Update on the Fungal Biofilm Drug Resistance and its Alternative Treatment

The mortality rate caused by fungal infections has been drastically increasing over the past few decades. The population affected consists mostly of immuno-compromised patients such as organ transplant patients, human immunodeficiency virus (HIV) positive patients or those suffering from leukemia. The two most common causative pathogens for mortality within the population of these affected patients are Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus). Life threatening diseases, for example, invasive aspergillosis, are caused by the opportunistic filamentous mold, A. fumigatus. It has been demonstrated that antifungal agents were not able to completely eradicate the disease, or that high doses were required to do so. This eventually resulted in severe side effects and patients` resistance to the drug became a major issue. C. albicans and A. fumigatus were both found to form a multicellular complex known as biofilm. This structure has the ability to resist conventional doses of antifungal drugs thus making treatment of such fungal infections problematic. This paper aims in reviewing the numerous factors involved in the formation of biofilm and their contributions to the resistance against antifungals. An urgent need for new treatment protocols capable of eliminating biofilms and reduce recurrences are required, therefore alternative possibilities in treating biofilms are also discussed here. Combination of conventional drugs were brought to scientists’ attention as a potential way of treating those difficult cases, however it comes with drawbacks. An alternative approach is to look into natural products, such as traditional Chinese medicine (TCM), which comes with multiple benefits and were demonstrated to possess antifungal properties. Although several reports showed TCMs having beneficial effects on C.albicans and dermatophytes, only a few papers reported its effects on A. fumigatus, and no studies were done on their biofilms. Research which consisted of combining standard antifungal agents with TCM have shown good synergism against Candida species and dermatophytes, further investigations for its application in the clinical setting need to be undertaken.


INTRODUCTION
In the last few decades, infection due to fungi has risen drastically with one of the most common causative agent being Aspergillus spp, it comes second after Candida albicans (C.Albicans).Aspergillus species are an increasingly widespread opportunistic filament forming molds consisting of more than 180 species [1].Aspergillus fumigatus (A.fumigatus) is the most common species which targets mainly the lungs forming a variety of life threatening systemic diseases such as invasive aspergillosis.Research shows that immunocompromised patients, such as organ transplant recipients, people who are human immunodeficiency virus (HIV) positive, those suffering from leukemia or those under long term therapy with glucocorticoids are mostly affected.
Mortality rate among these individuals has risen to 50% or higher over the past few years [2].Despite recent advances in the development of new antifungal agents, clinical resistance is still a problem that is hard to resolve.A fascinating discovery showed pathogenic fungi capable of producing a multicellular 2/7 complex known as biofilm, which is able to resist conventional doses of antimicrobial drugs.This discovery has lead researchers to believe that these structures could make clinical treatment of infectious diseases increasingly problematic [3].It was clearly established in the past, that bacterial biofilms predominate in nutrient-sufficient environments [4], and researchers were persuaded that fungal resistance to drugs was also associated with a biofilm formation [5].
Before biofilm in pathogenic fungi became a major issue, there was a generalized perception that microorganisms existed as free floating (planktonic) organisms in liquid culture [6].However, there were instances of increased resistance to antifungal agents which called into question the theory of this planktonic feature.C. albicans was the most popular fungal pathogen studied, and it was observed to adhere to a variety of biotic and abiotic surfaces, where they form an organized structure of sessile cells that differ immensely from their planktonic type.Such communities of cells are referred to as biofilms.The fact that there is a discrepancy in the in vitro MICs of planktonic and sessile form of C. albicans reinforce the premise that this fungi exists as biofilm and can possibly explain the lack of absolute correlation between clinical and in vitro resistance.Our focus in this review is to highlight that A. fumigatus, like C. albicans, can exist in the form of biofilm and this phenomenon contributes to the resistance against many antifungal agents.We also make recommendations for future work in order to improve the situation.

