-enylphenol) -5-prop - 2-enylphenyl)-4- prop-2-enylphenol) and carvacroland carvacrol against against phenylpropanoid.phenylpropanoid. prop-2-enylphenol) MBIC80 againstMBIC80 C.against C. 5 terpenes and one phenylpropanoid. MBIC80 against C. MBEC80 of thymolC. and neoformansMBECC. Theneoformans compounds80were of thymol reduced were Sources. 5 terpenes and one C. neoformans NCIM 3541 and neoformans and C. laurentii: carvacrol against C. MBECEPM,80 cellular of thymol density and Sources5.Sources terpenes . and one neoformansneoformans and C. lau- and C. lau-MBEC80 of thymol 2017 EOs from Origanum vulgare, Mentha piperita, C. laurentii NCIM 3373 for thymol: 32 and 16 neoformans were 128128 and and 256 and 128256altered carvacrol andµg/mL, the 256 surface µg/mL, againstThe compounds The[148 compounds] reduced reduced Thymus vulgaris5phenylpropanoid. Cinamomum terpenes verum and one µg/mL; for carvacrol, 64 andMBICµg/mL,80 against respectively, C. and and carvacrol against EOs fromEOs Origanum from Origanum rentii: forrentii thymol:80: for thymol:32 32 morphology of biofilm cells Cymbopogon citratusphenylpropanoid.Syzygium aromaticum 32 µg/mL and for citral, 128MBICagainst againstC. laurentii C.64 and 128andC. neoformans carvacrol wereagainst phenylpropanoid.and C. neoformansC. neoformans NCIM 3541 NCIM and 3541 C. and C. MBIC80 against respectively,C. C.respectively, neoformans and wereandEPM, cellularEPM, cellulardensity anddensity and vulgare,Sources vulgareMentha., Mentha thymolthymol carvacrolcarvacrol and 64 µg/mL, respectively.and 16 neoformansµg/mL;andµ 16g/mL, forµg/mL; respectively. andcar- C.for lau- car- Sources. thymol carvacrol neoformans and C. lau- C.128 neoformans and 256 µg/mL, were The compounds reduced 2017 2017Sources . laurentii laurentiiNCIM 3373 NCIM 3373 neoformansMBEC80 for and citral againstC. was lau- 256 128 C.against laurentii and 256C. laurentii 64µg/mL, altered 64 The thealtered surfacecompounds the mor-surface reduced[148] mor- [148] piperita,EOs piperitaThymus from, Thymusvul- Origanum vul- vacrol, rentii64vacrol, andµ:g/mL for 32 64 thymol: for and both fungi32 32 128 and 256 µg/mL, The compounds reduced EOs from Origanum C. neoformans NCIM 3541 and C. rentii: for thymol: 32 respectively,and 128 µg/mL, and re- EPM,phology cellular of biofilm density cells and EOsvulgare from, Mentha Origanum thymol carvacrol C. neoformans NCIM 3541 and C. rentiiand 16: for µg/mL; thymol: forand 32 car- 128 respectively, µg/mL, re- andphology EPM, of biofilm cellular cells density and garis Cinamomumvulgaregaris Cinamomum, Mentha thymol carvacrol C. neoformans NCIM 3541 and C. µg/mL and µg/mL and 16 for µg/mL; cit- and for for cit- car-respectively, and EPM, cellular density and 2017 vulgare, Mentha thymol carvacrol laurentii NCIM 3373 and 16 µg/mL; forspectively. car-againstspectively. C. laurentii 64 altered the surface mor- [148] verum2017 Cymbopogon piperitaverum ,Cymbopogon Thymus vul- laurentii NCIM 3373 ral, 128vacrol, andral, 12864 64 µg/mL, and and 64 32 µg/mL, against C. laurentii 64 altered the surface mor- [148] piperita, Thymus vul- vacrol, 64 and 32MBEC 80and MBECfor 128citral80 µg/mL, for was citral re- wasphology of biofilm cells citratusgaris andcitratus SyzygiumCinamomum and Syzygium respectively.µg/mLrespectively. and for cit- and 128 µg/mL, re- phology of biofilm cells garis Cinamomum µg/mL and for256 cit- µg/mLspectively.256 for µg/mL both for both aromaticumverumaromaticum Cymbopogon ral, 128 and 64 µg/mL, verum Cymbopogon ral, 128 and 64 µg/mL, spectively.MBEC80 for citral was verumcitratus Cymbopogon and Syzygium cinnamaldehydecinnamaldehyde ral,respectively. 128 and 64 µg/mL,fungi MBECfungi 80 for citral was citratus and Syzygium respectively. MBEC256 µg/mL80 for forcitral both was citratusaromaticum and Syzygium respectively. 256 µg/mL for both aromaticum 256fungi µg/mL for both aromaticum cinnamaldehyde fungi cinnamaldehyde fungi Antibiotics 2021, 10, x FOR PEER REVIEW 16 of 33

Table 4. More relevant results of natural compounds showing a capacity for inhibiting fungal biofilms.

In Vitro Type of Studies Type of Compound/ Year Structure and Name Strains Inhibition of Cell Adhesion Eradication of Ma- Studies of Mechanisms Reference Natural Source Biofilm Formation or Hyphal Formation ture Biofilms of Action

At 8 and 16 µg/ml EE re- Formyl-phloroglu- EE inhibited 60 and C. albicans SC5314 and ATCC 24433; EE inhibited C. albicans yeast- duced by 9.2% and 65.3% cinol meroterpenoid 73% biofilm formation 10 Fluconazole-resistant C. albicans to-hyphal transition in both EE eradicated mature the ergosterol production 2017 Source at 16 and 32 µg/mL, [145] and 8 Fluconazole-susceptible C. al- liquid and solid hypha-in- biofilm at 128 µg/mL and increased by 3.95- Eucalyptus spp. and respectively and 100% bicans ducing media and 5.43-fold the farnesol Psidium spp. at >32 µg/mL production, respectively eucarobustol E (EE)

C. albicans SC5314; C. parapsilosis ATCC 22019; C. gla- Isoquinoline alkaloid BBR inhibited Candida bio- brata ATCC 15126; C. tropicalis 2017 Source film formation with MBIC [146] ATCC 750 and other C tropicalis Not informed values of 64–256 µg/mL strains (2203, 2317, 2006, 2718, 333, 087, 20026) berberine (BBR)

Magnolol inhibited 35.6%–69.5% pre- 3-3’-Neolignan C. dubliniensis CDC 27897; C. albi- formed biofilms of 2017 Source cans CDC 27907 and ATCC 24433; [147] the three fungi at 32 Magnolia officinalis C. glabrata CDC 28621 magnolol. µg/mL (2-(2-hydroxy-5-prop-2-enylphenyl)-4- prop-2-enylphenol)

MBEC80 of thymol Antibiotics 20215, 10terpenes, 1053 and one 16 of 31 Antibiotics 20202121,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW and carvacrol against 1717 ofof 3333 phenylpropanoid. Antibiotics 2021 , 10, x FOR PEER REVIEW MBIC80 against C. 17 of 33 C. neoformans were Sources. neoformans and C. lau- 128 and 256 µg/mL, The compounds reduced EOs from Origanum rentii: for thymol: 32 Antibiotics 2021, 10, x FOR PEER REVIEW C. neoformans NCIM 3541 and C. respectively, and EPM, cellular density and 17 of 33 vulgare, Mentha thymol carvacrol Table 4. Cont. and 16 µg/mL; for car- 2017 laurentii NCIM 3373 against C. laurentii 64 altered the surface mor- [148] piperita, Thymus vul- vacrol, 64 and 32 Year Type of Compound/Natural Source Structure and Name Strains In Vitro Type of Studies and 128 µg/mL, re- phology of Referencebiofilm cells AntibioticsgarisAntibiotics Cinamomum 2021, 10 20, x21 FOR , 10 ,PEER x FOR REVIEW PEER REVIEW µg/mL and for cit- 17 of 3317 of 33 Antibiotics 2021, 10, x FOR PEER REVIEW spectively. 17 of 33 verumAntibiotics Cymbopogon 2021, 10, x FOR PEER REVIEW ral, 128 and 64 µg/mL, 17 of 33 MBEC80 for citral was citratus and Syzygium respectively. 256 µg/mL for both aromaticum citral cinnamaldehydecitralcitral menthol fungi cinnamaldehyde menthol

