ArticlePDF AvailableLiterature Review

Biofilm formation in clinically relevant filamentous fungi: a therapeutic challenge

Critical Reviews in Microbiology

June 2021
48(2)

DOI:10.1080/1040841X.2021.1950121

Authors:

Maryam Roudbary

The University of Sydney

Roya Vahedi Shahandashti

Medizinische Universität Innsbruck

André Luis Souza dos Santos

Federal University of Rio de Janeiro

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Biofilms are highly-organized microbial communities attached to a biotic or an abiotic surface, surrounded by an extracellular matrix secreted by the biofilm cells. The majority of fungal pathogens contribute to biofilm formation within tissues or biomedical devices, leading to serious and persistent infections. The clinical significance of biofilms relies on the increased resistance to conventional antifungal therapies and suppression of the host immune system, which leads to invasive and recurrent fungal infections. While different features of yeast biofilms are well-described in the literature, the structural and molecular basis of biofilm formation of clinically related filamentous fungi has not been fully addressed. This review aimed to address biofilm formation in clinically relevant filamentous fungi.

The schematic stepwise phases of the development of filamentous fungi biofilms on biotic and abiotic surfaces. The Figure has been made by Biorender.com.

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QSMs and their known roles in clinically relevant filamentous fungi.

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Summary of filamentous fungal biofilm and their clinical relevance.

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Biofilm formation in clinically relevant filamentous

fungi: a therapeutic challenge

Maryam Roudbary, Roya Vahedi-Shahandashti, André Luis Souza dos

Santos, Shahla Roudbar Mohammadi, Peyman Aslani, Cornelia Lass-Flörl &

Célia F. Rodrigues

To cite this article: Maryam Roudbary, Roya Vahedi-Shahandashti, André Luis Souza dos Santos,

Shahla Roudbar Mohammadi, Peyman Aslani, Cornelia Lass-Flörl & Célia F. Rodrigues (2021):

Biofilm formation in clinically relevant filamentous fungi: a therapeutic challenge, Critical Reviews in

Microbiology, DOI: 10.1080/1040841X.2021.1950121

To link to this article: https://doi.org/10.1080/1040841X.2021.1950121

Published online: 06 Aug 2021.

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REVIEW ARTICLE

Biofilm formation in clinically relevant filamentous fungi: a

therapeutic challenge

Maryam Roudbary

, Roya Vahedi-Shahandashti

, Andr

e Luis Souza dos Santos

Shahla Roudbar Mohammadi

, Peyman Aslani

, Cornelia Lass-Fl€

orl

and C

elia F. Rodrigues

Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran;

Institute of

Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria;

Department of General Microbiology,

Microbiology Institute Paulo de G

oes, Federal University of Rio de Janeiro (UFRJ), Brazil;

Department of Mycology, Faculty of Medical

Science, Tarbiat Modares University, Tehran, Iran;

Department of Parasitology and Mycology, Faculty of Medicine, Aja University of

Medical Sciences, Tehran, Iran;

LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of

Engineering, University of Porto, Porto, Portugal

ABSTRACT

Biofilms are highly-organized microbial communities attached to a biotic or an abiotic surface,

surrounded by an extracellular matrix secreted by the biofilm-forming cells. The majority of fun-

gal pathogens contribute to biofilm formation within tissues or biomedical devices, leading to

serious and persistent infections. The clinical significance of biofilms relies on the increased

resistance to conventional antifungal therapies and suppression of the host immune system,

which leads to invasive and recurrent fungal infections. While different features of yeast biofilms

are well-described in the literature, the structural and molecular basis of biofilm formation of

clinically related filamentous fungi has not been fully addressed. This review aimed to address

biofilm formation in clinically relevant filamentous fungi.

ARTICLE HISTORY

Received 2 May 2021

Revised 17 June 2021

Accepted 19 June 2021

Published online 5 August

2021

KEYWORDS

Biofilm; filamentous fungi;

antifungal resistance

mechanisms; quo-

rum sensing

1. Introduction

Invasive fungal infections are one of the major causes

of morbidity and mortality, being attributed to enor-

mous healthcare-related costs, worldwide (Brown et al.

2012; Arastehfar et al. 2020). Species belonging to

yeasts, like Candida and Cryptococcus, and filamentous

fungi, such as Aspergillus, are considered as the most

clinically relevant fungal species, being able to cause an

array of infection in humans, ranging from superficial to

deep-seated systemic infections (Brown et al. 2012).

Unlike bacterial infections, for which there are numer-

ous classes of antibiotics with a wide range of mecha-

nisms of action, the number of antifungal agents

available in clinical settings is limited to four major

classes, namely azoles, polyenes, echinocandins, and

flucytosine (Gintjee et al. 2020). Echinocandins and

mould-active triazoles are mainly used to treat invasive

yeast and mould infections, respectively, while the use

of amphotericin B, a polyene agent, is limited due to its

nephrotoxicity (Arastehfar et al. 2020).

Similarly to bacteria, fungal species inherently pro-

duce complex structures, biofilms. These are a

community of cells embedded with extracellular matrix

(ECM) (Ramage et al. 2009; Pitangui et al. 2012). This

ECM is mostly composed of extracellular polysacchar-

ides, proteins, DNA, and lipids (e.g. ergosterol), which

shields the embedded cells from hostile host conditions

and renders them to be extremely resistant to various

chemical compounds, such as antifungals, disinfectants

used in the hospital settings, and other agents found in

natural niches (Ramage et al. 2009; Bridier et al. 2011;

Pitangui et al. 2012; Rodrigues et al. 2017). Biofilms are

also seen as a key virulence factor, which, after colon-

isation, allow the pathogen to stabilise and conse-

quently resulting in persistent infections (Desai et al.

2014). Finally, these features let the pathogenic fungi to

survive in hospital environment and seed future out-

breaks (Schelenz et al. 2016; Sherry et al. 2017).

Although an accurate estimation is lacking in clinic-

ally important fungi, it has been calculated that

approximately 80% of the recurrent and chronic bacter-

ial infections (Davies 2003) and 500,000 annual deaths

(Sharifi et al. 2018) are attributed to biofilms. Since bio-

films can be formed on both biotic and abiotic surfaces,

CONTACT C

elia F. Rodrigues c.fortunae@gmail.com LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty

of Engineering, University of Porto, Porto, Portugal

ß2021 Informa UK Limited, trading as Taylor & Francis Group

CRITICAL REVIEWS IN MICROBIOLOGY

https://doi.org/10.1080/1040841X.2021.1950121

and the fact that the number of implanted devices

(Raad 1998; Costerton et al. 1999; Donlan 2002;

Vlamakis 2011), such as central venous catheters,

indwelling bladder catheters, joint prostheses, are

increasingly being used in clinical settings, it would be

plausible to assume a growing trend towards biofilm-

related infections. The clinical relevance of biofilms is

further compounded by the fact that recalcitrant bio-

film-related infections mostly require removal of the

implant devices and affected tissues (Desai et al. 2014),

which not only increases the economic burden, but

also complicate the patient’s outcomes.