Biofilms and their resistance to antifungal agents
Chronic A. fumigatus infection starts off by human beings inhaling the conidia from the surrounding air.Histology and microscopic examination of bronchopulmonary lavage samples from aspergillosis patients have revealed that A. fumigatus forms multiple intertwined hyphae gathered into a complex multicellular mycetoma structure, similar to Candida species [7].Although the life cycle of C. albicans and A. fumigatus are significantly different [8], cellular differentiation and the ability to form filamentous growth are similar key aspects to their potential to produce biofilm.However, there were some controversies on the true definition of biofilms; where Chandrasekar PH et al. argued that adherence to plastic microscopic coverslips or polystyrene bottoms of microtiter plates do not assure the production of biofilm.Such as in one such case, where Saccharomyces cerevisiae can adhere to biotic or abiotic surfaces without producing a biofilm [3].The presence of an extracellular matrix (ECM) along with other mechanisms such as efflux pump activity, persister cells, stress response, overexpression of drug targets and the general physiology of the cells all contribute in making A. fumigatus biofilm resistant to antifungal therapy.This is reflected in its unaffected growth at the standard dose of concentration of antifungal agents.At times, resistance can even reach up to 1000 fold higher than planktonic morphologies [10].Therefore, conventional therapeutic approach of invasive aspergillosis is far from ideally eradicating fungal biofilms.
Chronic infection with A. fumigatus is dependent on the ability of its conidia to germinate and evolve into mycelia.This mycelial structure eventually invades the pulmonary epithelial and endothelial cells.In terms of kinetics, the development of A. fumigatus biofilms is divided into initial adherence at 4 hours of incubation, conidial germination at approximately 6 hours, and maturation and differentiation at 8 hours onwards [11,12].The presence of ECM satisfies the simple definition of a biofilm, it is composed of galactomannan, ɑ-1, 3 glucans, galactosaminogalactan, monosacharides, polyols, melanin, proteins [13].ECM of microbial biofilms has also been shown to contain extracellular DNA(eDNA); eDNA is the key structural component of the ECM, which in turn is known to be a self-manufactured extracellular matrix essential for maturation of biofilm.Protecting against external stress, ECM binds to specific classes of antifungal agents and limits penetration of the drugs.Although little is known concerning the presence of eDNA in fungal biofilms, C. albicans biofilm's structural and architectural integrity were associated with an increase in eDNA as the biofilms matured [14].It was demonstrated by Margarita Martins et al., where after being treated with DNase, C.albicans biofilm underwent a substantial inhibitory reaction.Researchers suggested that it might be due to the direct effect on the ECM, compromising its architectural structure and maintenance.They thus concluded that eDNA played an essential role in matured biofilms rather than young developing ones.Rajendran et al. were the first to report the presence of eDNA in A.fumigatus.In their study, they were able to demonstrate that as A.fumigatus biofilm matures, eDNA, a by-product of autolysis, increases.This is believed to be part of a mechanism of antifungal resistance when compared to planktonic cells.eDNA might be a genomic DNA released from A.fumigatus during biofilm maturation.The addition of DNase remarkably improved the antifungal effects of amphotericin B or caspofugin.This suggests that inhibiting eDNA production can offer a potential management therapy of A. fumigatus infections [15].There were however no particular changes in biofilm when tested against the combination of azoles and DNase; the reason is that eDNA forms part of the ECM and is involved in biofilm stability rather than ergosterol biosynthesis pathway targeted by azoles.
Conidial seeding density is also essential in overall structural integrity of the biofilm and resistance to antifungal agents, a concentration of 1x10 5 conidia/ml demonstrated the most robust filamentous structure, resistant to mechanical disruption [1].The presence of persister cells is crucial in chronic fungal infection, they form a subpopulation of dormant cells which are highly tolerant to antifungal agent and they ensure the survival of biofilm even after extensive and long-term course of antifungal therapy, thus promoting recurrence.There is a hypothesis that persister cells are present in C.albicans biofilm even after treatment, this is explained by a biphasic killing pattern post treatment, where a portion of fungal cells are left unaffected after a dose-response activity is observe [16].Generation of miconazole-resistant persisters in C.albicans biofilms are induced by Superoxide dismutases (SODs).Through their inhibition by the Cu/Zn-Sod inhibitor, N, N-diethyldithiocarbamate(DDC) persister cells were found to be less [17].