citral menthol

eugenol eugenol eugenol citral citral citralcitral mentholmenthol Eudesmane sesquiter- citral menthol EudesmaneEudesmane sesquiter-sesquiter- menthol C. albicans DSY654. Genotype: Ent-iLL inhibited the yeast- pene C. albicans DSY654. Genotype: EntEnt--iLLiLL inhibitedinhibited thethe yeastyeast-- Ent-iLL reduced ergoste- penepene eugenol Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG- to-hyphal switch at 4 or 8 EntEnt--iLLiLL reducedreduced ergoste-ergoste- Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG- to-hyphal switch at 4 or 8 2017 Source Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG- to-hyphal switch at 4 or 8 rol content by inhibiting [151] 20172017 Source Source URA3-hisG/ µg/mL in agar plate tests or rolrol contentcontent byby inhibitinginhibiting [151][151] TritomariaEudesmane quin- sesquiter- URA3--hisG/hisG/ µµg/mLg/mL inin agaragar plateplate teststests oror Erg11 and Erg6 TritomariaTritomaria quin-quin- Dcdr2::hisG liquid medium, respectively Erg11Erg11 andand Erg6Erg6 eugenol eugenol C.Dcdr2::hisG albicans DSY654. Genotype: Entliquid-iLL medium, inhibited respectively the yeast- quedentata eugenol Dcdr2::hisG liquid medium, respectively quedentatapenequedentata eugenol Ent-iLL reduced ergoste- ent -isoalantolactoneeugenol (ent-iLL) Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG- to-hyphal switch at 4 or 8 2017 Source entent--isoalantolactoneisoalantolactone ((entent--iLL)iLL) rol content by inhibiting [151] EudesmaneEudesmane sesquiter- sesquiter- URA3-hisG/ µg/mL in agar plate tests or Cur at 50 μg/mL down- C. albicans DSY654. Genotype:Ent-iLL inhibitedEnt the-iLL inhibited the yeast- Cur at 50 μg/mL down- TritomariaEudesmane quin- sesquiter- C. albicans DSY654.C. albicans DSY654.Genotype: Genotype: Ent-iLL inhibited the yeast- CurErg11 at and50 μg/mL Erg6 down- Eudesmane sesquiterpeneEudesmanepene sesquiter- yeast-to-hyphal switch at 4 or Ent-iLL reduced ergosterol Ent-iLL reduced ergoste- pene Dcdr2::hisGC. albicansDcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG- DSY654. Genotype: liquidEnt-iLL medium, inhibited respectively the yeast - Ent-iLL regulatedreduced ergoste- 2017 Source pene C.Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisG albicans DSY654. Genotype:8 µg/mL in agar-Entto plate-hyphal-iLL tests inhibited switch theat 4 yeastor 8 - content by inhibiting Erg11 regulatedregulatedEnt[151-iLL] reduced ergoste- penequedentata Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisGURA3-hisG/ - to-hyphal switch at 4 or 8 Ent-iLL reduced ergoste- 2017Tritomaria Source2017 quinquedentata Source ent-isoalantolactone (ent-iLL) Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisGor liquid medium,Cur- to -athyphal 50 µg/mL switch reduced at 4 or the8 and Erg6 rol contenttherol contentbyadhesin inhibiting by ALS3, inhibiting [151]with [151] 2017 Source Dcdr1::hisG/Dcdr1::hisG/Dcdr2::hisGURA3Dcdr2::hisG-hisG/ - toCurµg/mL-hyphal at 50 in µg/mLswitchagar plate reducedat 4 tests or 8 orthe thetherol adhesinadhesin content ALS3,ALS3, by inhibiting withwith [151] 2017 SourceTritomaria--diketonediketone quin- diphenoldiphenol URA3-hisG/ respectivelyµg/mL in agar plate tests or rolErg11 content and Erg6by inhibiting [151] Tritomaria-diketone quin- diphenol URA3URA3-hisG/-hisG/ µcapacityg/mLµg/mL in ofinagar C.agar albicansplate plate tests totests at- or or Erg11 andminimalCur Erg6 at 50 impact μg/mL on down ALS1.- SourceTritomaria quin- Dcdr2::hisG capacityliquidcapacity medium, ofof C. albicans respectively toto at-at- The MBIC80 for Cur was ≥200 μg/mL for ses-minimalErg11 and impact Erg6 on ALS1. TritomariaSource quin- Dcdr2::hisG liquid medium, respectively The MBIC80 for Cur was ≥200 μg/mL for ses-Erg11 and Erg6 2017 Sourcequedentata ent-isoalantolactone (ent-iLL) C.Dcdr2::hisG albicans SC5314 tachliquid to polymethyl medium, respectively methacry-The MBIC80 for Cur was ≥200 μg/mL for ses-The clustered aggregative [152] quedentata2017 ent-isoalantolactone (ent-iLL) Dcdr2::hisGC. albicans SC5314 liquidtach to medium, polymethyl respectively methacry- regulatedThe clustered aggregative [152] 2017quedentata Curcumaquedentata longa (Zingi-(Zingi-ent-isoalantolactone (ent-iLL) C. albicans SC5314 tach to polymethyl methacry-silesile cellscells Cur at 50 µg/mL The clustered aggregative [152] Curcuma longa (Zingi- ent-isoalantolactone (ent-iLL) lateCur (PMMA)at 50 µg/mL denture reduced base the sile cells down-regulated andthe adhesinflocculation ALS3, genes with entcurcumin-isoalantolactone (Cur) (ent-iLL) Cur at 50 µg/mLlatelate reduced (PMMA)(PMMA) denturedenture basebase andCurand flocculationatflocculation 50 μg/mL genesgenesdown - beraceae)-diketone diphenol curcumincurcumin (Cur)(Cur) the adhesin ALS3,Cur with at 50 μg/mL down- β-diketone diphenolberaceae)beraceae) the capacity of C. albicans to Cur at 50 μg/mL down- materialcapacity of C. albicans to at- minimal impact on ALS1. CurAAF1minimal at, 50 impactμg/mL ondown ALS1.- 2017 Source C. albicans SC5314 attach to polymethylmaterial The MBIC for Cur was ≥200 µg/mL for sessile cells AAF1regulated[152,,] Source 80 The MBIC80 for Cur was ≥200The clustered μg/mL aggregative regulatedfor ses- Curcuma2017 longa (Zingiberaceae) C. albicans SC5314 methacrylate (PMMA)tach to polymethyl methacry- EAP1Theregulated clustered, and ALS5 aggregative tran- [152] Cur at 50 µg/mL reduced the and flocculation genes AAF1,regulatedtheEAP1 adhesin, and ALS5 ALS3, tran- with Curcuma longa (Zingi- dentureCur base at material50 µg/mL reduced the sile cells the adhesinEAP1 ALS3,, and withALS5 tran- -diketone diphenol Cur at 50 µg/mL reduced the EAP1, and ALS5 transcripts the adhesin ALS3, with -diketone diphenol curcumin (Cur) Curlate at(PMMA) 50 µg/mL denture reduced base the thescriptsandscripts adhesin flocculation werewere ALS3, upup--regulated regulatedgenes with -diketone diphenol curcumin (Cur) capacitycapacity of C. albicans of C. albicansto at- to at- were up-regulatedminimalscriptsminimal impact were onimpact ALS1. up- regulatedon ALS1. Sourceberaceae)-diketone diphenol The MBIC80 for Cur was ≥200 μg/mL for ses- Source materialcapacity of C. albicansThe to MBICat- 80 for Cur was ≥200 μg/mL for ses- AAF1minimal, impact on ALS1. 2017 Source C. albicans SC5314 capacitytach to polymethyl of C. albicans methacry- to at- The MBIC80 for Cur was ≥200 μg/mL for ses-minimalThe clustered impact aggregative on ALS1. [152] 2017 SourceCurcuma longa (Zingi- C. albicans SC5314 tach to polymethyl methacry- Thesile MBICcells 80 for Cur was ≥200 μg/mL Thefor ses- clustered aggregative [152] Curcuma20172017 longa (Zingi- C. C.albicans albicans SC5314 SC5314 tachtach to topolymethyl polymethyl methacry- methacry-sile cells TheEAP1The clustered, clusteredand ALS5 aggregative aggregative tran- [152] [152] Curcuma longa (Zingi-curcumin (Cur) late (PMMA)late (PMMA) denture denture base base sile cells and flocculation6and6 gg andand flocculation 66 genes--SS significantlysignificantly genes beraceae)Curcumaberaceae) longa (Zingi-curcumin (Cur) late (PMMA) denture base sile cells 6 gand and flocculation 6-S significantly genes Phenolsberaceae) with a varia- curcumin (Cur) latematerial (PMMA) denture base andalteredAAF1scripts flocculation, werethe expressions up- regulatedgenes of beraceae)PhenolsPhenols withwith aa varia-varia- curcumin (Cur) material AAF1, alteredaltered thethe expressionsexpressions ofof materialmaterial 66--SS atat 10,10, 50,50, andand 100100 AAF1AAF1, , bleble lengthlength inin thethe lat-lat- 6-S at 10, 50, and 100 EAP1, andsomeEAP1some ALS5 ,hyphahypha and tran- ALS5--specificspecific tran- ble length in the lat- 6-gingerol (6-G) µg/mL inhibited 85, 6 g and 6-S significantly someEAP1 hypha, and- specificALS5 tran- eral chain (6 or 8 car- 66-gingerol6--gingerolgingerol (6-G) (6(6--G) µg/mLµg/mL inhibitedinhibited 85,85, altered the expressions of EAP1(scripts6HWP1 g and, and were and6-S ALS5 significantlyECE1 up- regulatedtran-), bio- eral chain (6 or 8 car- some hypha-specificscripts (HWP1 were(HWP1 up -andregulated ECE1), bio- eral chain (6 or 8 car- 6-S at 10, 50, and 100 µg/mL94, and 94% biofilm (HWP1scripts and were ECE1 up-),regulated bio- bons) bearing a keto 6-S at 10 µg/mL was more ef- 94,94, andand 94%94% biofilmbiofilm and ECE1), biofilm-related scriptsfilm-related were up(HWP1-regulated Phenols with aPhenolsbons) variable lengthbearing with in the a laterala varia- keto 6-S at 10 µg/mLinhibited was 85, 94,more and 94% ef- alteredfilm-related the expressions (HWP1 of bons) bearing a keto Fluconazole-resistant C. albicans6-S at 10 µg/mL6- wasS at more 10 µg/mL was more ef- formation, respec- (HWP1 film-related (HWP1 chain (6 or 8 carbons) bearing a keto FluconazoleFluconazole-resistant-resistantC. C. albicans biofilm formation, 6formation,-S at 10, 50, respec- and 100 substituentble length in and the a lat- - Fluconazole-resistant C. albicanseffective in suppressingfective in suppressing hyphal formation, respec- and RTA3) and multidrug andsome RTA3 hypha) and-specific multidrug 2018 substituent andsubstituent a β-OH (gingerols) and or a ∆ a-5 - albicans DAY185 fective in suppressingrespectively. 6 g andhyphal 8 g at 50 6and g and[153 RTA3] 6-S) andsignificantly multidrug 2018 substituent and a - DAY185 hyphal formationfective than 6 g in at suppressing hyphal tively.µg/mL 6 inhibited g and 8 g 85, at transporter (CDR16 andg and and6-S significantlyRTA3) and multidrug [153] double2018 bond2018 (6- shogaol) 6-gingerol (6-G) DAY185 µg/mL inhibited by 88 andtively.tively. 66 gg andand 88 gg atat [153][153] OHeral (gingerols)chain (6 or or8 car- a - 50 µg/mL formation than 6 g at 50 CDR2) related genes. 80% of transporter(HWP16 g and and 6- S(CDR1ECE1 significantly), andbio- Source: ZingiberOHPhenols officinale (gingerols) with a varia-or a -- formationformation thanthan80%, 6 respectively6 gg atat 5050 the biofilm50 µg/mL inhibited by 6transporteralteredtransporter g and 6the-S significantly expressions (CDR1(CDR1 andand of Phenols with a varia- 94,50 µg/mLand 94% inhibited biofilm by C. elegans infectedaltered with C. the expressions of 5 doublePhenols bond with (6 a -varia- µg/mL formation 506-S µg/mL at 10, 50, inhibited and 100 by CDR2)altered related the expressions genes. 80% of Phenolsbons)5 double bearing with bond a a varia-(6 keto- 6µg/mL-S at 10 µg/mL was more6-S at ef- 10, 50, and 100 albicans alteredfilmCDR2)-related therelated expressions (HWP1 genes. 80% of ble length5ble double in length the lat-bond in the (6 lat-- 88-gingerol8--gingerolgingerol (8-G) (8(8--G) Fluconazole-resistant C. albicans µg/mL 88formation,6 and-S at 80%, 10, respec-50, respec- and 100 survivedsome in the hyphaCDR2)some-specific hypha related- specific genes. 80% shogaol)substituentble length andin the a  lat-- 86-gingerol (8(6-G) fective in suppressingµg/mL hyphal 6inhibited88 µg/mL88-S andandat 10, 80%,inhibited80%, 50,85, andrespec-respec- 10085, presence of both compounds ofandsome C. RTA3 elegans hypha) and infected-specific multidrug with bleshogaol)eralshogaol) length chain in(6 theor 68 -lat- gingerolcar- (6-G) tivelyµg/mL the inhibited biofilm for- 85, at 50 µg/mL someof(ofHWP1 C. eleganshypha and - ECE1infectedspecificinfected), bio- withwith eral2018 chain (6 or 8 car- 6-gingerol6-gingerol (6 -(6G)-G) DAY185 µg/mL94,tively.tively and the6inhibited 94% g biofilmand biofilm 8 g85, for-at (HWP1 and ECE1), bio- [153] eralSource:OHSource:eral chain(gingerols) chain Zingiber (6 (6or or8 or offici-car- 8 a car- - formation than 6 g at 5094, and tively94% biofilm the biofilm for- (C.transporterHWP1( albicansHWP1 and and survivedsurvived ECE1(CDR1 ECE1), bio-and ), inin bio- thethe bons) bearingSource:bons) bearinga Zingiberketo a ketooffici- 6-S at 106 µg/mL-S at 10 wasµg/mL more was ef- more ef- ma5094, µg/mLtion and 94% inhibited biofilm by film-relatedC.film albicans -(relatedHWP1 survived (HWP1 in the nale5 doublebons) bearing bond (6 a- keto FluconazoleFluconazole-resistant-resistant C. albicans C. albicans µg/mL6-S at 10 µg/mL wasformation, more ef-94,maformation, andtionrespec-tion 94% respec- biofilm presenceCDR2)film-related related of both ( genes.HWP1 com- 80% substituentbons)nalesubstituentnale and bearing a  -and a keto a - 8-gingerol (8-G) Fluconazole-resistant C. albicansfective in6fective- Ssuppressing at 10 in µg/mL suppressing hyphal was more hyphal ef- 88formation, and 80%, respec-respec- and RTA3filmpresenceandpresence) and- relatedRTA3 multidrug ofof) and both(bothHWP1 multidrug com-com- 2018 2018 shogaol)substituent and a - DAY185FluconazoleDAY185 -resistant C. albicans fective in suppressingtively. hyphal 6formation,tively. g and 68 gg andatrespec- 8 g at poundsofand C. elegans RTA3 at 50) infected andμg/mL multidrug[153] with [153] 2018substituentOH (gingerols) and ora  a- - DAY185 fectiveformation in suppressing than 6 g at 50hyphal tively. 6 g and 8 g at andpoundstransporterpounds RTA3 atat) 5050and (CDR1 μg/mLμg/mL multidrug and [153] OH2018 (gingerols) or a -  DAY185 formation than 6 g at 50 tively.50tively µg/mL the6 g andbiofilminhibited 8 g atfor- by transporter (CDR1 and [153] OHSource:OH (gingerols) (gingerols) Zingiber or offici- ora  a- 6-shogaol- (6-S) formationformation than than 6 g 6 atg at5050 50 µg/mL inhibited by transporterC.transporter albicans survived (CDR1 (CDR1 and in and the 5 double5 bonddouble (6 -bond (6- 686--shogaolgingerolshogaol (6 (6(8---S)S)G) µg/mL µg/mL ma50tion µg/mL inhibited by CDR2) relatedCDR2) genes.related 80% genes. 80% nale5 double bond8- (6gingerol- 6-shogaol (8-G) (6-S) µg/mL 88 and 80%,5088 µg/mLand respec- 80%, inhibited respec- by presenceCDR2) relatedof both genes.com- 80% shogaol)5shogaol) double bond (6- 8-gingerol8-gingerol (8 -(8G)-G) µg/mL 88 and 80%, respec- of C. elegansCDR2)of C. infectedelegans related infected with genes. with80% shogaol) tively the88tively biofilmand the80%, biofilmfor- respec- for- poundsof C. elegans at 50 μg/mL infected with Source: shogaol)Source:Zingiber Zingiber offici- offici- tively the biofilm for- C. albicansofC. C. albicanssurvived elegans survived infectedin the inwith the Source: Zingiber offici- mation tivelyma tion the biofilm for- C. albicans survived in the nale Source:nale Zingiber offici- 6-shogaol (6-S) mation presenceC.presence of albicans both com-of survived both com- in the nale mation presence of both com- nale poundspresence poundsat 50 μg/mL at of 50 both μg/mL compounds at 50 μg/mL pounds at 50 μg/mL 6-shogaol6-shogaol (6-S) (6-S) 6-shogaol6-shogaol (6 -(6S)- S) Antibiotics 2021, 10, x FOR PEER REVIEW 18 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 18 of 33