Apart from the knowledge gap on the clinical impact

of fungi, studying biofilms structures, and the dynamics

and complex circuits regulating biofilm formation have

been mainly focussed on a major yeast, Candida albi-

cans. On the other side, there is a very limited informa-

tion on other fungal species, such as Aspergillus,

Zygomycetes,Scedosporium,Histoplasma,Fusarium, and

black-yeast like fungi. These fungi can successfully col-

onise surfaces and produce strong biofilms. Therefore,

the scope of this review is to discuss the biofilm forma-

tion of clinically relevant filamentous fungi that have

been stagnant, in comparison with bacteria and other

fungi. Additionally, we highlight the structural and

molecular basis of biofilms and their role in the devel-

opment of resistance to antifungal agents.

2. Biofilms in the medically prevalent

filamentous fungi

2.1. Aspergillus spp.

Aspergillus fumigatus, the causative agent of invasive

respiratory infections in immunocompromised patients

and colonisation in the airways of cystic fibrosis (CF)

patients (Oxford Academic 2020a), has demonstrated to

be involved in biofilm formation (Chandrasekar and

Manavathu 2008; Loussert et al. 2010; Morisse et al.

2013; Beauvais and Latg

e2015). The primary establish-

ment of chronic aspergillosis involves the germination

of conidia into mycelia, following the invasion into pul-

monary cells (Filler and Sheppard 2006). Scanning elec-

tron microscopy (SEM) has indicated that the aerial

hyphae of the mycelial colony developed over pulmon-

ary cells are embedded in a dense hydrophobic ECM

(Beauvais et al. 2007). The resistance of colonies

embedded in ECM to amphotericin B, compared with

the planktonic state, supports the evidence of biofilm

formation during pulmonary aspergillosis (Beauvais

et al. 2007; Ader et al. 2009; Escande et al. 2011).

Subsequently, in vivo, the presence of mature A. fumi-

gatus biofilms has been confirmed in both aspergilloma

and during the development of disseminated aspergil-

losis (Loussert et al. 2010). Bronchopulmonary lavage

(BAL) histopathologic examination of patients with

aspergillosis has revealed a complex multicellular myce-

toma structure (Microbiology Society 2020). The high

percentage of failure of antifungal therapy in the treat-

ment of invasive aspergillosis may be associated with

Aspergillus spp. pulmonary biofilms. In several studies,

the biofilm formation of A. fumigatus occurs under in

vitro conditions on polystyrene microtiter plates

(Mowat et al. 2007; Seidler et al. 2008; Singhal et al.

2011) and biomaterial devices such as catheters, cardiac

pacemakers, joint replacements, heart valves, and

breast augmentation implants have been extensively

reported (Escande et al. 2011; Jeloka et al. 2011; Sardi

et al. 2014). Despite the lack of clinical in vivo studies,

the ability of biofilm formation in vitro by other

Aspergillus spp., of Aspergillus flavus,Aspergillus terreus,

and Aspergillus niger has been reported (Fiori et al.

2011; Chatterjee and Das 2020).

Remarkably, a high percentage of biofilms consist of

mixed-species communities of bacteria and fungi that

are profoundly common and clinically important (Amin

et al. 2010; Peleg et al. 2010). Aspergillus fumigatus and

non-fumigatus Aspergillus are frequently co-colonising

with Pseudomonas aeruginosa, a common Gram-nega-

tive bacterium, in the airways of CF patients, open skin

wounds in burn patients, and cardiac implants (Amin et

al. 2010; Peleg et al. 2010; Manavathu et al. 2014). The

structural and functional in vitro properties of mixed

biofilms of P. aeruginosa and A. fumigatus vary from

those of monomicrobial biofilms, occurring in a severe

clinical problem in CF patients and other patient groups

at risk of airway infection with these two species

(Manavathu et al. 2014). For example, phenazine-

derived metabolites, redox-active small molecule sec-

ondary metabolites, produced by P. aeruginosa, can

serve as interspecies signals in co-culture biofilm with

A. fumigatus (Zheng et al. 2015). Biofilm formation of A.

fumigatus and Aspergillus nidulans has been differen-

tially modulated by P. aeruginosa phenazine-derived

metabolites, shifting from weak vegetative growth to

induced asexual sporulation (Zheng et al. 2015). A wide

range of diseases are related to the co-infection of fungi

and bacteria at various tissue have been described in

numerous studies. As an example, we note P. aerugi-

nosa,S. aureus,Burkholderia cepacia,Acinetobacter bau-

manii,Haemophilus influenzae mixed with Candida

albicans,A. fumigatus, and Scedosporium spp. (Dixon

and Hall 2015), and in burn wounds and trauma sites,

Candida,Aspergillus,Mucorales, and Fusarium spp. co-

colonized with Pseudomonas and Staphylococcus spp.

2 M. ROUDBARY ET AL.

(Peleg et al. 2010). The increasing co-infections of fungi

and bacteria explain the necessity of understanding the

mechanisms and to outcomes of polymicrobial bio-

film formation.

2.2. Zygomycetes

Zygomycosis, a fulminant opportunistic fungal infec-

tion, is caused by fungi belonging to the class

Zygomycetes and the order Mucorales, including

Rhizopus,Rhizomucor,Mucor,Lichtheimia,

Apophysomyces,Cunninghamella, and Saksenaea genus

(Ribes et al. 2000). Opportunistic infections caused by

Zygomycetes, can be characterised by angioinvasion

and fungal-ball formation (Chakrabarti et al. 2006).

Mucormycosis is categorised into rhino-orbito-cerebral

(ROC), pulmonary, gastrointestinal, cutaneous, and dis-

seminated types, depending upon the clinical presenta-

tion and the anatomical sites involved (Chakrabarti et

al. 2006). The biofilm-formation potential of common

pathogenic zygomycetes including Rhizopus oryzae

(Almeida et al. 2018), Lichtheimia corymbifera,

Rhizomucor pusillus, and Apophysomyces elegans has

been previously addressed (Singh et al. 2011). Phase-

contrast and confocal laser scanning micrographs dem-

onstrated the existence of highly compact adherent

hyphal structures encased in an ECM composed primar-

ily of N-acetylglucosamine and glucosamine in all the

examined species, excluding A. elegans (Singh et al.

2011). It has also been shown that the biofilm forma-

tion may be involved in angio-invasive infections

caused by Mucorales, including paranasal fungal balls,

chronic rhinosinusitis, and catheter-based infections

(Singh et al. 2011; Ezzat et al. 2020).

2.3. Black yeast-like fungi

Black fungi members include melanized species with

either slimy conidia or dry conidia (Chowdhary et al.

2014). Melanized species with wet conidia are nearly

common etiologic agents of human infections, in con-

trast, to dry conidia (Wang et al. 2018). Black fungi spe-

cies belonging to the genera Exophiala,Aureobasidium,

Ochroconis,Coelomycete and Pyrenochaeta are associ-

ated with human infections (Lugauskas et al. 2004; Lian

and de Hoog 2010). Humid conditions such as bath-

rooms, swimming pools, and sauna serve as favoured

environments for black fungi including from the

Exophiala genus (e.g. Exophiala equina,Exophiala leca-

nii-corni), which are the most common causative agents

of superficial mycoses in humans (Hilmarsdottir et al.