Efflux pumps are transport proteins helping in the removal of toxic substrates from within the cells into the external environment, they help in maintaining homeostasis within the complex structure of biofilm.Increased efflux of the drug is

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mediated by ATP binding cassette (ABC) and the major facilitator superfamily (MFS) transporters.Genetic elements encoding efflux pumps contribute in antifungal resistance. A. fumigatus was predicted to have at least 49 ABC family transporter and 278 MFS genes [18].Analysis of biofilm resistance to voriconazole and efflux pump activity showed a consistent increase in efflux pump activity as biofilm matures from 8 hours to 24 hours [19].Candida has at least 5 different drug resistance genes (CDR1 to CDR5) associated with drug efflux [20].In previous reports, itraconazole resistance in A. fumigatus was due to overexpression of Atr1 clones, the gene being an ABC transporter drug resistant gene [21].
Ergosterol is the major sterol found in fungal membranes, its main function is to maintain fungal growth and is the main target for the three classes of antifungals commonly used.Overexpression of ergosterol increases the level of resistance towards drugs such as azoles or polyenes and all polyenes have high affinity for ergosterol.For example, in Candida species, the sterol binds with the hydrophilic ring of amphotericin B subsequently leading to formation of pores in the membrane, causing small ions such as potassium to leak out and disrupting the oxidative enzymes of the target cells, this action does not necessarily result to death [22].There is evidence showing that the immediate killing of cells was of the interactions between ergosterol and amphotericin B but instead is a result of the polyene acting as an oxidizing agent with the formation of reactive oxygen species (ROS).ROS is an additional mode of action inducing apoptosis in fungal pathogens such as A.fumigatus and C.albicans [23].Azoles, however, function by blocking ergosterol biosynthesis at the C-14 demethylation stage.Triazoles bind to lanosterol 14-ɑ-demethylase (14-ɑ-DM), and this leads to ergosterol depletion as lanosterol and other toxic 14-ɑ methylated sterols keep accumulating.Azole resistance mechanism in Candida involves overexpression of the target 14-ɑ-DM enzyme and possibly a downstream mutation in the ergosterol pathway [24].Azole derivatives, with a reduced binding affinity for ERG11 gene product, 14-ɑ-DM, is an important mechanism of azole resistance in yeasts and fungi.This reduction in affinity is caused by modification of 14-ɑ-sterol demethylase (CYP51p).It was also demonstrated that in vivo and in vitro itraconazole resistant A. fumigatus strains have point mutations in CYP51A, a gene encoding CYP51p [25].Over expression of CYP51A gene was also shown to be responsible for azole resistance in clinical isolates of A. fumigatus.
Other mechanisms involved in fungal biofilm resistance include heat shock protein 90 (Hsp90).Research on its implication with C.albicans demonstrated that it functions mainly by stabilizing phosphatase calcineurin and MaPk Mkc1 in planktonic cells.Inhibiting Hsp90 in planktonic C.albicans would cause a reduction in the levels of the above, however, none of these phenomena were observed in C.albicans biofilms.This led to the theory that Hsp90 regulates drug resistance through different mechanisms in biofilm.Inhibiting Hsp90 in C.albicans made the effect of azoles shift from ineffectual to highly efficacious in eradicating biofilms both in vitro and in vivo.When gendanamycin, an Hsp90 inhibitor was combined with fluconazole, MIC values were observed to be reduced by more than 30-fold.A reduction of almost 60% in matrix glucan level was observed with depletion of Hsp90, providing a link between Hsp90, glucan production and how they regulate biofilm drug resistance in C.albicans.Experiments were performed on A.fumigatus biofilms with a combination of azoles (fluconazole, voriconazole) or echinocandins (caspofungin, micafungin) and Hsp90 inhibitor, gendanamycin.The results showed good synergy between voriconazole or echinocandins and gendanamycin, but not with fluconazole.Upon observation with confocal scanning microscopy, multiple broken and burst hyphae were scattered all over the biofilm when tested with caspofungin and gendanamycin; whereas hyphae were defined as flat and ribbon-like upon addition of both voriconazole and gendanamycin.These results confirmed the hypothesis that inhibition of Hsp90 induces changes in the morphologies of A.fumigatus biofilms, and thus improving the effect of azoles and echinocandins against A.fumigatus biofilms [26].Targetting Hsp90 may provide a positive therapeutic strategy for biofilm infections caused by both C.albicans and A.fumigatus.