Antibiotics 2021, 10, 1053 17 of 31 The three compounds inhibit biofilm formation and eradicate mature Antibiotics 2021, 10, x FOR PEER REVIEW 18 of 33 biofilms by the following mechanisms: (i)The ergosterol three compounds biosynthesis inhibit inhibition biofilm and formation selectively and interactioneradicate mature via er- Antibiotics 2021, 10, x FOR PEER REVIEW 18 of 33 AntibioticsMonoterpenesAntibiotics 2021, 10 20, x21 FOR, 10 ,PEER x FOR REVIEW PEER REVIEW gosterolbiofilms binding,by the following mechanisms: 18 of 3318 of 33 Table 4. Cont. Source Origanum vul- (ii)(i) ergosteroldisruption biosynthesis of the biofilm inhibition cell surface and with selectively reduction interaction in cell height, via er-

Year Type of Compound/NaturalgareMonoterpenes Source Structure and Name Strains In Vitro Type of Studies (iv)gosterolThe alterationsthree binding, compounds in the fatty inhibit acid biofilm profile formation which attenuate andReference eradicate the cell mature mem- Inhibition of Cell Adhesion Eradication of Mature Studies of Mechanisms of thymol carvacrol C. neoformans NCIM 3541 (equiva- Biofilm Formation 2019 CinamomumSource Origanum verum vul- or Hyphal Formation brane(ii)biofilms disruptionBiofilms fluidity by the withof following the enh biofilmanced mechanisms:Action cell permeability, surface with resulting reduction in porein cell for- height, [150] lent to ATCC 32045) (i)The ergosterol three compounds biosynthesis inhibit inhibition biofilm and formation selectively and interactioneradicate mature via er- Cymbopogongare citratus mationThe(iv) threealterations andThe compounds effluxthree in compoundstheof the fattyinhibit K+/intracellular acid biofilminhibit profile biofilmformation which content, formation attenuate and eradicate andthe cell eradicate mature mem- mature Monoterpenes thymol carvacrol C. neoformans NCIM 3541 (equiva- gosterolbiofilms binding,by the following mechanisms: 2019Mentha Cinamomum piperita verum and (v)biofilmsbrane mitochondrial fluiditybiofilms by the with following by depolarization enhthe followinganced mechanisms: permeability, causedmechanisms: higher resulting levels in of pore ROS. for- Then, [150] lent to ATCC 32045) (i) ergosterol biosynthesis inhibition and selectively interaction via er- ThymusCymbopogonSource Origanum vulgaris citratus vul- themation(ii) disruptionoxidative (i)and ergosterol efflux stress of the of caused biofilmthebiosynthesis K+/intracellular a cellsignificant surface inhibition declinewith content, and reduction in selectively the amount in cell interaction ofheight, EPM via er- Monoterpenes C. neoformans NCIM 3541 The three compounds inhibit(i) biofilm ergosterol formation andbiosynthesis eradicate mature biofilmsinhibition by the following and selectively interaction via er- 2019 gareMonoterpenes (iv)gosterol alterations binding, in the fatty acid profile which attenuate[150] the cell mem- Source OriganumMonoterpenesMentha vulgare Monoterpenespiperita and thymol carvacrol C. neoformans(equivalent to NCIM ATCC 32045) 3541 (equiva- mechanisms: andgosterol(v) mitochondrial capsulegosterol binding, sugars binding, depolarization (mann ose, xylose, caused and higher glucuronic levels acid), of ROS. leading Then, to Cinamomum2019 verum CinamomumSource Origanum verum vul- (i) ergosterol biosynthesis inhibitionbrane(ii) disruption andfluidity selectively withof interaction the enh biofilm viaanced ergosterol cell permeability, binding, surface with resulting reduction in porein cell for- height, [150] Cymbopogon citratusSourceThymus Origanum Sourcevulgaris Origanum vul- vul- lent to ATCC 32045) (ii) disruption of the biofilma(ii)the cell reduced surface disruptionoxidative(ii) with capsule disruption reduction stress of the in size cell caused biofilm height,of and the ana biofilm cellsignificant overall surface cell negative declinesurfacewith reduction charge inwith the reduction amounton in the cell cell height,of in sur-EPM cell height, Mentha piperita and Thymus vulgaris alterations in the fatty acid profile which attenuate the cell membrane fluidity with enhanced Cymbopogongare gare citratus mation(iv) alterations (iv)and effluxalterations in theof the fatty in K+/intracellular the acid fatty profile acid whichprofile content, attenuate which attenuate the cell themem- cell mem- gare citral thymol thymol thymol carvacrol carvacrol carvacrol C. neoformans NCIM 3541 (equiva- permeability, resulting in poreface(iv)and formation alterationscapsule and efflux sugars ofin the the K+/intracellular(mann fattyose, acid content, xylose, profile and which glucuronic attenuate acid), the cell leading mem- to 2019 Cinamomum verum thymol carvacrol C. neoformansC. neoformans NCIM 3541 NCIM (equiva- 3541 (equiva- (v) mitochondrial depolarizationbrane caused fluidity higher levels with of ROS. enh Then,anced the oxidative permeability, stress caused a resulting in pore for- [150] Mentha2019 Cinamomumpiperita and verum a(v) reduced mitochondrialbrane capsule fluidity depolarizationsize with and enhan overallanced caused permeability, negative higher charge levels resulting ofon ROS. the incell Then, pore sur- for- [150] 2019 Cinamomum verum lent to ATCClent to 32045) ATCC 32045) significant decline in the amountbrane of EPM fluidity and capsule with sugars enh (mannose,anced xylose, permeability, and glucuronic resultingThe expression in pore of for- genes [150] ThymusCymbopogon vulgaris citratus lent to ATCC 32045) acid), leading to a reduced capsulethemation oxidative size and an efflux overall stress negative of caused the charge K+/intracellular ona thesignificant cell surface decline content, in the amount of EPM CymbopogonCymbopogon citratus citratus citral SKmationface at 4 µg/mLandmation efflux inhib-and of efflux the K+/intracellular of the K+/intracellular content,involved content, in hyphae for- Mentha piperita and and(v) mitochondrial capsule sugars depolarization (mannose, xylose, caused and higher glucuronic levels acid),of ROS. leading Then, to 1,4Mentha-naphtoquinone Menthapiperita andpiperita de- and ited(v) mitochondrial biofilm(v) mitochondrial formation depolarization depolarization caused highercausedmationThe levels higherexpression and of levels adhesion,ROS. of of Then, genesROS. Then, Thymus vulgaris athe reduced oxidative capsule stress size caused and aan significant overall negative decline charge in the amounton the cell of EPMsur- rivativeThymus Thymusvulgaris vulgaris C. albicans SC5314 and 10 clinical The filamentation in Lee’s bytheSK 65.4%, atoxidative 4 µg/mLthe while oxidative stress inhib- the caused stressSK at acaused 32 significant µg/mL a significant de- declineECE1,involved declinein the HWP1, amountin in hyphae the EFG1, amount of for-EPM of EPM faceand capsule sugars (mannose, xylose, and glucuronic acid), leading to 2019 Source1,4-naphtoquinone de-citral isolates from Changhai Hospital of media was completely inhib- biofilmandited capsulebiofilm growthand capsuleformationsugars was (mannsugars stroyedose, (mann maturexylose,ose, andxylose,bio- glucuronicCPH1,RAS1,mation and glucuronic and acid), adhesion, ALS1, leading acid), leadingto [154] to a reduced capsule size and an overall negative charge on the cell sur- a reduceda reduced capsule capsulesize and size an overalland an negativeoverallThe negative charge expression oncharge the of cell on genessur-the cell sur- (rivativeLithospermum erythro- Shanghai,C. albicans China.SC5314 and 10 clinical itedThe byfilamentation 0.5 µg/mL ofin SKLee’s almostby 65.4%, totally while inhib- the filmsSK at by32 92.8%µg/mL de- ALS3ECE1, and HWP1, CSH1 EFG1,were citral SKface at 4 µg/mL inhib- involved in hyphae for- 2019rhizon Source) citral citral isolates from Changhai Hospital of media was completely inhib- itedfacebiofilm when facegrowth exposed was to stroyed mature bio- downregulatedCPH1,RAS1, ALS1, while [154] 1,4-naphtoquinone de-shikonin (SK) ited biofilm formation The expression of genes mationThe expression and adhesion, of genes involved in hyphae The expression of genes (Lithospermum erythro- Shanghai, China. ited by 0.5 µg/mLSK at 4 µ ofg/mL SK inhibited 32almost µg/mL totally of SK inhib- films by 92.8% TUP1,TheALS3 expression and NRG1 CSH1, and of were BCR1 genes C. albicans SC5314 and 10 The filamentation in Lee’s formation and adhesion, 1,4-naphtoquinonerivative derivative C. albicans SC5314 and 10 clinical The filamentationbiofilm formation in Lee’s by 65.4%, bySK 65.4%,at 4 µg/mL while inhib- the SK at 32 µg/mL de- ECE1,involved HWP1, in hyphae EFG1, for- rhizon) clinical isolates from media was completely ited whenSK atSK 32 µ exposedatg/mL 4 µg/mL destroyed to inhib-ECE1, HWP1, EFG1, weredownregulated upregulatedinvolved while in hyphae for- 2019 Source while the biofilm growth wasSK at 4 µg/mL inhib- involved[154] in hyphae for- 1,4-naphtoquinone de-shikonin (SK) Changhai Hospital of inhibited by 0.5 µg/mL of ited biofilmmature biofilms formation by 92.8% CPH1,RAS1, ALS1, ALS3 andmation and adhesion, (Lithospermum2019 Source erythrorhizon 1,4) -naphtoquinone de- isolates from Changhai Hospital of media was completelyalmost totally inhibited inhib- whenbiofilm itedgrowth biofilm was formationstroyed mature bio- CPH1,RAS1,mation ALS1,and adhesion, [154] 1,4-naphtoquinone de- Shanghai, China. SK With Van at 62.5 µg/mL, C. ited32 µg/mL biofilm of formation SK