2005; Heinrichs et al., 2013a). Exophiala castellanii,

Exophiala equina,Exophiala lecanii-corni,Knufia epider-

midis, and Ochroconis spp. have been detached from

black pigmented biofilms happening in indoor wet cells

(Heinrichs et al., 2013a,2013b). A comparative metage-

nomic analysis of the dark pigmented fungal biofilms of

the water-air interphase of water taps, revealed that E.

lecanii-corni is a dominant component of these biofilms

(Heinrichs et al., 2013b). Of note, the biofilm of

Exophiala dermatitidis (caused severe phaeohyphomy-

cosis), has also exhibited higher resistance to the tested

antimycotic agents than the planktonic form (Gao et al.

2016;2017; Kirchhoff et al. 2017). Further investigation

showed that the biofilm formation and virulence of E.

dermatitidis were influenced by co-cultivation with P.

aeruginosa. For example, N-acyl-L-homoserine lactone

(AHL), P. aeruginousa quorum sensing molecule, inhibits

biofilm and filaments formation of E. dermatitidis

(Kirchhoff et al. 2020). Therefore, further research is

required to evaluate the potential pathogenesis of the

darkly pigmented biofilms.

2.4. Histoplasma capsulatum

Histoplasma capsulatum, a thermally dimorphic fungus

with yeast and filamentous phases, causes a respiratory

and systemic mycosis termed histoplasmosis (Sil and

Andrianopoulos 2014). As H. capsulatum has a high dis-

persion ability to adapt to various conditions by

expressing specific genes, the filamentous form of that

might present in medical settings (L

opez 2006; Carreto-

Binaghi et al. 2015). The adherence pattern to pneumo-

cytes as well as biofilm formation ability of H. capsula-

tum yeasts has been assessed (Pitangui et al. 2012). The

results revealed efficient adherence to host cells and

biofilm formation, which could be a possible adapted

survival strategy to avoid the immune cells (Pitangui et

al. 2012). Furthermore, a reduced activity of amphoteri-

cin B and itraconazole against biofilm of H. capsulatum

has also been shown (Brilhante et al. 2015). Farnesol

showed promising outcomes, as an adjuvant, by

enhancing the susceptibility to antifungal drugs in both

planktonic and biofilm structure (Brilhante et al. 2015).

Additionally, the development of H. capsulatum biofilms

in different nutritional conditions and oxygen concen-

trations, clarified that lower oxygen concentrations and

medium components are associated with a denser bio-

film and higher polysaccharide content (Gonc¸alves et

al. 2020). Currently, the filamentation of H. capsulatum

in the biofilm is unknown. Nevertheless, analysing the

reports, it can be deduced that the limitation of crucial

nutrients might be a stressor that stimulates the fila-

mentation transition from yeast (Gonc¸alves et al. 2020).

CRITICAL REVIEWS IN MICROBIOLOGY 3

2.5. Scedosporium spp.

Scedosporium spp.,cause a wide spectrum of life-threat-

ening infections with broad clinical manifestation, rang-

ing from colonisation of the respiratory tract,

sinopulmonary, extrapulmonary, and disseminated

infections (Cortez et al. 2008). Biofilm formation and

adherence ability of Scedosporium apiospermum,

Scedosporium aurantiacum, and Scedosporium minutis-

porum have been described in both polystyrene and

lung epithelial cells (Mello et al. 2016; Kitisin et al.

2020). In the biofilms, the dense compact of the myce-

lial biomass with internal channels explains the biofilm

formation ability by these fungal pathogens (Mello et

al. 2016). Further examination on the matured

Scedosporium spp. biofilms proved that presence of

ECM consisted of carbohydrate-rich molecules and

eDNA (Mello et al. 2020). Scedosporium minutisporum

and S. aurantiacum manifested a slight higher amount

of ECM composition than S. apiospermum (Mello et al.

2020). This genus has shown adherence to epithelial

lung cells with different avidity (de Mello et al. 2020).

The forming monolayer of mycelia over the cells have

demonstrated the ability to differentiate into hyphae,

resulting in irreversible damage to the epithelial cell

and cell death (de Mello et al. 2020).

2.6. Fusarium spp.

Fusarium spp. have a high genetic diversity and resist-

ance to systemic antifungals, causing a broad spectrum

of infections dependent on the immune status of the

host (Sav et al. 2018). The biofilm-forming capacity of

clinical isolated Fusarium spp. showed that Fusarium

solani,Fusarium petroliphilum, and Fusarium keratoplas-

ticum can produce biofilms (Sav et al. 2018). As it hap-

pens with other genus, the increased antifungal

minimum inhibitory concentrations (MICs) of biofilm-

forming Fusarium isolates, compared with their plank-

tonic counterparts, indicate the possible correlation of

biofilm formation and increased resistance (Sav et al.

2018). Furthermore, the compacted architecture of

Fusarium oxysporum f. sp. cucumerinum with extracellu-

lar polysaccharide materials revealed less susceptibility

to fungicides and environmental stress, including tem-

perature, UV light (Peiqian et al. 2014). Fusarium kerato-

plasticum, a common species of human infections can

develop a dense biofilm, with an increased matrix with

the presence of extracellular DNA, RNA, polysacchar-

ides, and proteins (Kischkel et al. 2020). The anti-biofilm

activity of farnesol on the preformed biofilm resulted in

the destruction of hyphae and the ECM (Kischkel et al.

2020), preventing the adhesion of conidia,

filamentation, and the formation of biofilm (Kischkel et

al. 2020). Elucidating the different stages of biofilm for-

mation in F. solani showed a profound structure with

increased resistance to natamycin, voriconazole, caspo-

fungin, and amphotericin B (C

ordova-Alc

antara et al.

2019). On contact lenses and fingernails (Burkhart et al.

2002), the biofilm formation of Fusarium (Mukherjee et

al. 2012) showed to be a strong contributor to patho-

genicity, following the high virulence and mortality rate

in invasive infections.

2.7. Dermatophytes

Dermatophytes are fungi responsible for dermatophyt-

osis, which include superficial fungal infections of the

skin and its appendages (Danielli et al. 2017).

Dermatophytosis is the most common mycosis world-

wide, affecting 20–25% of the general population.

Azole and allylamines are the most common drugs

used as treatment; however dermatophytes are

described to be resistant to most of them, particularly

biofilms, in addition to their considerable toxicity

(Costa-Orlandi et al. 2020). Trichophyton rubrum, C. albi-

cans, and Candida parapsilosis are some of the most

prevalent species related to this disease. Numerous

reports indicate a variable predominance of Candida

spp. in relation to dermatophytes, especially in onycho-

mycosis and the possibility of isolating both from the

same site (Garcia et al. 2020).

Costa-Orlandi et al. (2014) described, for the first

time, the characteristics of T. rubrum and Trichophyton

mentagrophytes biofilms. The authors indicated that

both species can form a mature biofilm within 72 h, and

T. rubrum biofilm produces more biomass and exopoly-

saccharide (EPS) and is denser than T. mentagrophytes

biofilm. Additionally, the results demonstrated a coordi-

nated network of hyphae in all directions, embedded

within EPS in some areas (Costa-Orlandi et al. 2014).

There is presently no evidence on the involvement

of filamentous fungi in multi-species biofilms. A recent

report revealed that, notwithstanding the predomin-

ance of Candida spp., the presence of T. rubrum seems

to inhibit C. albicans filamentation and C. parapsilosis

development, which confirmed an antagonistic inter-

action, and susceptibility to terbinafine and efinacona-

zole (Garcia et al. 2020).