Eilidh Mowat et al. demonstrated in a study that amphotericin B was the most effective against mature A. fumigatus biofilms [27].However, Lass-Florl et al. reported that minimum inhibitory concentration (MIC) of amphotericin B higher than 2 µg/ml were highly associated with a fatal outcome [28].Voriconazole was effective mostly in early stages of the biofilm, but showed poorer ability to counteract fully grown intertwined hyphae.Caspofungin was, on the other hand, consistently ineffective against all stages of biofilms [27].Because of the above reasons leading to high resistance to common antifungal drugs, new and improved therapeutic strategies have to be established.An approach could be to look into natural products such as traditional Chinese medicine and its combination with other antifungals.

Traditional Chinese medicine as an alternative antifungal agent?
Recently, research has shown great interest on natural products with potential antifungal properties.Traditional Chinese medicines (TCMs) or their extracts have gathered a lot of attention due to their easy availability and minimal side effects.More than 300 herbs have been known to have "pesticidal" activities, some even have antifungal potential and have been used in the clinical world for centuries [29].Although studies on the effect of TCM on A. fumigatus are very scarce, there is a large quantity of research based on Candida spp.and how TCM inhibits their growth in vitro.In an attempt to screen 56 widely used dried Chinese medical plants for their antifungal properties against A. fumigatus, C. albicans, Geotrichum candidum and Rhodoturula rubra, Blaszczyk and his team demonstrated that Flos Carthami (Carthamus tinctorius L.) was specifically effective against A. fumigatus with a growth inhibitory area of diameter 7cm after 4 days.R. et rh.Rhei (Rheum palmatum L.) also proved effective against A. fumigatus as well as C. albicans [30].Another study that included A. fumigatus as their test pathogens demonstrated that chrysophanol, an isolated compound of Rheum emodi rhizomes had significant antifungal actions on A. fumigatus with an MIC of 50 µg/ml [31].
With an increase in immunocompromised patients, it was shown that non-albicans candida such as Candida krusei or Candida glabrata contributed to more and more infections.C.

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of crude extracts of Rhizoma Coptidis and Cortex phellodendri Chinesis against C. krusei and C. glabrata with MIC values of 50µg/ ml and 100 µg/ml respectively.Berberine is the alkaloid found in Rhizoma Coptidis that contributed largely in its antifungal activities [32].Their results concurred with previous works on the fact that berberine hydrochloride showed weak inhibition against C. albicans but instead demonstrated significant effect on C. krusei as well as C. glabrata.In another study where 40 TCMs were investigated against 8 superficial fungal strains, they demonstrated that Melaphis chinensis, Polygonum cuspidatum, Punica granatum and Schisandra chinensis were the 4 TCMs to inhibit fungal growth with MIC value of 50µg/ ml; the most susceptible fungi being trichophyton violaceum and tricophyton tonsurans [33].They also hypothesized that variance in MICs compared to previous studies might be because of the usage of different parts of the plants thus inducing different pharmacological effects.For instance, Melia azedarach showed no changes in the growth of Trichophyton rubrum and Microsporum gypseum whereas in previous reports, the MIC value was significantly low (16µg/ml) [34].Punica granatum, however, showed similar inhibitory results on all 8 fungi tested with MIC of 50µg/ml compared to reports on dermatophytes, A. niger, C. albicans and even Cryptococcus ssp [35].

Berberine was found effective against Trichophyton mentagrophytes, Trichophyton rubrum, and
Microsporum gypseum with a 50% inhibitory concentration (IC 50 ) of 2.1 to 26.6 µg/ml.Berberine can be found in many herbs reputed to possess antifungal capabilities, and hence it might be a direct candidate for future therapeutic treatments.Berberine is also one of the main alkaloids of Mahonia aquifolium, however in a study undertaken by Volleková et al., they demonstrated that among the protoberberines of M. aquifolium (berberine, jatrorrhizine, palmative and columbamine), jatrorrhizine also had significant inhibitory actions especially against dermatophytes such as Trichophyton and Microsporum with MIC values of 62.5 to 125 µg/ml or 250 to 500 µg/ml for Candida spp [36].The antifungal effects of jatrorrhizine on dermatophytes were almost the same as that of fluconazole and biconazole.It has been previously reported that the mechanism of action of protoberberines, berberine and palmative, against C. albicans was believed to be caused by the inactivation of sterol 24-methyl transferase (24-SMT) and chitin synthase, both important enzymes in ergosterol and chitin biosynthesis [37].Jatrorrhizine also showed similar antifungal properties, therefore, is believed to have similar mode of actions.However, according to the structural model of jatrorrhizine, it possesses an additional polar 2-methoxy-3-hydroxy substitution compared to berberine and palmative.This free hydroxy group, together with inactivation of 24-SMT and chitin synthase, are reasons to consider jatrorrhizine as a potential target for future antifungal therapy.