CSH1 were downregulated mationTUP1, NRG1 and adhesion,, and BCR1 exposed to 32 µg/mL of SK (rivativeLithospermum erythro- Shanghai,C. albicans China.SC5314 and 10 clinical itedThe filamentationby 0.5 µg/mL ofin SKLee’s almostby 65.4%, totally while inhib- the filmsSK at by32TUP1, 92.8%µg/mL NRG1 de- ALS3ECE1, and HWP1, CSH1 EFG1, were Phenolicrivativerivative aldehyde C. albicansC. albicansSC5314 SC5314and 10 clinicaland 10 clinicalalbicansThe filamentation Thewere filamentation unable in toLee’s ex- in Lee’sAdherenceby 65.4%, by while 65.4%, to polysty- the while SK the atwhile 32SK µg/mL at 32 , de-µg/mL and C.ECE1,were de- albicans upregulated HWP1,ECE1, biofilms HWP1, EFG1, were EFG1, 2019 Source C.isolates albicans from SC5314, Changhai MRC10 Hospital (Δicl1) of media was completely inhib- biofilm growth was MaturestroyedBCR1 werebiofilmmature upregulated eradi-bio- CPH1,RAS1, ALS1, [154] Sourcerhizon2019) Source shikonin (SK) isolates from Changhai Hospitalpress of filaments media was and completely pre- reneited inhib- whensurfacebiofilm exposed growth to was stroyed matureabsentdownregulated bio- orCPH1,RAS1, negligible while in ALS1,C. [154] 2019 Source shikonin (SK) isolates from Changhai Hospital of mediaWith Van was at completely 62.5 µg/mL, inhib- C. biofilm growth was stroyed mature bio- CPH1,RAS1, ALS1, [154] 2020 (Lithospermum erythro- andShanghai, MRC11. China. Consult the genotypesWith Van at 62.5 itedµg/mL, byC. 0.5 µg/mL of SK 32almost µg/mL totally of SK inhib- cationfilms by (52%) 92.8% was ob- TUP1,ALS3 and NRG1 CSH1, and were BCR1 [155] ManyPhenolic plants(Lithospermum aldehyde includ- erythro- Shanghai, China. albicans were unablesentedalbicans to a itedwere normal by unable 0.5 morphology µg/mL to ex- of SK andAdherence biofilmalmost toformation polysty- totally inhib- films by 92.8%elegansC. albicans wormsALS3 biofilms and treated CSH1 were were (Lithospermum erythro- Shanghai,C. albicans China.SC5314,MRC10 ited by 0.5 µg/mLAdherence of to SK polystyrene almost totally inhib- filmsC. albicansby 92.8%biofilms were ALS3 and CSH1 were Phenolic aldehyderhizon) inC. thealbicans original SC5314, reference MRC10 express(Δicl1) filaments and ited when exposed to servedMature biofilm eradi-downregulated while rhizon) (∆icl1) and MRC11. Consult surface Matureited biofilm when eradication exposedabsent to or negligible in C. were upregulateddownregulated while 2020 Source ingrhizonSource Melia) azedarach shikonin (SK) presented a normalwithpress few filaments or no adherence and pre- to wereitedrene when surfacereduced exposed by 49% to withdownregulatedabsent[155 125] or µg/mL negligible whileof Van in C. vanillin shikonin(Van) (SK) the genotypes in the original and biofilm formation were (52%) was observed elegans worms treated with Many plants2020 including Melia azedarach shikonin (SK) and MRC11. Consult the genotypesmorphology with With few or Van no at 62.5 µg/mL, C. 32 µg/mL of SK cation (52%) was ob- TUP1, NRG1, and BCR1 [155] Many plants includ- reference buccalsented epitheliala normalreduced morphologycells. by 49% 32and µg/mL biofilm32 ofµg/mL formationSK of SK 125 µg/mL of Van TUP1,elegans NRG1wormsTUP1,, and NRG1treated BCR1, and BCR1 Phenolic aldehyde in the original reference adherence to buccalalbicans were unable to ex- Adherence to polysty-served C.were albicans upregulated biofilms were ing Melia azedarach vanillin (Van) C. albicans SC5314, MRC10 epithelial(Δicl1) cells. with few or no adherence to FSwere reduced reduced the by num- 49% Mature biofilm eradi-werewith 125upregulatedwere µg/mL upregulated of Van Source vanillin (Van) pressWith Vanfilaments at 62.5 and µg/mL, pre- C. rene surface absent or negligible in C. 2020 and MRC11. Consult the genotypes Withbuccal Van epithelialWith at 62.5 Van cells.µg/mL, at 62.5 µg/mL,C. ber C.of viable cells and cation (52%) was ob- [155] Phenolic aldehyde albicans were unable to ex- Adherence to polysty-FS was able to modu- C. albicans biofilms were Many plantsPhenolic includ- aldehyde sented aalbicans normalFS reduced were morphology the unable number of to theFSand ex- reducedtotal biofilmAdherence biofilm theformation num- bio- to polysty- elegans wormsC. albicans treated biofilms were Phenolic aldehyde inC. thealbicans original SC5314, reference MRC10 (Δicl1) albicans wereviable unable cells and to the ex- total Adherence to polysty- servedMature biofilm eradi-C. albicans biofilms were SesquiterpeneSource alcohol C. albicansC. albicansSC5314, SC5314, MRC10 MRC10(Δicl1) (Δicl1)press filaments and pre- rene FSsurface was able to modulate lateMature preformed Maturebiofilm bio-biofilmeradi- absent eradi- or negligible in C. ing Melia azedarach with few or nobiofilm adherence biomass. to were reduced by 49% with 125 µg/mL of Van Sesquiterpene2020 Source alcohol Source vanillin (Van) and MRC11. Consult the genotypesFS prevented theFSpress adhesion prevented filaments ofpress filamentsthe and adhesion pre- and of pre- mass.reneber of preformedsurface Theviablerene metabolic biofilms, surfacecells and cation (52%) was ob- absent orabsent negligible or negligible in C. [155] in C. 2020 Fusariumand keratinoplaticum MRC11. Consult the genotypes The metabolic activity was cation (52%) was ob- [155] 2020 Source2020 SourceMany plants includ- Fusariumand MRC11. keratinoplaticum Consult the genotypesATCCconidia and filamentation buccalsented epithelial fora normal morphologycells. and biofilmdecreasing significantlyformation the films,cationFS was decreasing (52%) able towas modu- sig-ob- elegans[156] worms treated [155] 2020 Many plants includ- ATCC 36031 conidia sentedand filamentationonly a reduced normal at 500 morphologyµ forM. At activity andwas biofilmonly re- formation elegans worms treated[156] Dimorphic fungusManyC. albicans plants includ- in the original reference biofilm formationsented a normal morphology andthe total biofilmnumber biofilm of viableformation cells, bio- in served elegans worms treated DimorphicSesquiterpene fungus alcohol C. 36031 in the original reference 700 µM, FS completely nificantlylate preformedserved the number bio- ing Meliaing azedarach Melia azedarach in the original reference with fewwith or no few adherence or no adherence to FSwere reduced toparticular reduced were atthe >600reduced by num-µ M49% byserved 49% with 125with µg/mL 125 ofµg/mL Van of Van ing Melia azedarach vanillinvanillin (Van) (Van) biofilmwithFS prevented few formation or noprevented the adherence adhesion the biofilm to of ducedweremass. reduced Theat 500 metabolic μ byM. 49%At with 125 µg/mL of Van albicansSource vanillin (Van) Fusarium keratinoplaticum ATCC buccal epithelialformation cells. ber of viable cells and offilms, viable decreasing cells, in par-sig- 2020 buccalconidia epithelial buccaland filamentation epithelial cells. cells. for 700activity μM, wasFS com- only re- FS was able to modu- [156] Dimorphic fungus C. 36031 theFS reduced total biofilm the num- bio- ticularnificantly at >600 the number μM Sesquiterpene alcohol farnesolfarnesol (FS) (FS) biofilm formation pleteFSduced reducedly atpreventedFS 500 reduced the μM. num- Atthe the latenum- preformed bio- albicans FS prevented the adhesion of mass.ber of Theviable metabolic cells and of viable cells, in par- Source Fusarium keratinoplaticum ATCC biofilmber700 ofμM, viable formationber FS ofcom- cells viable and cells films,FS andwas decreasing able to modu- sig- 2020 conidia and filamentation for activitythe total was biofilm only bio- re- FSticular was atableFS >600 was to μ modu-ableM to modu- [156] DimorphicSesquiterpene fungus alcohol C. farnesol (FS) 36031 theplete totally preventedthe biofilm total biofilmbio- the nificantlylate bio- preformed the number bio- SesquiterpeneSesquiterpene alcohol alcohol biofilmFS prevented formation the adhesion of ducedmass. The at 500 metabolic μM. At late preformedlate preformed bio- bio- albicansSource Fusarium keratinoplaticum ATCC FS preventedFS prevented the adhesion the adhesion of mass.biofilm of The mass.formation metabolic The metabolic offilms, viable decreasing cells, in par-sig- 2020 Source Source FusariumFusarium keratinoplaticum keratinoplaticum ATCC ATCCconidia and filamentation for 700activity μM, was FS com- only re- films, decreasingfilms, decreasing sig- sig- [156] 2020 Dimorphic2020 fungus C. 36031 conidia conidiaand filamentation and filamentation for activity for activitywas only was re- onlyticularnificantly re- at >600 the number μM [156] [156] DimorphicDimorphic fungus C.fungus C. 36031 36031 biofilm formation pleteducedly at prevented 500 μM. Atthe nificantlynificantly the number the number albicans farnesol (FS) biofilm biofilmformation formation duced atduced 500 μ atM. 500 At μM.of viableAt cells, in par- albicans albicans biofilm700 μM, formation FS com- of viableof cells, viable in cells,par- in par- 700 μM,700 FS μM,com- FS com-ticular at >600 μM farnesol (FS) pletely prevented the ticular atticular >600 μatM >600 μM farnesolfarnesol (FS) (FS) pletely preventedpletely prevented the the biofilm formation biofilm biofilmformation formation Antibiotics 2021, 10Antibiotics, 1053 2021, 10, x FOR PEER REVIEW 18 of 31 19 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 19 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 19 of 33 Antibiotics 2021, 10, x FOR PEER REVIEW 19 of 33 Table 4. Cont. HSc-D decreased the