Unprecedentedly, it was described the biofilm forma-

tion by Microsporum canis, responsible for tinea capitis

and categorised by a reduced therapeutic response

(Danielli et al. 2017). The study indicated a highly struc-

tured network of hyphae growing in all directions, with

compacted EPS regions, but typically with a porous and

4 M. ROUDBARY ET AL.

thin consistency, concentrated in the superficial region

of the mycelium (Danielli et al. 2017). An ex vivo model

for dermatophyte biofilm growth, using hair from dogs

and cats, was developed by Brilhante et al. (2019). With

some variations among species, that work showed

the appropriateness of this model to develop

structured biofilms with good biomass of M. canis

and Microsporum gypseum,T. mentagrophytes and

Trichophyton tonsurans. Furthermore, the microscopic

analysis of the biofilms showed hyphae colonising and

perforating the hair shaft, profuse fungal conidia, bio-

film EPS and biofilm water channels (Brilhante et

al. 2019).

Several recent studies have showed the potential anti-

fungal effect of novel drugs, against dermatophytes. A

recent work incorporated nonyl 3,4-dihydroxybenzoate

(fungicide compound against planktonic cells) and derm-

atophyte biofilms in nanostructured lipid systems, to

decrease toxicity in high concentrations, increase its

solubility and maintain its effectiveness against T. rubrum

and T. mentagrophytes biofilms. The formulation showed

a good efficacy against biofilms and a lower toxicity

(Costa-Orlandi et al. 2020). Similarly, the anti-dermato-

phytic and anti-biofilm activity of 2-hydroxychalcone

(2-chalcone) in the dark and photodynamic therapy

(PDT)-mediated against T. rubrum and T. mentagrophytes

biofilms. It was showed that the drug targets ergosterol

in the cell and promotes the generation of ROS, leading

to cell death by apoptosis and necrosis. Early-stage bio-

film and mature biofilms were inhibited by 2-chalcone,

indicating the potential therapeutical use of this com-

pound (Bila et al. 2021).

Formation of biofilm by dermatophytes is a key fac-

tor of fungal virulence, which whitethorn be tangled to

the persistence of infections. The characterisation of

biofilms formed by dermatophytes can surely contrib-

ute to the search of new drugs, doses and duration of

the treatment of these mycoses.

2.8. Paracoccidioides,Sporothrix and

Coccidioides

Paracoccidioides species are dimorphic fungi that firstly

infect the lungs, but can also spread through the body

(e.g. oral cavity, systemic colonisation, vascular pros-

thesis), most likely due to the formation of a biofilm

that makes it hard for the host to eradicate the infec-

tion (Sardi et al. 2015). These species are associated to

paracoccidioidomycosis, an endemic mycosis limited to

Latin America arising with the chronic form in 90% of

cases (Cattana et al. 2017).

The characterisation of Paracoccidioides brasiliensis

biofilms can help understanding the pathogenesis of

paracoccidioidomycosis, and the search for new and

effective drugs. Paracoccidioides brasiliensis biofilm and

gene expression of adhesins and hydrolytic enzymes

was reported by Sardi et al. (2015). The authors indi-

cated that the presence of a biofilm is associated with a

growth in the expression of adhesins and enzymes (e.g.

GP43, enolase, GAPDH and aspartyl proteinase). On

contrary, phospholipase was down-regulated in biofilm

(Sardi et al. 2015). Moreover, the dynamics of mono-

and dual-species biofilm growth of Paracoccidioides bra-

siliensis and C. albicans and their in vivo infectivity was

evaluated by Oliveira et al. (2020). The results indicated

that dual-species biofilm with P. brasiliensis plus C. albi-

cans presented a higher biomass, higher metabolic

activity and CFUs (colony forming unit) than their

mono-species biofilms. Galleria mellonella larvae

showed a reduction in the survival rate when infected

with P. brasiliensis and C. albicans, when compared with

those infected with single species, which finally indi-

cated that P. brasiliensis and C. albicans co-existence is

likely to occur on oral mucosal biofilms (Oliveira et

al. 2020).

Observing other species, it is known that Sporothrix

schenckii complex can origin a benign subcutaneous

mycosis, and biofilm formation may be one of the sig-

nificant elements associated to its virulence. S

anchez-

Herrera et al. (2021) reported that throughout the

development, S. schenckii biofilms are surrounded by

EPS, particularly glycoprotein (mannose rich), carbohy-

drates, lipids, and nucleic acids. The later was consid-

ered as a key component to structural integrity and

antifungal resistance (S

anchez-Herrera et al. 2021).

Finally, recurrent coccidioidal meningitis has also been

related to biofilms (on the tip of ventriculo-peritoneal

shunt tubing). The formed biofilm was the probable

responsible for a 4-year persistence of Coccidioides

immitis, notwithstanding the fact that the patient took

an adequate dosage of fluconazole (Davis et al. 2002).

Studies fungal species biofilm contributes to a better

understanding of growth biofilm and physiology, add-

ing new insights into the mechanisms of virulence and

persistence of pathogenic microorganisms.

An overview of clinically relevant filamentous fungi

that can form biofilm is given in Table 3.

3. Biofilm structures in filamentous fungi

Biofilms produced by budding yeasts are more compar-

able to bacteria rather than those formed by filament-

ous fungi, with hyphal tip growth (Beauvais et al. 2007;

CRITICAL REVIEWS IN MICROBIOLOGY 5

Harding et al. 2009; Mowat et al. 2009). Filamentous

fungal biofilms are thought to have unique morpho-

logical traits, since they lack the binary fission and the

growth dynamics observed in the asexual phase in

yeasts (Biesebeke et al. 2002; Beauvais et al. 2007; Gulis

et al. 2008; Harding et al. 2009). These distinct morpho-

logical properties may have contributed to a specialised

liquid-air interface structures in filamentous fungi,

which thought to be involved in host penetration and

nutrient acquisition (Harding et al. 2009).

Some studies have explored the biofilm properties of

the most common filamentous fungi, namely A. fumiga-

tus (Beauvais et al. 2007; Mowat et al. 2008,2009), A.

niger (Villena and Gutierrez-Correa 2006), F. solani, and

F. oxysporum (Imamura et al. 2008; Smith and Mcginnis

2005), through microscopic and antifungal susceptibil-

ity testing approaches.

Based on all the descriptions, a step-wise model for

filamentous fungal biofilms has been proposed

(Harding et al. 2009; Costa-Orlandi et al. 2017). The sug-

gested model provides a better understanding of the

nature of filamentous fungal biofilms development

(Figure 1): 1. adsorption: contacting of propagules such

as spores, sporangia, or hyphal fragments of the organ-

ism to a biotic or abiotic surface; 2. fixed adhesion:

attachment of spores or other reproductive structures

to surface via secreting adhesins during germination or

hydrophobins production, involved proteins in the

adhesion of hyphae to hydrophobic surfaces (W€

osten

2001); 3. initial microcolony colonisation: formation

microcolonies through apical elongation and hyphal

branching. This step following by monolayer formation

and ECM production, resulting in tenacious adherence

to the substrate; 4. preliminary maturation: formation

condensed hyphal networks or hypha-hypha adhesion,

resulting in layering covered by ECM, and water chan-

nel formation via hydrophobic repulsion among

hyphae; 5. maturation: formation survival structures

(fruiting bodies, sporogenous cells, sclerotia, and other)

depending on the fungi; 6. dispersion or planktonic

phase: releasing conidia or hyphae fragments, resulting

in a new cycle.