Another promising alternative would be pseudolaric acid B, extracted from "tujingpi" or Cortex pseudolaricis; they have been known for their antifertility, cytotoxic, antigiogenic and antifungal activities.Pseudolaric acid B was observed to be effective against six different species of Candida namely, albicans, glabrata, krusei, tropicalis, dubliniensis and parapsilosis with MIC values ranging from 16 to 128 µg/ml.These values were approximately similar to that of fluconazole.It has also been pointed out that pseudolaric acid B had clear, non-trailing endpoints, demonstrating the fungicidal instead of fungistatic characteristics of this plant extract, against Candida species [38].Pseudolaric acid B could be a potential answer to the problematic drug resistance where non-albicans candidas have become infection-causing pathogens, especially when C. krusei is naturally resistant to fluconazole.
Ginseng as a well-known herb, is widely used in herbal medicines across East Asia.Ginseng stem-leaf saponins, which is often chosen over Ginseng roots due to its lower cost and abundance, contain active ingredients with similar pharmacological functions.Many saponins have antifungal properties and provide protection against potential pathogens [39].The mechanism of saponin fungitoxicity is believed to originate from its ability to disrupt cellular membranes by binding with membrane sterols and causing loss of membrane integrity [40].Ginseng stem-leaf saponins comprise of ginsenosides, polysacharides, triterpenoids, and flavonoids.Many of its medicinal effects are mostly attributed to ginsenosides; it also has multiple pharmacological actions affecting the central nervous system (CNS), cardiovascular system, growthmetabolism system and immune system, as well as anti-fatigue, anti-hyperglycemic, anti-obesity, anti-cancer, anti-oxidant and anti-aging activities.Ginseng stem-leaf saponins was shown to also have some antifungal effects on planktonic C. albicans [41].In a study done by Woo Sang Sung et al. they demonstrated this effect with Korean Red Ginseng saponins.During in vitro drug susceptibility testing, the MIC value for ginsenosides ranged from 50 to 100 µg/ml, showing antifungal activity against human pathogenic fungal strains.However, it showed less potent activity than amphotericin B which acted as a control with an MIC of 5µg/ ml on all fungal strains [42].
Although TCMs have been proven effective on Candida spp and dermatophytes, very few is known about its action on A. fumigatus, let alone on biofilms.Further investigations are required for screening of TCMs against Aspergillus spp.and more importantly, the safety of these compounds must be established before clinical use.

What are the options?
As discussed above, there is a clear need to develop new strategies to overcome resistance of A. fumigatus biofilms.Discovery of new antifungal agents to be used in clinical settings has been lagging behind; although recently, efforts in drug combination have been made to overcome resistance to antifungal agents.
Since amphotericin B stays the preferred choice of antifungal therapy despite its high risk of nephrotoxicity, recent studies have been undertaken to lessen its drawbacks, in the meanwhile, reducing the effective dose of amphotericin B to avoid toxicity in renal tissues remains the main priority.A possibility is to accentuate its anti-biofilm activity by combining this polyene with another drug.About 47% of transplant patients used a combinational antifungal therapy course in hope of expanding the antifungal spectrum [43].In a study on the treatment of cryptococcal meningitis [44], the combination of flucytosine and amphotericin B resulted in faster sterilization of the

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cerebrospinal fluid compared to treatment with amphotericin B alone.In this study, recurrences were reduced, however, high cost and worrisome side effects limit the use of combinations of common antifungal drugs.In another study, renal dysfunction was induced by amphotericin B, leading to rapid accumulation of flucytosine and raising the patient's risk of hematological toxicity [45].Hence, it is important to master the mechanism of each class of antifungal agents to avoid negative interactions or complications.