Year Type of Compound/Natural Source Structure and Name Strains In Vitro Type ofHSc Studies-D restricted the for- transcriptionalReference levels of Inhibition of Cell Adhesion Eradication of Mature Studies of Mechanisms of HSc-D decreased the Labdane diterpenoid mation of hyphaeBiofilm Formation at 4 μg/ml, the genes ALS3, HWP1, or Hyphal FormationHSc-D restricted the for- Biofilms Action transcriptional levels of Source CDR1 and CDR2 efflux pumps de- but showed no activity HSc-D completely HScand- DECE1 decreased encoding the adhe- Labdane diterpenoid mation of hyphae at 4 μg/ml, theHSc genes-D decreased ALS3, HWP1 the , 2020 liverwort Heteroscy- ficient strain C. albicans DSY654 andHSc against-D restricted SC5314. HScthe for--D de- prevented biofilm for- transcriptionalsins and affected levels the of [157] Source CDR1 and CDR2 efflux pumps de- butHSc showed-D restricted no activity the for- HSc-D completely andtranscriptional ECE1 encoding levels adhe- of Labdanephus diterpenoid the wild type C. albicans S5314 mationcreased of the hyphae adherent at 4 C.μg/ml, albi- mation at 8μg/mL theRas1 genes-cAMP ALS3-Efg1, HWP1 path-, 2020 liverwortLabdane diterpenoidHeteroscy- ficient strain C. albicans DSY654HSc-D restrictedand againstmation the SC5314.of hyphae HSc at- 4D μg/ml, de- prevented biofilm for- HSc-D decreased the sinsthe genesand affected ALS3, HWP1 the , [157] Sourcecoalitus CDR1 and CDR2 efflux pumpsformation de- of hyphaebutcans atshowed cells 4 on no A549 activity cancer cell HSc-D completely transcriptional levels of the andway, ECE1 NRG1 encoding and UME6 adhe- to Labdane diterpenoidSource CDR1CDR1 and and CDR2 CDR2 efflux efflux pumpsµg/ml, de- but showedbut noshowed no activity HSc-D completely genes ALS3, HWP1, and and ECE1 encoding adhe- phusSource theCDR1 wild and type CDR2 C. albicans efflux pumpsS5314 de- creasedbut showed the adherent HSc-Dno activity completely C. albi- preventedmationHSc-D completelyat 8μg/mL Ras1and -ECE1cAMP encoding-Efg1 path- adhe- Source 2020 liverwort Heteroscy- ficientpumps strain deficient C. strainalbicansC. DSY654activity againstand against SC5314. SC5314. HSc-D de- prevented biofilm for- ECE1 encoding adhesins andsins and affected the [157] 2020 2020 liverwort Heteroscy- ficient strain C. albicans DSY654 and againstmonolayers SC5314. biofilmfrom HSc formation 1 µg/mL-D de- at prevented biofilm for- sinsretard[ 157and] the affected yeast- tothe-hyphal [157] liverwort2020Heteroscyphus liverwort Heteroscy- ficientalbicans strainDSY654 C. andalbicans the wild DSY654HSc-D decreased and against the SC5314. HSc-D de- prevented biofilm for- affected the Ras1-cAMP-Efg1sins and affected the [157] coalitusphus the wild type C. albicans S5314 canscreased cells the on adherent ≥A5498µg/mL cancer C. albi- cell mation at 8μg/mL way,Ras1- NRG1cAMP -andEfg1 UME6 path- to coalitus phus heteroscyphin D (HSc-D) the wildtype C. type albicans C.S5314 albicans S5314adherent C. albicanscreasedcells on the adherent C. albi- mation at 8μg/mL pathway, NRG1 and UME6 toRas1transition.-cAMP -Efg1 path- coalitusphus the wild type C. albicans S5314A549 cancer cellmonolayerscanscreased monolayers cells the on adherent fromA549 1 cancer µg/mL C. albi- cell mation at 8μg/mL retard the yeast-to-hyphal retardway,Ras1 -NRG1cAMP the yeast and-Efg1- toUME6 path--hyphal to coalitus from 1 µg/mLcans cells on A549 cancer cell transition. way, NRG1 and UME6 to coalitus cans cells on A549 cancer cell transition.way, NRG1 and UME6 to heteroscyphin D (HSc-D) monolayers from 1 µg/mL Ar was more effec- retard the yeast-to-hyphal monolayers from 1 µg/mL retard the yeast-to-hyphal Artemisinin sesquiter- tive in disrupting the transition. heteroscyphin D (HSc-D) transition. pene lactone heteroscyphin D (HSc-D) D (HSc-D) C. albicans 1372; Arpreformed was more EPM effec-- transition. Artemisinin sesquiter- tive in disrupting the Scopoletin: coumarin C. dubliniensis 1470, C. tropicalis Arstructure was more and effec- in kill- Ar and Sc promoted the pene lactone C. albicans 1372; FS and Sc reduced preformedAr was more EPM effec-- Artemisininderivative sesquiter- 1368, tiveing inthe disrupting sessile cells the as accumulation of intracel- Scopoletin:Artemisinin coumarin sesquiter- C. dubliniensis 1470, C. tropicalis biofilm biomass and structuretive in disrupting and in kill- the Ar and Sc promoted the 2020pene Sources lactone C.C. albicanskrusei 779, 1372 ; FS and Sc reduced preformedcompared toEPM Sc -at lular ROS by increasing [158] pene lactone C. albicans 1372; metabolic activity and preformed EPM- derivativeScopoletin:Ar: Artemisia coumarin 1368,C.C. dubliniensisglabrata 1374 1470, and C. tropicalis Ar was more effective in ingstructuretheir the respective sessile and incells kill- as accumulationAroxidative and Sc promotedstress of atintracel- their the Scopoletin: coumarin C. dubliniensisC. albicans 1372; 1470, C. tropicalis biofilmdisrupting biomass the preformed and structure and in kill- Ar and Sc promoted the Artemisinin sesquiterpeneScopoletin: lactone coumarin artemisinin (Ar) C. dubliniensis 1470, C. tropicalis FSled and to non Sc reduced-viable cells structureAr and Sc and promoted in thekill- Ar and Sc promoted the 2020 Sourcesderivative C.1368, kruseiC. dubliniensis 779, 1470, C. FS and Sc reduced biofilm FS andEPM- Sc structure reduced and in killingcompareding the sessile to Sc cells at as lularaccumulation ROS by increasing of intracel- [158] Scopoletin: coumarinannua derivative C. guilliermondii 808 metabolicFS and Sc activityreduced and MBECaccumulation10 . of intracellular respective MBEC10 derivative 1368,tropicalis 1368, biomass and metabolic biofilmthe sessile biomass cells as comparedand ing the sessile cells as accumulation of intracel- 2020 Sources Ar:derivative Artemisia C.1368, glabrata 1374 and biofilm biomass and theiring ROSthe respective by sessile increasing cells oxidative as oxidativeaccumulation[158] stress of at intracel- their 2020 Sources C. kruseiC. krusei 779,779, activity and led to non-viable to Sc at their respective compared to Sc at lular ROS by increasing [158] Ar: Artemisiaannua2020 SourcesSc: from several plantsartemisinin (Ar) C. krusei 779, ledbiofilm to non biomass-viable and cells comparedin C.stress albicans, at their to respectiveSc C. at lular ROS by increasing [158] 2020 Sources C. kruseiC. glabrata 779,1374 and cells metabolicMBEC .activity and compared to Sc at lular ROS by increasing [158] Sc: from severalannua plants including A. annua C. guilliermondii 808 metabolic10 activity and MBECMBEC10 . respective MBEC10 Ar:including Artemisia A. annua C. glabrataC. guilliermondii 1374808 and metabolicIn C. albicans, activity C. dubliniensis andtheir dubliniensis respective10 and C. oxidative stress at their Ar: Artemisia artemisinin (Ar) (Ar) C. glabrata 1374 and led to non-viable cells their respective oxidative stress at their Sc:annua from several plants artemisinin (Ar) C. guilliermondii 808 led toand nonC. glabrata-viable cells inMBEC C. albicans,10 . C. respective MBEC10 annua artemisinin (Ar) C. guilliermondii 808 led to non-viable cells MBECglabrata10 . respective MBEC10 includingannua A. annua scopoletin (Sc) C. guilliermondii 808 dubliniensisMBEC10 . and C. respective MBEC10 Sc: from several plants in C. albicans, C. Sc: from several plants At 4× MIC (200 glabratain C. albicans, C. including A. annua dubliniensis and C. includingScopoletin A.: coumarin annua scopoletin (Sc) µg/mL), Sc produced dubliniensisAt its MIC (50and C. glabrata scopoletin (Sc) Fluconazole, itraconazol and am- At 4× MIC (200 glabrata derivative scopoletin (Sc) a great reduction of glabrataµg/mL), Sc reduced 2020Scopoletin : coumarin scopoletin (Sc) phothericin--resistant C. tropicalis µg/mL), Sc produced At its MIC (50 [159] At 4× MIC (200 µg/mL), ScAt 4× MIC (200 Scopoletin: coumarinSource derivative Fluconazole,Fluconazole, itraconazol and and am- Atthe 4 ×areaAt MIC its MICoccupied (200 (50 µ g/mL), by Sc preformed Candida produced a great reduction of 2020 Source derivative ATCCamphothericin–resistant 28707 C. aAt great 4×reduced MIC reduction preformed (200 Candidaof µg/mL), Sc reduced [159] ScopoletinMitracarpus: coumarin frigidus . scopoletin (Sc) the area occupied by biofilmsµg/mL),biofilms Scon produced the sur- Atbiofilms its MIC (50 Mitracarpus2020 frigidus Scopoletin. : coumarin phothericinFluconazole,tropicalis ATCC-- resistantitraconazol 28707 C. tropicalisand am- µg/mL),biofilms Sc produced At its MIC (50 [159] SourcederivativeScopoletin : coumarin Fluconazole, itraconazol and am- on the surface of coverslipstheaµg/mL), great area reduction occupiedSc produced ofby preformedµg/mL),At its MIC Sc Candida(50reduced derivative ATCCFluconazole, 28707 itraconazol and am- aface great of reductioncoverslips of µg/mL), Sc reduced 2020 Mitracarpusderivative frigidus . scopoletinscopoletin (Sc) (Sc) phothericin--resistant C. tropicalis biofilmsa great reduction on the sur- of biofilmsµg/mL), Sc reduced [159] Source theWarburganal: area occupied MBIC by 50 preformed Candida 2020Source ATCCphothericin 28707-- resistant C. tropicalis the area occupied by preformed Candida [159] Source ATCC 28707 facethe areaof coverslips occupied by preformed Candida Mitracarpus frigidus . scopoletin (Sc) ATCC 28707 biofilms= 4.5 and on 50 the µg/ml sur- biofilmsWarburganal and Warburganal: MBIC = 4.5 Mitracarpus frigidus . scopoletin (Sc) 50 Warburganal:biofilmsWarburganal on the and MBIC polygodial:sur- 50 biofilms Drimane sesquiter- and ~50 µg/ml against C. faceagainst of coverslips C. albicans and polygodial: MBEC50 = Drimane sesquiterpene dialdehydes C. albicans SC5314; C. glabrata face ofMBEC coverslips= ~ 16 µg/ml albicans and C. glabrata, =face 4.5 ofand coverslips50 50 µg/ml Warburganal and 2020 Source pene dialdehydes C. albicansATCC 2001 SC5314; and C. glabrata C. glabrata ATCC Warburganal:C. glabrataagainst C., albicansrespec- MBICbut did50 not  16 µg/ml against C. [160] respectively. Polygodial: Warburganal: MBIC50 Warburgia2020 ugandensis BG2 eradicate the C. glabrata 50 [160] Drimane sesquiter- MBIC = ~10 and and ~50againstWarburganal: C. albicans MBIC and polygodial: MBEC50 = Source 2001 and C. glabrata BG2 50 =tively. 4.5 biofilmand Polygodial: 50 µg/ml Warburganalalbicans but did and not µg/mL, respectively = 4.5 and 50 µg/ml Warburganal and peneDrimane dialdehydes sesquiter- C. albicans SC5314; C. glabrata ATCC C.against glabrata C. ,albicans respec- and polygodial: 16 µg/ml against MBEC 50C. = 2020 DrimaneWarburgia sesquiter- ugandensis againstMBIC50 C. = albicans10 and and polygodial:eradicate the MBEC C. gla-50 = [160] SourceDrimane sesquiter- (+)-warburganal(+)-warburganal R=OH R=OH 2001 and C. glabrata BG2 tively.against Polygodial: C. albicans andalbicans polygodial: but didMBEC not50 = pene dialdehydes (-)-polygodial R=H C. albicans SC5314; C. glabrata ATCC C.50 glabrata µg/mL,, respec- respec- brata 16 µg/ml biofilm against C. 2020 pene dialdehydes C. albicans SC5314; C. glabrata ATCC C. glabrata, respec-  16 µg/ml against C. [160] 2020 Warburgiapene dialdehydes ugandensis (-)-polygodial R=H C. albicans SC5314; C. glabrata ATCC MBICC. glabrata50 = ,10 respec- and and eradicate 16 µg/ml the against C. gla- C. [160] 2020Source 2001 and C. glabrata BG2 tively.tively Polygodial: albicans but did not [160] Source (+)-warburganal R=OH 2001 and C. glabrata BG2 tively. Polygodial: albicans but did not Warburgia ugandensis MBIC50 µg/mL,50 = 10 respec- and and brataeradicate biofilm the C . gla- WarburgiaDammarane ugandensis-type gly- (-)-polygodial R=H MBIC50 = 10 and and eradicate the C. gla- Warburgia ugandensis (+)-warburganal R=OH tivelyMBIC 50 = 10 and and eradicate the C. gla- cosides (gypenosides) (+) -warburganal R=OH 50 µg/mL, respec- brata biofilm ((+)No-)--polygodial warburganaldescription R=H of R=OH the compounds Flu-resistant C. albicans CA10 and Gypenosides50 µg/mL, respec- showed brata biofilm Dammarane-type gly- (-)-polygodial R=H 50 µg/mL, respec- brata biofilm 2020 Source (-)-polygodial R=H tively [161] cosides (gypenosides) tested CA16 tivelyMBIC 80 > 128 µg/mL DammaraneGynostemma- pentaphyl-type gly- No description of the compounds Flu-resistant C. albicans CA10 and Gypenosides showed 2020 SourcecosidesDammarane (gypenosides)-type gly- [161] cosideslum (gypenosides) testedNo description of the compounds CA16Flu-resistant C. albicans CA10 and MBICGypenosides80 > 128 µg/mLshowed Gynostemmacosides (gypenosides) pentaphyl- No description of the compounds Flu-resistant C. albicans CA10 and Gypenosides showed 2020 Source No description of the compounds Flu-resistant C. albicans CA10 and Gypenosides showed [161] 2020 Source tested CA16 MBIC80 > 128 µg/mL [161] 2020lum Source tested CA16 MBIC80 > 128 µg/mL [161] Gynostemma pentaphyl- tested CA16 MBIC80 > 128 µg/mL Gynostemma pentaphyllum lum Antibiotics 2021, 10, x FOR PEER REVIEW 20 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 20 of 33 Antibiotics 2021, 10 , 1053 19 of 31