4. Molecular basis governing filamentous

fungal biofilm development

The application of “omics”technologies have revealed a

distinct underlying regulatory system and gene expres-

sion patterns during biofilm phases related to plank-

tonic counterparts, which reflect their unique

phenotypes (Bruns et al. 2010; Muszkieta et al. 2013). It

is known that biofilm and planktonic phases have dis-

tinct growth demands and this is reflected by the differ-

entially regulation of many genes and proteins such as

secondary metabolism, cell wall structure, transcription

factors, stress responses, and the translation machinery

(Gibbons et al. 2012).

Proteomic and transcriptomic analyses of A. fumiga-

tus biofilm have showed a stage dependent pattern

with different energy requirements, depending on the

age of the biofilm (Bruns et al. 2010). Young biofilm

(24 h), has higher demands of energy through upregu-

lation of primary metabolism genes (Bruns et al. 2010),

which are required processes for growth, maintenance,

Figure 1. The schematic stepwise phases of the development of filamentous fungi biofilms on biotic and abiotic surfaces. The

Figure has been made by Biorender.com.

6 M. ROUDBARY ET AL.

and survival of the organism (Drew and Demain 1977).

Comparatively, the metabolic activity of matured bio-

film (48 h), has been diminished (Bruns et al. 2010). In

contrast, the hydrophobins family, including RodAp and

RodBp (Paris et al. 2003), and secondary metabolites

such as gliotoxin, an immunosuppressive toxin (Sugui

et al. 2007), have been upregulated in the matured bio-

film (Bruns et al. 2010). The enhanced production of

gliotoxin in the matured biofilm stage may protect A.

fumigatus from the host immune system and enable its

survival and persistence in chronic lung infections such

as aspergilloma (Bruns et al. 2010). The substantial up-

regulation of secondary metabolism genes in A. fumiga-

tus biofilms could be associated with the upregulation

of LAEA, a secondary metabolism regulator (Gibbons et

al. 2012) which has also been reported to regulate the

synthesis of gliotoxin (Sugui et al. 2007). Two types of

proteins, hydrophobins and adhesins,confer a hydro-

phobic character to fungal morphotypes (Sunde et al.

2008; Gibbons et al. 2012). Hindering the immune rec-

ognition is another role attributed to hydrophobins,

RodAp, which absence is associated with a reduction of

virulence in A. fumigatus (Aimanianda et al. 2009; Bruns

et al. 2010). The upregulated hydrophobin expression

in biofilm cells, including several Rod genes (RodA,

RodB, and RodD) (Beauvais et al. 2007; Gibbons et al.

2012) supports the need for further studies to elucidate

the roles of hydrophobins in biofilm.

Adhesins have not been deeply investigated bio-

chemically in filamentous fungi; however, it is reported

an upregulation of 11 of the 25 genes with the highest

adhesion probability score (Afu1g09510, Afu1g14430,

Afu3g00420, Afu3g01150, Afu3g09690, Afu4g03240,

Afu6g13720, Afu7g00580, Afu7g02460, Afu7g05340, and

Afu8g01970)inA. fumigatus genome (Upadhyay et al.

2009; Gibbons et al. 2012). Since adhesion is the first

and one of the most crucial steps in the colonisation of

a substrate by a fungus (Sheppard 2011), this ability

should be further explored. On the other side, the role

of mitogen-activated protein kinases (MAPK) and phos-

phatases in adhesion and biofilm formation of A. fumi-

gatus, as well as the cell wall and ECM production, has

been explained by the loss of the MAPK genes (MpkA,

MpkC, and SakA), and phosphatase genes (sitA and

ptcB) (Manfiolli et al. 2018). Indeed, the loss of particular

MAP kinases and phosphatases cascades, which operate

different responses such as cell growth, differentiation,

and environmental stress adaptation based on changes

in certain environmental conditions (Chen and Thorner

2007), influences the cell wall carbohydrate exposure

and the ECM during biofilm formation and, subse-

quently, diminished adherence of A. fumigatus to

polystyrene and fibronectin-coated plates. Considering

the role of MpkC and SakA in A. fumigatus, the associ-

ation of MAPK and the high-osmolarity glycerol (HOG)

pathway involved in osmotic and oxidative stress

response, a biofilm regulation could be proposed (Chen

and Thorner 2007; Nascimento et al. 2016). It has also

been showed that the deletion of ptcB (one of the

phosphatases which promotes Hog1 phosphorylation),

in A. fumigatus, results in the increased B-1,3-glucan

and chitin content but more sensitivity to cell-wall dam-

aging drugs, reduced adhesion, biofilm formation, and

ECM production, which directly implies the association

of the HOG pathway in the regulation of biofilm forma-

tion (Ribes et al. 2000).

In A. niger, the time-dependent regulation manner of

HOG1 pathways genes, revealed the role of HOG path

in upregulation of genes of at different times in biofilm

state (Sun et al. 2020). Besides, biofilm cells and free

mycelia of A. niger demonstrated an enhanced activity

regarding the extracellular polysaccharide synthesis

pathway, and amino acid metabolisms (Sun et al. 2020).

It was also indicated the downregulation of the tricarb-

oxylic acid cycle (TCA cycle) at 24 h, which resulted in

the acceleration of amino acid accumulation, also

reflects the higher activity of biofilm (Sun et al. 2020).

The role of the Ca

2þ

mediated calcineurin signalling

pathway (CSP) on the biofilm formation of A. niger has

also been investigated (Liu et al. 2020). CSP is an essen-

tial signalling pathway regulating Ca

2þ

balance in the

mycelium of filamentous fungi. Knocking out several

genes involved in CSP, including the Ca

2þ

channel

MidA and CchA, calcineurin catalytic subunit CnaA, and

transcription factor CrzA, led to the reduction of both

2þ

levels in the mycelium and biofilm formation. The

deletion of the mentioned genes affected the biofilm

formation by decreasing the expression of hydrophobin

RodA and, consequently, the hydrophobicity and adhe-

sion of conidia. Actually, the hydrophobic ability of the

conidia influences the adsorption, which is a critical

step in the early stage of formation, immobilisation and

development of biofilm (Liu et al. 2020). Additionally,

those authors revealed that there is an increased sensi-

tivity of mutant strains to cell wall disrupters, compared

to wild-types. This study brought up the possibility of

different extracellular polysaccharides and cell surface

components, such as b-1,3-glucan and chitin, and their

influence on flocculation ability of hyphae, cell-cell

adhesion, and aggregation ability (Liu et al. 2020). The

lost mycelial flocculation ability of mutant strains, rela-

tive to the wild-types, provided proof for changes in

mycelium adhesion and aggregation, proposing an

impact of CSP on biofilm formation by altering the

CRITICAL REVIEWS IN MICROBIOLOGY 7

composition of mycelial cell carbohydrates in the mid-

dle stage of biofilm formation (Liu et al. 2020). In the

following, the altered expression of more than 50% of

cell wall genes in A. fumigatus biofilm (Fanning and

Mitchell 2012), and the absence of cell wall in human

hosts suggest the cell wall biogenesis pathways as a

potential target for specific disrupter. Moreover, the

role of the cspA gene, encoding a glycophosphatidyli-

nositol-anchored protein in the cell wall, in biofilm

development, and antifungal drug resistance in A. fumi-

gatus has been described (Fan et al. 2015). It was indi-

cated that the cspA deletion resulted in a reduced

biofilm formation, decreased resistance to antifungal

agents, and increased internalisation by A549 human

lung epithelial cells. Also, the disruption of cspA

resulted in an irregular arrangement of hyphae, circle-

like connections instead of the classic bundles of paral-

lel filaments forming acute angles, which might affect

biofilm construction. Comparing with the wild-type

cells, the absence of ECM in the mutant strain, sug-

gested that cspA is linked to the ECM production, and,

subsequently, with drug resistance (Fan et al. 2015).