Controversies over the interaction of azoles-polyenes have been discussed in a few articles.Azoles function as inhibitors of ergosterol synthesis, however, polyenes need ergosterol to function.Azoles damaging part of fungus is essential for the activity of polyenes, making these two antifungal agents antagonistic.Another proposed mechanism for this interaction was that amphotericin B interferes with the cell membraneassociated protease, enabling the passage of itraconazole into the cell [46].It is however difficult to replicate the amphotericin Bazole interaction in vitro because the two drugs possess different time course actions; amphotericin B has rapid fungicidal activity and tends to obscure the effect of slower azoles [47] whereas amphotericin B shows good synergy with echinocandin, caspofungin [48].In an experiment undertaken by Weixia Liu and his team, they showed that the combination of caspofungin and amphotericin B had a synergistic inhibitory activity against 8 out of 11 A. fumigatus biofilms, with fractional inhibitory concentration index (FICI) of below 0.5 [10].
The focus of new research has been based on the combination of antifungals with non-antifungals.This research focus has helped reduce the high costs and serious side effects that come along with combination of antifungal drugs.Baicalein, a popular Chinese herb showed growth inhibitory actions on C. albicans when combined with Amphotericin B [49].Pseudolaric acid B (PSB) with fluconazole also demonstrated significant synergism when tested against fluconazole-resistant C. albicans; with an MIC value reduced by almost 64-fold [38,50].Anti-biofilm mechanism of these TCMs is believed to include reduction in drug efflux or alteration in sterol biosynthesis [51].Berberine, as mentioned above showed weak activity against C. albicans.In a study where berberine chloride (BBR) was combined with fluconazole to test against fluconazole resistant isolates of C. albicans, they noticed a significant reduction in MIC of either of the individual agent with MIC 80 of fluconazole dropping from ≥64µg/ml to ≤ 0.125-2 µg/ ml whereas the MIC of BBR was reduced by at least 4 fold.100% of the isolates showed synergism in terms of MIC 80 with median FICI of 0.034 [52].They concluded that the combination of these two drugs could be a good option to treat fluconazole resistant C. albicans in vitro.However, the mechanism behind this interaction is still not completely understood, they hypothesized that it may be due to the inhibition of sterol 24-methyl transferase by BBR, together with active efflux of the azole with overexpression of CDR, MDR1, or FLU1 and also alterations of target enzymes.Due to poor permeability across the fungal membrane, amphotericin B must be administered at a high dosage making the rise in side effects problematic; combining it to berberine was considered relatively safe considering that its fatal dose had to exceed 23 mg/kg of body weight in mice [53].The study of Han et al. was the first to investigate the combination effect of berberine and amphotericin B on C. albicans infected mice.He used the enumeration of colony forming units (CFU) to determine growth inhibition and demonstrated that berberine alone was able to inhibit growth of C. albicans by approximately 60% compares to its control culture.Combining 50µg/ml berberine with 0.5µg/ml amphotericin B reduced CFU from 1.211x 10 8 CFU/ml to 1.1 x 10 4 CFU/ml, doubling the concentration of amphotericin B resulted to >90% CFU reduction.In vivo synergistic effect was observed when infected mice were given a mixture of the 2 drugs with a survival rate of 23 days longer.This rate of survival was similar to mice given 4 times the average amphotericin B dosage.

CONCLUSION
Medical scientists came to the conclusion that fungal infections caused by the formation of biofilms are one of the many diseases whose pathogenesis and progression are becoming more complicated.A Single drug may not be effective enough or may be hampered by severe side effects or resistances to the drug.There is therefore a serious need of new treatment protocol, with minimum side effects, to combat these life threatening diseases and reduce recurrences.Combination of antifungal agents with TCM has proven to be relatively effective against planktonic dermatophytes and Candida species but rare are the reports on A. fumigatus.Further investigations need to be done to understand the mechanism of action of TCMs and how they can inhibit the growth of A. fumigatus as well as its biofilms.