Antibiotics 2021, 10, x FOR PEER REVIEW 20 of 33 Table 4. Cont. AntibioticsAntibiotics 20202121,, 1010,, xx FORFOR PEERPEERdammarane REVIEWREVIEW skeleton 2020 ofof 3333

Year Type of Compound/NaturalPolyciclic Source compound Structure and Name Strains In Vitro Type of Studies Reference Inhibition of Cell Adhesion Eradication of Mature TBMStudies inhibited of Mechanisms the of with a lipophilic side Biofilm Formation Ten μg/mL of TBM elimi- or Hyphal Formation Biofilms biofilmAction extracelular chain No description of the compounds tested C. albicans SN250, C. tropicalis 98- At 2 and 4 µg/mL, nated C. albicans C. tropi- vesicle (EV) produc- 2021 Source dammarane skeleton 234, C. glabrata 4720, C. auris B11220 TBM reduced the bio- calis, C. glabrata, C. auris [162] tion and, thus, elimi- PolyciclicSea squirt compoundmicrobiome and A. fumigatus 293 films by 50% and A. fumigatus, biofilms Dammarane-type glycosides (gypenosides) Flu-resistant C. albicans CA10 Gypenosides showed TBMnated inhibited the EPM theas- 2020 Source [161] withconstituent a lipophilic Micromon- side and CA16 MBIC > 128 µg/mL Tenfrom μg/mL catheters of TBM elimi- Gynostemma pentaphyllum 80 biofilmsembly extracelular chainospora sp. turbinmicin (TBM) C. albicans SN250, C. tropicalis 98- At 2 and 4 µg/mL, nated C. albicans C. tropi- vesicle (EV) produc- 2021 SourceOrganosulfur com- dammarane skeleton 234, C. glabrata 4720, C. auris B11220 TBM reduced the bio- calis, C. glabrata, C. auris [162] tionAllicin and, eradicated thus, elimi- PolyciclicSeapound. squirt compoundmicrobiome dammarane skeleton skeleton and A. fumigatus 293 films by 50% and A. fumigatus, biofilms dammarane skeleton TBMnated50% C.inhibited the albicans EPM theas-bio- 2021with constituentPolyciclicDiallylPolyciclic a lipophilic thiosulfinate compoundcompound Micromon- side C. albicans ATCC 14053 Tenfrom μg/mL catheters of TBM elimi- [164] biofilmsemblyTBMfilmsTBM inhibitedatinhibited extracelularsub-MIC thethe = 4 chainosporawithSourcewith a a sp. lipophiliclipophilic sideside turbinmicin (TBM) C. albicans SN250, C. tropicalis 98- At 2 and 4 µg/mL, natedTenTen μg/mLμg/mL C. albicans ofof TBMTBM C. tropi- elimi-elimi- Polyciclic compound with a lipophilic side allicin TBM inhibited the biofilm vesiclebiofilmTen µ (EV) g/mLextracelular ofproduc- TBM C. albicans SN250, C. tropicalis µg/mLbiofilm extracelular chain 2021 SourceOrganosulfurchain(Allium sativum com-) 234,C. albicans C. glabrata SN250, 4720, C. C.tropicalis auris B11220 98- TBMAt 2 extracelularand reduced 4 µg/mL, vesicle the (EV) bio- eliminated C. albicans C. calis,nated C. C. glabrata, albicans C.C. auristropi- [162] chain C. albicans98-234, C. SN250, glabrata 4720, C.C. tropicalis 98- At 2 and 4 µg/mL, TBM At 2 and 4 µg/mL, nated C. albicans C. tropi- 2021 Source production and, thus, tionAllicinvesicletropicalis, and, eradicated(EV) thus, C. glabrata, produc- elimi- C. auris [162] Sea squirt microbiome and A.auris fumigatusB11220 and A. 293 fumigatus reduced the biofilms by 50%films by 50% vesicle (EV) produc- and A. fumigatus, biofilms Sea squirt2021 microbiome pound.Source constituent 234, C. glabrata 4720, C. auris B11220 TBMeliminated reduced the EPMthe bio- and A. fumigatus, biofilms calis,CBD C.showed glabrata, a multitar- C. auris [162] 2021 Source 234, 293C. glabrata 4720, C. auris B11220 TBM reduced the bio-nated50%tion and,C. the albicans thus,EPM elimi-as-bio- calis, C. glabrata, C. auris [162] Micromonospora2021 constituentDiallylSeasp. squirt thiosulfinate microbiome Micromon- C.and albicans A. fumigatus ATCC 29314053 filmsassembly by 50% tionfrom and, catheters thus, elimi- from andget mode A. catheters fumigatus of action , biofilms with [164] Sea squirt microbiome and A. fumigatus 293 films by 50% semblyfilmsnated at the sub EPM-MIC as- = 4 and A. fumigatus, biofilms osporaSourceconstituent sp. Micromon- turbinmicin (TBM) nated the EPM as- fromup- regulation catheters of yeast- constituent Micromon-allicin µg/mLsembly from catheters Organosulfur(osporaAllium sp. sativum com-) turbinmicinturbinmicin (TBM) (TBM) sembly ospora sp. turbinmicin (TBM) Allicin eradicated associated genes and Organosulfur compound. pound.Organosulfur com- CBDAllicin at 25 eradicated µg/mL 50% C. At 1.56 and 3.12 CBDdownregulation showed a multitar- of hy- Diallyl thiosulfinateOrganosulfur com- 50%Allicin C. eradicatedalbicans bio- 2021 Terpenphenol C. albicansC. albicans SC5314ATCC 14053 C. albicans albicans biofilms at sub-MIC Allicin eradicated [164] Source2021 Diallylpound. thiosulfinate C. albicans ATCC 14053 caused a pronounced µg/mL, mature bio- getphae mode-specific of action genes. with C. al- [164] pound. = 4 µg/mL films50% C. at albicanssub-MIC bio- = 4 (Allium sativum______) SC5314 carrying the green fluores- 50% C. albicans bio- 2021 SourceDiallyl thiosulfinate C. albicans ATCC 14053 up - regulation of yeast- [164] 20212021 Diallyl thiosulfinate allicinallicin C. albicans ATCC 14053 inhibitory biofilm for- µg/mLfilmsfilm decreased at sub-MIC 28% = 4 bicans virulence genes de- [165][164] Source cent protein (GFP) filmsCBD at showed sub- aMIC multitarget = 4 (SourceAllium sativum) mation effect. MBIC90 and 44%, respec- associatedcreased. CBD genes increases and Source allicin µg/mLmode of action with up- Cannabis sativa allicin reporter gene (C. albicans–GFP) µg/mLregulation of yeast-associated (Allium sativum) CBD= at 25 µg/mL At 1.56 and 3.12 CBDdownregulation showed a multitar- of hy- (Allium sativum) 100 µg/mL tively.genes and downregulation ofROS production, reduces Terpenphenol C. albicansC. albicans SC5314SC5314 C. albicansC. albicans Terpenphenol cannabidiol (CBD) CBD at 25 µg/mL caused a hyphae-specific genes. C. SC5314 carrying the green causedAt 1.56 a pronounced and 3.12 µg/mL, µg/mL, mature bio- getphaeCBDthe modeintracellular -showedspecific of action genes.a multitar- ATP with C. lev- al- ______pronounced inhibitory albicans virulence genes CBD showed a multitar- 2021 ______SC5314fluorescent carrying protein the (GFP) green fluores- mature biofilm decreased [165] Source 2021 biofilm formation effect. inhibitory biofilm for- filmdecreased. decreased CBD increases 28% upbicansget- moderegulation virulence of action of genes yeast with de-- [165] reporter gene (C. 28% and 44%, respectively. els,get modifiesmode of actionthe cell with wall, Cannabis sativaSource cent protein (GFP) MBIC 100 µg/mL ROS production, reduces the albicans–GFP) 90 = mation effect. MBIC90 and intracellular44%, respec- ATP levels, associatedcreased.upandup-- regulationincreasesregulation CBD genes increasesthe ofof and yeastplasmayeast - - Cannabis sativa reporter gene (C. albicans–GFP) modifies the cell wall, and CBD= 100 atµg/mL 25 µg/mL Attively. 1.56 and 3.12 downregulationROSassociatedmembrane production, genes permeability ofandreduces hy- Terpenphenol cannabidiol (CBD) (CBD) C. albicans SC5314 C. albicans increases the plasma associated genes and causedCBD at a25 pronounced µg/mL µg/mL,At 1.56membrane andmature permeability 3.12 bio- phaethedownregulation intracellular-specific genes. ATPof hy- C. lev- al- ______TerpenphenolMonoterpenes SC5314C. albicans carrying SC5314 the C. green albicans fluores- CBD at 25 µg/mL At 1.56 and 3.12 downregulation of hy- 2021 Terpenphenol C. albicans SC5314 C. albicans inhibitorycaused a pronounced biofilm for- filmµg/mL, decreased mature 28% bio- bicansels,phae modifies-specific virulence the genes. genes cell C.wall, deal- [165] Source______Sources centSC5314 protein carrying (GFP) the green fluores- Camphor at 0.125 mg/mL Camphorcaused a pronouncedand euca- µg/mL, mature bio- phae-specific genes. C. al- ______C.SC5314 albicans carrying 475/15, the C. albicansgreen fluores- 503/15, Camphor at 0.125 mg/mL 20212021Antibiotics EO from 20 21many, 10, speciesx FOR PEER REVIEW and eucalyptol at 23 mg/mL mationinhibitorylyptolinhibitory inhibited effect. biofilmbiofilm MBIC C. for-al-for-90 andfilmfilm 44%, decreaseddecreased respec- 28% 28% creased.andbicansbicans increases virulencevirulence CBD increasesthe genesgenes plasma de- de- [165][165] 21 of 33 CannabisSource sativa reportercentC. albicans protein gene ATCC (GFP) (C. albicans1023,; C.–GFP) krusei reduced ROS by 52% 2021 ofSource genera such as Cin- cent protein (GFP) induced a notable reduction =mationbicans, 100 µg/mL C. effect. tropicalis, MBIC C.90 tively.and 44%, respec- ROSmembranecreased. production, CBD permeability increases reduces [166] Cannabis sativa cannabidiol (CBD) reporterH1/16; C. gene tropicalis (C. albicans ATCC– 750GFP) and mation effect. MBIC90 and 44%, respec- whilecreased. eucalyptol CBD increases was in- MonoterpenesCannabis sativa reporter gene (C. albicans–GFP) = 100 µg/mL tively. theROS intracellular production, ATP reduces lev- Monoterpenesnamomum, Eucalyptus, C. albicans 475/15, C. albicans Camphor at 0.125in mg/mLthe number of hyphal cells parapsilosis= 100 µg/mL and C. tively. ROS production, reduces cannabidiol (CBD) Camphor and eucalyptol Sources cannabidiol (CBD) C. parapsilosis503/15, C. albicans ATCCATCC 22019 and eucalyptol at 23 mg/mL Camphor at 0.125 mg/mL active SourcesArtemisia , Salvia and camphor Camphorin C. albicans at inhibited0.125 475/15 mg/mLC. albicans, C. Camphorkrusei biofilm and biomasseuca- els,the intracellularmodifies the ATPcell wall, lev- 2021 EO from many species of genera such as camphor 1023,; C. krusei H1/16; C. induced a notable reduction reduced ROS by 52% while the [166intracellular] ATP lev- C. albicans 475/15, C. albicans 503/15, tropicalis, C. parapsilosis and Camphor at 0.125 mg/mL Cinnamomum, Eucalyptus, Artemisia, Salvia tropicalis ATCC 750 and C. in the number of hyphal cells eucalyptol was inactive and increases the plasma EOThuja from many species and eucalyptolC. krusei at biofilm23 mg/mL biomass lyptol inhibited C. al- els, modifies the cell wall, and Thuja C. albicansparapsilosis ATCCATCC 22019 1023,; C. kruseiin C. albicans 475/15 reducedels, modifies ROS theby 52%cell wall, 2021 of genera such as Cin- induced a notable reduction bicans, C. tropicalis, C. membraneand increases permeability the plasma [166] H1/16; C. tropicalis ATCC 750 and whileand increases eucalyptol the was plasma in- Monoterpenesnamomum, Eucalyptus , in the number of hyphal cells parapsilosis and C. membrane permeability C. parapsilosis ATCC 22019 activemembrane permeability SourcesArtemisiaMonoterpenes , Salvia and Camphorin C. albicans at 0.125 475/15 mg/mL Camphorkrusei biofilm and biomasseuca- Monoterpenes camphor C. albicans 475/15, C. albicans 503/15, Camphor at 0.125 mg/mL EOThujaSources from many species andCamphor eucalyptol at 0.125 at 23mg/mL mg/mL lyptolCamphor inhibited and euca- C. al- Sources eucalyptol CC.. albicans ATCC475/15, 1023,; C. albicans C. krusei 503/15, Camphor at 0.125 mg/mL Camphor and euca- reducedCamphor ROS at 0.125 by 52% mg/mL 2021 ofEO genera from many such asspecies Cin- eucalyptol C. albicans 475/15, C. albicans 503/15,inducedand eucalyptol a notable at 23reduction mg/mL bicans,lyptol inhibitedC. tropicalis, C. al-C. Camphor at 0.125 mg/mL [166] EO from many species H1/16;C. albicans C. tropicalis ATCC 1023,; ATCC C. 750 krusei and and eucalyptol at 23 mg/mL lyptol inhibited C. al- whilereduced eucalyptol ROS by 52%was in- ATCC: American2021 namomumof type ATCC:genera cultuire , such AmericanEucalyptus collection as Cin-, type (Manassas, cultuire VA, collection USA); CDC: (Manassas, comprehensive CVA,. albicans dental USA); care ATCC CDC: number, 1023,; comprehensive provided C. kruseiby Kuwaitininduced thedental number University a carenotable of number, hyphal Dental reduction Cliniccells provided parapsilosisbicans, (KUDC); C.by EOs:tropicalis, andKuwait essential C. C. Univer oils; EPM:sity Dental extracellular Clinicreduced polymeric (KUDC); ROS matrix; byEOs: 52% essential [166] oils; MBEC: minimum2021 of effective genera concentration;such as Cin- MBIC: minimum biofilm inhibitory concentration;C.H1/16; parapsilosis C. tropicalis NCIM: ATCC National ATCC 22019 750 collection and ofinduced industrial a notable microorganisms, reduction Pune; bicans, ROS: C. reactivetropicalis, oxygen C. species; SC: obtainedactivewhile from eucalyptol professor D. was in- [166] ArtemisianamomumEPM: extracellular, ,Salvia Eucalyptus and , polymeric matrix; MBEC: minimumH1/16; effective C. tropicalis concentration; ATCC 750 MBIC:and in minimumC.the albicans number 475/15 biofilm of hyphal inhibitory cells kruseiparapsilosis concentration; biofilm and biomass C. NCIM: National collectionwhile eucalyptol of industrial was microorgan-in- Sanglard, Centre Hospitaliernamomum, UniversitaireEucalyptus, camphor Vaudois, Lausanne, Switzerland; SMIC:C. sessile parapsilosis minimum ATCC inhibitory 22019 concentration.in the Other number culture of hyphal collection cells acronyms parapsilosis can and be found C. in the respective reference.active ThujaArtemisiaisms, Pune;, Salvia ROS: and reactive oxygen species; SC: obtainedC. from parapsilosis professor ATCC D. 22019 Sanglard, Centin C.re albicans Hospitalier 475/15 Universitaire krusei Vaudois, biofilm biomass Lausanne, Switzerland; SMIC:active sessile minimum inhibitory Artemisia, Salvia and camphorcamphor in C. albicans 475/15 krusei biofilm biomass ThujaThujaconcentration. Other culture collection acronyms can be found in the respective reference. Antibiotics 2021, 10, 1053 20 of 31