Recently, the influence of oxygen on the biofilm

morphotypes of A. fumigatus exhibited a hypoxia-typic

morphotype (Kowalski et al. 2019), which has been gen-

erated by the expressed sub-telomeric gene cluster,

containing genes that modify the hyphal surface and

disturb inter-hyphal interactions. Hypoxia-morphotype

leads to increased host inflammation, rapid disease pro-

gression, and mortality in a murine model of invasive

aspergillosis, suggesting a direct impact of filamentous

fungal biofilm morphology on fungal-host interactions

(Kowalski et al. 2019).

Besides, on specific pathways, biofilms display a

reduced susceptibility to antifungal drugs that can also

be involved in the poor in vivo drug efficacy, which

seem to be explicitly expressed (Bruns et al. 2010;

M€

uller et al. 2011; Rajendran et al. 2011; Gibbons et al.

2012; Muszkieta et al. 2013). For instance, MDR4, a mul-

tidrug resistance transporter gene in A. fumigatus,

showed to have a phase-dependent expression manner

in biofilm, upregulating in both 12- and 24-h, with max-

imal upregulation at 12-h. Furthermore, after treatment

with voriconzaole, A. fumigatus has shown an increased

resistance in the 12-h (16- to 128-fold) and 24-h (>512-

fold) phases, when compared with 8-h germlings, which

is in agreement with the phase expression pattern

(Rajendran et al. 2011). Given the expression profile of

MDR4, the efflux pump is likely to play an early function

in biofilm by regulating the internal environment

through removing antifungals. Since ECM is produced

during the maturation phase of biofilm, it is therefore

likely that A. fumigatus might use the ECM to decrease

the drug penetration, suggesting it as a reason for the

phase-dependent expression of MDR4. Additionally,

several mechanisms might be responsible for a high

resistance of biofilms (compared with the planktonic

state), as discussed later in section 5.

Biofilm formation is thought to be a single process

but it has become clear that biofilm development

involves a series of sequential molecular events.

Shedding more light on the underlying regulatory net-

work of intracellular signalling pathways involved in the

biofilm, will provide new avenues for research into the

identification of potential therapeutic targets.

5. Quorum-sensing molecules and their actions

in filamentous fungi

Apart from survival and reproduction competition,

microorganisms are no longer considered independent

cells (Kalia 2015; Barriuso et al. 2018; Padder et al.

2018). They have developed a communication manner -

quorum sensing (QS) - that allows them to orchestrate

an appropriate response to local environmental

changes (Kalia 2015; Barriuso et al. 2018; Padder et al.

2018). QS is a process that links gene expression to the

cell density of microbial populations by secretion of

small metabolites (Hogan 2006; Srinivasan et al. 2016).

The cell-cell communication in QS relies on the produc-

tion and release of small diffusible chemical signalling

molecules in the extracellular environment, referred to

as autoinducers or quorum-sensing molecules (QSMs)

(Ramage et al. 2009; Wongsuk et al. 2016). QSM serve

as transcriptional regulators; while their concentration

reaches a critical threshold, corresponding to an appro-

priate cell density, they bind to the receptors (Waters

and Bassler 2005; Padder et al. 2018; Zhang et al. 2019).

Subsequently, the target genes of QS and the genes

encoding QS signal synthesis are prompted by tran-

scriptional regulators, thereby generating self-inducing

feedback, rising the production of the corresponding

signalling molecules (Hogan 2006; Williams et al. 2013).

The existence of QS systems in fungi has been recog-

nised by farnesol, QSM of C. albicans (Hornby et al.

2001; Rodrigues and



Cern

akov

a2020). Many QSMs are

actively involved in fungal QS, contributing to various

vital mechanisms in microorganisms, including regula-

tion of virulence genes of pathogenesis and dissemin-

ation during the infection establishment, adaptation of

microorganisms to host, biofilm development, morpho-

genesis, secondary metabolite production, stress resist-

ance, and suppression of the cell population (Ramage

et al. 2005; Dixon and Hall 2015; Wongsuk et al. 2016;

8 M. ROUDBARY ET AL.

Barriuso et al. 2018; Bandara et al. 2020). However,

much research has focussed on QS in bacteria and

yeast, particularly C. albicans, but QS in non-unicellular

filamentous fungi remains controversial due to the

nature of these fungi that develop mycelial growth.

There are several QSMs in filamentous fungi, particu-

larly in species from the genera Aspergillus and

Penicillium. Among them, we can find oxylipins, butyro-

lactone I, linoleic acid, c-heptalactone, multicolic acid,

farnesol (Table 1).

Oxylipins are oxygenated polyunsaturated fatty

acids, made by oxygenase enzymes (Tsitsigiannis and

Keller 2007; Brodhun and Feussner 2011). In A. flavus,

oxylipins serve as a signal that regulates secondary

metabolisms (mycotoxin production), and morpho-

logical differentiation and spore development either

asexual spores or sclerotia via a density-dependent

mechanism resembling QS (Horowitz Brown et al. 2008;

Brown et al. 2009; Affeldt et al. 2012). Increased cell

density resulted in the lowest numbers of sclerotia, and

the highest numbers of conidia, suggesting density-

dependent phenomena (Horowitz Brown et al. 2008).

Moreover, deletion of Aflox, encoding an oxylipin-gen-

erating lipoxygenase (LOX), showed that there is a cor-

relation between LOX activity and density-dependent

development of both sclerotia and conidia, suggesting

the possible existence of a dense dependent factor,

maybe a LOX-derived metabolite stimulates the conid-

ium-to-sclerotium switch, depending on the high or

low density of cells (Horowitz Brown et al. 2008). In

order to elucidate how oxylipins, as signals, are

detected and transmitted by A. flavus and involved in

QS mechanisms, the role of G protein-coupled recep-

tors (GPCRs) has been investigated (Affeldt et al. 2012).

Depletion of GprC and GprD in A. flavus resulted in lock-

ing of fungus in a low-density state, which revealed the

necessity of these receptors to respond and transit to a

high-density development state (Affeldt et al. 2012). It

also has been revealed that oxylipin acts as develop-

mental signals through GPCRs and induces fungal cellu-

lar differentiation, including lateral branching (Niu et al.

2020), mediating the interhyphal communications by

fusion to other hyphae, providing more substrate inva-

sion by mechanical strength, and more molecular and

nutrient exchanges within the network (Fischer and

Glass 2019; Harris 2019). These findings propose a cas-

cade-like regulation process, which leads to the morph-

ology and metabolic differentiation, more branching

and nutrient exchange, that might be arranged for

effective competition and accessing multiple nutrients

in complex environments, such as biofilms.