Although the main objetive of this review is to provide published data on the in vitro or in vivo antibiofilm activity of natural products, mention should be made to the method reviewed by Jha et al., 2020 [17]. These authors performed a multiple target-based structural bioinformatic study to recognize molecules that have the capacity of targeting proteins with a vital role in wall synthesis (FKS2 and FKS1), ergosterol synthesis (ERG11, ERG1, ERG24 and ERG3), and drug transport, such as Flu1 and the kinases CST20, HST7 and CEK1 involved in CHP1 pathway. The Cph1 transcription factor is involved in biofilm and pseudohyphae formation [168]. According to the results obtained, 2-O-prenylcoumaric, acid, 4-coumaryl acetate, coniferaldehyde, coniferyl alcohol, cycloserinehybrid and 2- coumaric acid targeted all proteins, while 110 molecules bound to at least four of them [17]. Among the evaluated compounds, vanillin showed interactions with ERG11, ERG1, ERG24, FKS2, CST20, HST7 and CEK1 while BBR targeted all of these proteins except Flu1 [17]. Thymol interacted to all proteins, except ERG24 and ERG1, and carvacrol bound to ERG24, ERG1 HST7, CST20, CEK1 and Flu1. Likewise, eugenol targeted ERG24, ERG1, FKS1, FKS2, FUR1 and Flu1 and cinnamaldehyde interacted with all of the proteins except FKS1, FKS2, FUR1 and Flu1. These data would explain, at least in part, the antibiofilm activity of the mentioned small molecules, which has been reported above and also in Table4, while opening the possibility to test experimentally the effects on biofilms of the rest of the ligands.

4. Reported Antibiofilm Activities of Nanosystems Containing Natural Products The published literature related to the antibiofilm properties of nanosystems are shown in Table5. Quatrin et al. [ 169] prepared nanoemulsions (NE) containing 5% E. globulus EO and 2% sorbitan monooleate (Span 80) with the aqueous phase containing 2% Tween 80. EO-NE were tested for their capacity to inhibit biofilm formation of three Candida spp. in high density polyethylene substrates. EO-NE at 22.5 mg/mL reduced C. albicans, C. tropicalis and C. glabrata biofilm formation by 84.5%, 61.3% and 84%, respectively. The biofilms were quantified by CV staining and corroborated by ATM and the fluorescence CW technique. Due to M. alternifolia EO (tea tree oil (TTO)) showed antibiofilm activity against C. albicans spp. in previous studies but possesses poor solubility and high volatility [170], Souza et al. [171] prepared TTO-nanoparticles and tested them against C. albicans, C. glabrata, C. parapsilopsis, C. membranafaciens and C. tropicalis biofilms. The results obtained showed that TTO and TTO-nanoparticles decreased by 67% and 72% respectively the C. albicans and C. glabrata biofilm biomass visualized with CV and confirmed by CW and ATM. The antibiofilm activity was more evident against C. glabrata. TTO-NP decreased EPM and proteins in biofilms and, therefore, TTO-NP can penetrate more easily through the EPM, releasing the TTO into the biofilm and resulting in a better antimicrobial activity. TTO-NPs can disturb the fungal membrane by blocking the respiratory chain through the inhibition of the enzyme succinate dehydrogenase of the internal fungal cell mitochondrial membrane. Antibiotics 2021, 10, x FOR PEER REVIEW 23 of 33

Table 5. Nanososystems containing natural products.

Nanosystems formed with essential oils (EOs) Inhibition of EOs Included in Cell Adhesión or Eradication of Mechanisms of Action or Year Type of Nano System Fungal Strains Biofilm Formation Reference Nanosystems Hyphal For- Mature Biofilms Genes Expression mation Nanoemulsions (NE) C. albicans ATCC ______14053; (EO-NE) at 22.5 mg/mL re- Composition C. tropicalis ATCC duced C. albicans, C. tropi- 2017 Eucalyptus globulus EO [169] Oil phase: 5% EO and 2% sorbitan 66029; calis and C. glabrata biofilm monooleate Aqueous phase: 2% C. glabrata ATCC formation Tween 80 and 25 mL of water 66032 Antibiotics 2021, 10, 1053 21 of 31 C. albicans ATCC Nanoparticles (NP) 14053; ______C. glabrata ATCC Composition Table 5. Nanososystems containing natural products.TTO-NP at 15.6% decreased TTO-NP decreased EPM 66032; Nanosystems formed with essential oils (EOs) Proprietary method from Inven- the biofilm formation of all and protein content in bio- Melaleuca alternifolia EO C. parapsilopsisInhibition ATCC of Cell Adhesión or Mechanisms of Action or Genes Year EOs Included in Nanosystems® Type of Nano System Fungal Strains Biofilm Formation Eradication of Mature Biofilms Reference 2017 tiva (Porto Alegre, Brazil)), based Hyphal Formation strains tested. The antibio- Expression films, and TTO-NPs inhib- [171] (tea tree oil, TTO) Nanoemulsions (NE) 220190; on high______pressure homogenization. film activity was higher in ited the enzyme succinate Composition C. albicans ATCCC. 14053; tropicalis ATCC (EO-NE) at 22.5 mg/mL reduced 2017 Eucalyptus globulus EO 7.5% (Oilw/ phase:v) of 5% TTO, EO and 2% cetyl palmitateC. tropicalis ATCC 66029; C. albicans,C. C. tropicalisglabrataand C. dehydrogenase[169] sorbitan monooleate Aqueous C. glabrata ATCC66029; 66032 glabrata biofilm formation as thephase: solid 2% Tweenlipid 80 and 25 mLTween 80 as of water C. membranaefaciens the surfactantNanoparticles (NP) ______ATCC 2013770 Composition C. albicans ATCC 14053; Nanosystems formed with natural compoundsProprietary method from C. glabrata ATCC 66032; TTO-NP at 15.6% decreased the TTO-NP decreased EPM and ® C. parapsilopsis ATCC 220190; biofilm formation of all strains protein content in biofilms, and 2017 Melaleuca alternifolia EO (tea tree oil, TTO) Inventiva (Porto Alegre, [171] Brazil)), based on high pressure C. tropicalis ATCC 66029; Inhibition oftested. The antibiofilm activity TTO-NPs inhibited the enzyme AntibioticsType 20 of21 Compound, 10, x FOR PEER REVIEWhomogenization. 7.5% (w/v) of C. membranaefaciens ATCC was higher in C. glabrata succinate dehydrogenase 24 of 33 TTO, cetyl palmitate as the solid 2013770 Cell Adhesion or Eradication of Studies of Mechanisms of Year Name and Structure Nanosystemlipid and Tween 80 as the Fungal Biofilms spp. Biofilm Formation Reference surfactant Hyphal For- Mature Biofilms Action Nanosystems formedNatural with natural Source compounds Type of Compound mation Inhibition of Cell Adhesion or Studies of Mechanisms of Year Name and Structure Nanosystem Fungal Biofilms spp. Biofilm Formation Eradication of Mature Biofilms Reference Hyphal Formation Action Natural1,4-Diketone-Naphtoquinone Source diphenol deriv- PLGA-JU at Antibiotics βative-Diketone 20 21 diphenol, 10, x FOR PEER REVIEW PLGA-JU at 1.25 and 0.625 CSNPdoses -equivalentCur at 400 24 of 33 Positively charged chitosan nano- CSNP-Cur at 200 μg/mL in- Positively charged chitosan μg/mL eradi- Nanoemulsions PLGA-JU re-CSNP-Curmg/mL at 200 µg/mL inhibitedCSNP-Cur by 100% at 400 µ g/mLto 1.25 and 0.625 particlesnanoparticles (CSNP) (CSNP) hibited almost completely 2020 C. albicans DAY185 inhibited almost completely the eradicated the preformed C. PLGA-JU caused[172] membrane 2020 Cur was loaded on CSNP, C. albicans DAY185 cated the pre- [172] JU was loaded on poly (D,L-lactic- C. albicans, non-speci- duced the cellC. albicans ad-thebiofilm C. formation albicans biofilmalbicans biofilms for- mg/mL of JU 2020 Cur wasforming loaded CSNP-Cur on CSNP, forming the C. albicans biofilm for- depolarization of biofilm [173] curcumin (Cur) (Cur) co-glycolic acid) (PLGA) forming fied voucher hesion at 1.25 mation, being more effec- formedcompletely C. albi- inhib- Source1,4-Naphtoquinone deriv- CSNP-Cur mation PLGA-JU at cells CurcumaSourcejuglone longa (JU) PLGA-JU and 0.625 mg/mL tive than free JU and Flu- cansited prebiofilms-estab- 1,4-Naphtoquinoneative derivative PLGA-JU at 1.25 and 0.625 doses equivalent Curcuma longa conazole lished C. albicans Source Nanoemulsions PLGA-JU re- mg/mL inhibited by 100% to 1.25 and 0.625 PLGA-JU at 1.25 and 0.625 PLGA-JU at doses equivalent to Nanoemulsions biofilms PLGA-JU caused membrane Juglans regia PLGA-JU reduced the cell mg/mL inhibited by 100% the C. 1.25 and 0.625 mg/mL of JU JU wasJU wasloaded loaded on on poly poly (D,L-lactic- C. albicans, non-speci- duced the cell ad-the C. albicans biofilm for- mg/mL ofPLGA-JU JU caused membrane 2020 C. albicans, non-specified voucher adhesion at 1.25 and 0.625 albicans biofilm formation, being completely inhibited [173] 2020 (D,L-lactic-co-glycolic acid) depolarization ofdepolarization biofilm cells of biofilm [173] mg/mL more effective than free JU and pre-established C. albicans Four major transcriptional co-glycolic(PLGA) forming acid) PLGA-JU (PLGA) forming fied voucher hesion at 1.25 mation, being more effec- completely inhib- Fluconazole biofilms cells juglone-Diketone (JU) diphenol com- genes that promote biofilm Sourcejuglone (JU) PLGA-JU andCU- SL0.625 at submg/mL-in- tive than free JU and Flu- ited pre-estab- Juglanspound regia conazole lished C. albicans formation, ROB1, EFG 1, βSource-Diketone diphenol compound Cur-SL hibitory concen- Four major transcriptionalTEC1, genesBRG 1 and NDT80 Juglans regia NanocomplexCur-SL formed with the sur- trations of 9.37 CU-SL at 9.37 μg/mL inhib- biofilms that promote biofilm formation, Nanocomplex formed with the CU-SL at sub-inhibitory ROB1, EFG 1, TEC1, BRG 1 and CU-SL at 9.37 µg/mL inhibited were down-regulated, as surfactant sophorolipid (SL) concentrations of 9.37 µg/mL NDT80 were down-regulated, as 2021 2021 factant sophorolipid (SL) C. albicans DAY185C. albicans DAY185 μg/mL signifi-biofilm developmentited biofilm and development Four major transcriptional[174] [174] Cur was loaded to the significantly suppressed fungal well as the adhesin genes ALS1, maturation well as the adhesin genes -Diketone diphenol com- curcuminCur wasnanoparticle loaded prepared to with the SL nanoparticle adhesion cantly sup- and maturation SAP8 and EAP1,genesthe hyphal that promote biofilm (Cur)curcumin (Cur) forming Cur-SL CU-SL at sub-in- regulatory genesALS1,SAP4, HWP1 SAP8 and EAP1, the Sourcepound prepared with SL forming Cur-SL pressed fungal RAS1 and HYR1formation,and ERG11 ROB1, EFG 1, CurcumaSource longa Cur-SL hibitory concen- hyphal regulatory genes adhesion TEC1, BRG 1 and NDT80 ATCC:Curcuma American longa type cultuire collectionNanocomplex (Manassas, formed VA, USA); with EO:the essentialsur- oil; NE: nanoemulsions;trations NP: nanoparticles. of 9.37 CU Other-SL at culture 9.37 μg/mL collection inhib- acronyms can be found inSAP4, the respective HWP1 reference.RAS1 and were down-regulated, as 2021 factant sophorolipid (SL) C. albicans DAY185 μg/mL signifi- ited biofilm development HYR1 and ERG11 [174] well as the adhesin genes Cur was loaded to the nanoparticle cantly sup- and maturation curcuminATCC: American (Cur) type cultuire collection (Manassas, VA, USA); EO: essential oil; NE: nanoemulsions; NP: nanoparticles. Other culture collection acronymsALS1, can SAP8 be foundand EAP1, in the the respective reference. prepared with SL forming Cur-SL pressed fungal Source hyphal regulatory genes adhesion Curcuma longa SAP4, HWP1 RAS1 and HYR1 and ERG11 ATCC: American type cultuire collection (Manassas, VA, USA); EO: essential oil; NE: nanoemulsions; NP: nanoparticles. Other culture collection acronyms can be found in the respective reference. Antibiotics 2021, 10, 1053 22 of 31

Considering that free Cur at 200 µg/mL inhibited almost completely the C. albicans biofilm formation, Ma et al. [172] prepared Cur-chitosan nanoparticles (CSNP). However, CSNP-Cur exhibited a slightly less inhibitory effect than free Cur. On the contrary, 400 µg/mL of CSNP-Cur eradicated the preformed C. albicans biofilms by 91.38%, being more effective than free Cur. SEM and CLSM showed that CSNP-Cur reduced polymicrobial biofilm thickness as well as killed microbial cells embedded in biofilms on silicone surfaces. In a recent paper, Gumus et al. [173] prepared nanoparticles of juglone (JU) encapsulated in poly D,L-lactic-co-glycolic acid (PLGA). As determined by plate count technique, these PLGA-JU nanoparticles completely inhibited biofilm formation and pre-established biofilms, at doses equivalent to 1.25 and 0.625 mg/mL of JU, respectively, whereas fluconazole did not cause a significant inhibition, even at the highest dose applied (10 mg/mL). PLGA-JU were more active than free JU and fluconazole. The inhibition of biofilm formation seems to be due to PLGA-JU reducing the cell adhesion, and the number of viable cells and altered membrane structures at both mentioned doses. Rajasekar et al. [174] synthesized a nanocomplex formed by the surfactant sophorolipid (SL) derived from non pathogenic yeasts such as Starmerella/Candida bombicola and Cur, and tested it against C. albicans biofilms. Sub-inhibitory concentrations of Cur-SL (9.37 µg/mL) significantly decreased fungal adhesion to glass coverslips, as well as biofilm development, maturation, and filamentation. Concomitantly, a significant downregulation of a select group of biofilm adhesins and hyphal regulatory genes was observed.

5. Discussion In this review, the natural products that demonstrated activity against fungal biofilms from 2017 to May 2021 were collected, with the aim of offering an overview of the progress made in this area to curb difficult-to-eradicate fungal infections. The type of natural products that showed better antibiofilm activities, the fungal species of the target biofilms, the type of assays used and the mechanisms of action were analyzed in order to detect the advances performed in this period that can be the basis of future works. Regarding the type of natural products that showed antibiofilm activities, 17 of the 42 manuscripts involved natural mixtures such as EOs (seven manuscripts) propolis (two manuscripts) or extracts prepared with solvents (total eight manuscripts), six of them from plants, one from lichens and one from microalgae or cyanobacteria. Two papers dealt with nanosystems including EOs. The antibiofilm activities of the seven EOs do not add a great novelty to the already known antibiofilm activity of these products. However, the nanosystems prepared with EOs open a great avenue for further research. In addition, the paper authored by Quatrin et al. [169], which tested a nanosystem with E. gobulus EO (EO-NE), not only used C. albicans as in most publications on antibiofilm EOs, but also used C. tropicalis, C. glabrata and C. auris although the EO-NE was devoid of activity in the latter sp. [169]. Considering that C. glabrata is a fungus intrinsically resistant to most antifungal drugs [175], the activity found against this fungus is encouraging for further research. With respect to extracts obtained with solvents from different spp., no preference for a botanical family was observed, and in most cases the antibiofilm activities should be deepened since the mechanism to achieve this effect was not analyzed. Twenty three of the 42 retrieved articles tested pure natural compounds, twenty of them alone and three included in nanosystems. Regarding the main type of pure compounds that showed a capacity for inhibiting biofilms on its own, nine of them were terpenes with four monoterpenes derivatives, four sesquiterpenes and one diterpene, which is in accordance with previous reports [124]. Two manuscripts studied the coumarin Sc, and one evaluated dammarane-type glycosides (gypenosides). A phloroglucinol, an alkaloid, a neolignan and some diphenols were investigated along with four compounds belonging to other types of chemical families. Cur was included in two nanosystems. Most of the compounds (21 compounds) were derived from plants, with one compound obtained from fungi (farnesol) and one obtained from a sea squirt microbiome (turbinmicin). Antibiotics 2021, 10, 1053 23 of 31

With respect to the fungal spp. tested in the 42 papers included in this review, 38 of these involved Candida spp. biofilms, two comprised Fusarium spp., two involved Cryptococcus spp. and one, A. fumigatus plus Candida spp. Among Candida biofilms, 23 studies included only C. albicans and 12 used different non-albicans Candida spp., but also involved C. albicans. Two papers used only C. tropicalis biofilms. Unfortunately, only two papers used C. auris among the non-albicans Candida spp. The newly described yeast C. auris has emerged as a resistant fungal pathogen responsible for hospital outbreaks, especially in intensive care units [176], leading to high mortality rates [177]. C. auris is at present a worrisome healthcare problem and new antifungals that overcome its resistance are highly needed [177]. The protein NFAP2 and the highly functionalized polycyclic turbinmicin completely disrupted EV delivery during C. auris biofilm growth [164]. Turbinmicin also showed activity against A. fumigatus biofilms. Regarding anti-C. neoformans and C. laurentii biofilms, only thymol, carvacrol and citral proved to be effective for inhibiting the formation and eradication of the mentioned biofilms; however, importantly, their mechanisms of action were studied [150,152]. These are significant findings, since this fungus can colonize the central nervous system, highlighting its significance as a critical pathogenic yeast. Propolis from Brazil and farnesol were the sole natural samples which showed a capacity for inhibiting Fusarium spp. This mold causes mildly superficial to fatally disseminated fungal infections, being the second most common mold causing opportunistic invasive infections after Aspergillus [178]. Keratitis is still the most common infection produced by Fusarium spp. [75,179]. The 2005–2006 outbreak of keratitis due to contact lens infections was attributed partly to the ability of F. keratitis to form biofilms [180,181]. Regarding the type of tests performed, most papers used only in vitro assays. Of them, twelve articles studied the inhibition of cell-to-hyphal transition or cell adhesion, 31 reported inhibition of biofilm formation, and 25 described eradication capacity. It is worth taking into account that for the antibiofilm compounds to be useful in clinics, it is crucial to determine whether a drug is able to penetrate and eradicate the pre-formed biofilms [102]. The fact that the reported works have not assessed the biofilm eradication capacity but only inhibitory effects on biofilm formation might contribute to a poor correlation between biofilm susceptibility and clinical outcomes. Only 15 papers deepened the study by giving evidence of the mechanisms of action. Of them, 11 were performed with pure compounds, three with nanosystems, and only one with an extract. It is important to highlight that sometimes the mechanisms of antibiofilm action were demonstrated to be multifactorial [150,182]. Only five of the 42 analyzed papers used in vivo assays, of which four were performed with C. elegans and only one, used rats. This is worrying since animal models of Candida biofilm infections are relevant for identifying novel antifungals useful in clinics [105].

6. Conclusions and Perspectives From 2017 to May 2021, there has been an active research on natural products with antifungal biofilm activity. It is clear that great progress has been made and that the newly discovered natural antibiofilm agents could provide novel agents for biofilm-associated infections. Of them, the protein NFAP2 and the highly functionalized polycyclic turbinmicin have demonstrated interesting antibiofilm properties and deserve further research. However, several types of studies must be deepened. For example, fungal biofilms different from those of Candida spp. should be prepared and used as targets. Among them, it is necessary to investigate the behavior of natural samples on filamentous fungal biofilms, which are scarcely studied in comparison to Candida spp. such as those of the genera Aspergillus, Fusarium and Trichophyton [183,184]. To be useful for future development, all papers should analyze biofilm eradication capacities in addition to the study of the inhibition of biofilm formation and other properties. Besides, in vivo assays should be included in all papers dealing with activity against biofilms. Antibiotics 2021, 10, 1053 24 of 31

It is worth taking into account that several clinical trials involving natural antibioﬁlm agents are in progress, and some of them exhibited promising perspectives, as recently reviewed by Lu et al. [185].

7. Materials and Methods The search for suitable papers was performed in electronic databases by using the following keywords: “biofilm”, “fungal infections”, “sessile cells”, “secondary metabolites”, “natural products”, “repurposing”, “antifungal drugs”, and “antibiofilm”. Additional papers matching the search criteria were included after surveying the references from the selected articles. The information gathered was divided into three groups: (I) natural extracts including EOs, propolis and extracts from plants, lichens, algae and cyanobacteria, (II) pure natural compounds, and (III) nanosystems. This last section was sub-divided by EOs included in nanosystems and pure natural compounds included in nanosystems.

Author Contributions: Conceptualization, S.Z.; methodology, E.B., L.S., M.C.C. and T.E.; for- mal analysis, S.Z., M.C.C., T.E.; investigation, S.Z., E.B., L.S., M.C.C. and T.E.; resources, S.Z., L.S.; writing—review and editing, S.Z., M.C.C., T.E.; funding acquisition, S.Z., L.S., M.C.C., T.E. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by ANPCyT (PICT 2017-1381, PICT 2018-02034 and PICT 2019- 00721); ASaCTeL (IO-2019-139); UNR (8002019030001UR) and Universidad Católica de Córdoba. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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