Butyrolactone I, a secondary metabolite produced by

A. terreus, has been considered as a QSM, according to

its auto-stimulatory effect (Schimmel et al. 1998; Raina

et al. 2012). Butyrolactone I influences its own produc-

tion and induces morphological changes, increases

hyphal branching, spore formation (Schimmel et al.

1998; Raina et al. 2012). Also, further analysis uncovered

the regulatory role of butyrolactone I in the expression

level of lovastatin (Palonen et al. 2017). LaeA, a global

Table 1. QSMs and their known roles in clinically relevant filamentous fungi.

QSMs Organism Role References

Oxylipins Aspergillus flavus -Regulation of secondary metabolism (mycotoxin

production), morphological differentiation and

spore development.

-Cellular differentiation, like lateral branching.

(Horowitz Brown et al. 2008; Brown et

al. 2009; Affeldt et al. 2012; Niu et

al. 2020)

Butyrolactone I Aspergillus terreus -Auto-inducer effect on Butyrolactone I

production.

-Induction of morphological changes, increase in

hyphal branching, spore formation, and

regulation of lovastatin production.

(Schimmel et al. 1998; Raina et

al. 2012)

c-heptalactone Aspergillus nidulans -Shortening of the lag phase, earlier

deceleration phase, and enhanced

penicillin production.

(Williams et al. 2012)

Multicolic acid Penicillium sclerotiorum -Increase of sclerotiorin production. (Raina et al. 2010)

Farnesol (exogenous) Aspergillus fumigatus -Inhibition of the cell wall integrity pathway by

direct or indirect production of cell wall

stressor.

-Disorganisation of the apical actin cytoskeleton.

(Dichtl et al. 2010)

Farnesol (exogenous) Aspergillus nidulans - Induction of apoptosis. (Semighini et al. 2006)

Farnesol (exogenous) Aspergillus niger -Abolishion of conidiation.

-Increase of aerial hyphae formation.

-Decrease of the intracellular level of cyclic

adenosine monophosphate (cAMP).

(Lorek et al. 2008)

Farnesol (exogenous) Aspergillus falvus - Induction of apoptosis. (Wang et al. 2014)

Farnesol (exogenous) Fusarium graminearum - Induction of apoptosis. (Semighini et al. 2008)

Farnesol (exogenous) Histoplasma capsulatum -Synergistic effect with itraconazole on biofilm

and planktonic phases.

(Brilhante et al. 2015)

CRITICAL REVIEWS IN MICROBIOLOGY 9

regulator, is involved in conidiation and secondary

metabolism along with the velvet family.

The overexpression of LaeA, induced by butyrolactone I,

increased the biogenesis of lovastatin, supporting the

suggested role of butyrolactone I as a QS molecule in A.

terreus (Palonen et al. 2017). Moreover, lovastatin pro-

duction and the expression levels of lovastatin biosyn-

thetic genes, lovB and lovF,inA. terreus have been

increased by exogenous linoleic acid supplementation,

as an oxylipin precursors (Sorrentino et al. 2010).

However, supplementing linoleic acid to the low cell

density of A. terreus culture enhanced lovastatin pro-

duction to 1.8-fold. Nevertheless, lovastatin yield has

been impaired by adding at a later stage of growth,

suggesting the regulatory role of cell density in lovasta-

tin production at the time of linoleic acid supplementa-

tion (Sorrentino et al. 2010).

c-heptalactone is a lactone-containing QSM that reg-

ulates the A. nidulans’s behaviour at a low cell density

(Williams et al. 2012). The supplementation of low-dens-

ity cell cultures of A. nidulans with nanomolar concen-

trations of c-heptalactone resulted in the shortening of

the lag phase, earlier deceleration phase, and enhanced

penicillin production via increased ipnA::lacZ reporter

activity, a penicillin production marker (Williams et al.

2012). Further analysis in A. nidulans showed that the

oleic and linoleic acid-derived oxylipins production

depends on the regulation of three conserved oxylipin

biosynthetic enzymes, PpoA, PpoB, and PpoC. A. nidu-

lans oxylipin mutation (DppoA; DppoB; DppoC A)

resulted in the defective colonisation of seeds, reduced

lipase production, altered secondary metabolite profiles

and impaired mycotoxin sterigmatocystin production

(Tsitsigiannis and Keller 2006). The silenced mutation in

ppo genes, ppoA,ppoB, and ppoC,inA. fumigatus,

revealed that they are involved in hypervirulence in the

invasive pulmonary aspergillosis murine model and

increased tolerance to H

stress (Tsitsigiannis et al.

2005). These findings imply that Ppo products, oxylipins

(already mentioned above), serve as activators of host

defense mechanisms by hindering the development of

pulmonary and invasive aspergillosis (Tsitsigiannis et al.

2005). c-butyrolactone-containing compounds (multi-

colic acid and related derivatives) have been suggested

as the QSMs in Penicillium sclerotiorum, by increasing

sclerotiorin production, a secondary metabolite (Raina

et al. 2010).

The QSM farnesol, a precursor of sterol biosynthesis

in C. albicans, prevents the biofilm development and

morphological transition in Candida spp. (Hornby et al.

2001; Hogan 2006; Rodrigues and



Cern

akov

a2020).

Exogenous farnesol affects filamentous fungi such as A.

fumigatus (Dichtl et al. 2010), A. nidulans (Semighini et

al. 2006), A. niger (Lorek et al. 2008), A. flavus (Wang et

al. 2014), Fusarium graminearum (Semighini et al. 2008)

and Histoplasma capsulatum (Brilhante et al. 2015).

In A. fumigatus, exogenous farnesol has been impli-

cated as a cell wall perturbing agent that inhibits the

cell wall integrity (CWI) pathway, originating direct or

indirect cell wall stress (Dichtl et al. 2010). Mutants lack-

ing two essential kinases of CWI signalling, AfMkk2 or

AfMpkA, namely the MAP kinase kinase AfMkk2 or its

target MAP kinase AfMpkA, revealed high susceptibility

to farnesol (Dichtl et al. 2010). The higher susceptibility

of mutants compared with the wild type suggested

that the mode of action of farnesol is not only restricted

to CWI signalling inhibition and could be related to

interfering with prenylated proteins. The loss of hyphal

tip localisation of AfRho1 and AfRho3, two prenylated

Rho family GTPases involved in CWI and cytoskeleton

controlling, implied disorganisation of the apical actin

cytoskeleton, as complementary part in higher suscepti-

bility of mutant to farnesol (Dichtl et al. 2010). In A.

nidulans, farnesol has been shown to induce apoptosis,

a programmed cell death, through a mechanism that

demands functional mitochondria, ROS production, and

mediating a heterotrimeric G protein complex (GTP-

binding proteins), FadA (Semighini et al. 2006). Farnesyl

pyrophosphate (FPP), a protein prenylation precursor,

plays a role in the post-translation modification of pro-

teins. Farnesylated proteins include Ras and Ras-related

G proteins, control cell growth, differentiation, prolifer-

ation, and survival (Edwards and Ericsson 1999).

Additionally, co-cultivation of A. nidulans with C. albi-

cans revealed an impaired hyphal growth and colony

development in a farnesol-dependent manner

(Semighini et al. 2006). Since A. nidulans does not

secrete detectable amounts of farnesol, this may sug-

gest that it reacts to farnesol produced by other fungi

(Semighini et al. 2006). In addition to its QS function

(morphogenesis regulation), farnesol seems to be

employed by C. albicans to reduce the competition

with other microorganisms in a competitive situation.

Exogenous farnesol produced by C. albicans serves as a

conidiation inhibitor in A. niger (Lorek et al. 2008). The

high concentration of farnesol resulted in the abolished

conidiation and increased aerial hyphae formation with

a cotton-like appearance, and the diminished intracellu-

lar level of cyclic adenosine monophosphate (cAMP)

(Lorek et al. 2008). Moreover, A. flavus treated with far-

nesol had inhibition of germination and growth, and

induction of apoptosis markers, including nuclear con-

densation, phosphatidylserine (PS) externalisation, DNA

fragmentation, and ROS generation, metacaspase

10 M. ROUDBARY ET AL.

activation, and abnormal cellular ultrastructure (Wang

et al. 2014). Another study demonstrated that farnesol

in Fusarium graminearum triggers apoptosis through

impairing development and inhibition of macroconidial

germination and viability (Semighini et al. 2008).

Farnesol also affects filamentous fungus, H. capsulatum,

in biofilm and planktonic phases, particularly in com-

bination with itraconazole (Brilhante et al. 2015), sug-

gesting farnesol as a promising antifungal.

QSMs can affects the host immune system by several

means (Joo and Jetten 2008). For example, farnesol

induces the expression of some immune responses and

inflammatory genes in lung cells through the nuclear

factor kappa-B (NF-jB) signalling pathway, including IL-

6,CXCL3,IL-1a, and COX-2 (Joo and Jetten 2008). The

NF-jB pathway stimulation via MEK1/2-ERK1/2-MSK1-

dependent phosphorylation of p65 l leads to the pro-

duction of cytokines (interleukin-6 and interleukin-1a)

(Joo and Jetten 2008). Also, farnesol with yeast cell wall

components (zymosan), intensifies the expression of

proinflammatory cytokines in the murine macrophage

cell line but inhibits activation of cellular immunity, sug-

gesting farnesol as an immune-modulatory molecule

(Oxford Academic 2020b). On the other hand, the mat-

uration of monocytes to dendritic cells has been

affected by farnesol (Leonhardt et al. 2015). Matching

with the control, in the presence of farnesol, the cell

surface markers of immature dendritic cells were modi-

fied, including increased CD86 and reduced CD1a, low-

ering expression of multiple genes involved in cell

adhesion and migration (Leonhardt et al. 2015).

Therefore, there was a diminished ability of dendritic

cells to activate T cells, which eventually dampens the

adaptive immune response (Leonhardt et al. 2015).

Additionally, it has been concluded that QSMs

impact the stress resistance in fungi, by preserving the

fungus from ROS. In C. albicans, resistance to ROS has

been enhanced by the increased expression of protect-

ive catalase Cat1, due to the inhibition of the Ras1-

cAMP pathway and cross-talk with Hog1 regulators

(Westwater et al. 2005; Deveau et al. 2010).

Taken together, QS process seems to have been

adapted by fungi to regulate a range of developmental

processes gene expression and coordinate cell-to-cell

interactions, to optimise a successful cellular morph-

ology and metabolic differentiation for effective compe-

tition in a complex environment. Therefore, further

elucidation of the QS mechanisms in filamentous fungal

biofilm, as a tangled network, will lead to a better

understanding of fungal pathogenesis and facilitating

the development of novel antifungal tar-

get approaches.

6. Antifungal resistance phenomenon in

biofilm structure and possible mechanisms

Fungal biofilm resistance is a multifactorial phenom-

enon, including fundamental physical barriers and com-

plex regulatory processes (Ramage et al. 2012;

Shishodia et al. 2019; Kowalski et al. 2020). Different

resistance mechanisms in the fungal biofilm might be

developed to decrease the efficacy of antifungal treat-

ment, including ECM production, modifications or over-

expression of target molecules, compound expulsion by

efflux pumps, restricted diffusion, tolerance, and cell

density (Niimi et al. 2010; Ramage et al. 2012). The fol-

lowing sections will describe the mechanisms involved

in the increased antifungal resistance of the filamentous

fungal biofilms (Table 2).

6.1. Major cases of drug resistance

Biofilm structures are highly resistant to antimicrobial

agents and hence giving rise to persistent infections in

patients. Treating infections caused by biofilms require

a higher dosage of antifungal agents and different

therapeutic schemes, since structural attributes, such as

ECM, will dramatically reduce the efficacy of the drug

used (e.g. diffusion issues) (Rodrigues et al. 2017;

Shishodia et al. 2019). The inefficacy of several antifun-

gal agents against fungal biofilms has been frequently

reported in several studies (Mowat et al. 2007;2008;

Seidler et al. 2008; Fiori et al. 2011; Liu et al. 2012;

Zhang et al. 2012; Pierce et al. 2013; Wuren et al. 2014;

Kirchhoff et al. 2020; Nazir et al. 2020). Compared to

planktonic cells, biofilm or sessile cells of Aspergillus

spp. (A. fumigatus,A. flavus,A. terreus, and A. niger)

exhibited higher sessile minimum inhibitory concentra-

tions 90 (SMIC90) of amphotericin B, voriconazole, and

echinocandins, including anidulafungin and caspofun-

gin (Fiori et al. 2011). Further investigation on the

developed biofilm of A. fumigatus on bronchial epithe-

lial cells revealed a decreased antifungal drug suscepti-

bility towards azoles, polyenes, and echinocandins

(Seidler et al. 2008). For instance, the efficacy of itracon-

azole, voriconazole, amphotericin B, and caspofungin

against biofilm of A. fumigatus, was 1000 times higher

than that of the planktonic cells (Mowat et al. 2007).

Besides, exposure of Aspergillus spp. to serum proteins,

such as foetal bovine serum (FBS), fetuin-A, and bovine

serum albumin (BSA), led to the formation of a thick

ECM and increased resistance to antifungals (Wuren et

al. 2014). Similarly, F. solani and F. oxysporum biofilms

also displayed reduced susceptibility to all tested anti-

fungal agents, including amphotericin B, voriconazole,

CRITICAL REVIEWS IN MICROBIOLOGY 11

itraconazole and fluconazole (Zhang et al. 2012;

Machado Vila et al. 2015).

6.2. Physiological state and cell density

The physiological state of cells in biofilm communities

has also been associated with the susceptibility profiles

of biofilms. Different studies explained how environ-

mental stresses modify biofilm architecture and likely

antifungal sensitivity, including glucose and iron limita-

tion (Baillie and Douglas 1998), elastin addition

(Brand~

ao et al. 2018), growth rate (Beauvais and Latg

2015), oxygen availability (Dumitru et al. 2004; Kowalski

et al. 2019), pH (Kuchar



ıkov

a et al. 2011), and tempera-

ture (Pettit et al. 2010). It was shown that self-induced

hypoxic microenvironments in A. fumigatus during bio-

film maturation facilitate fungal survival in the presence

of antifungal treatments (Kowalski et al. 2020).

Decreasing oxygen levels in the biofilm of A. fumigatus

increase antifungal drug resistance (Kowalski et al.

2020). Therefore, it is more likely that multiple factors

are implicated in the physiological state of the cell,

Table 2. Biofilm associated resistance mechanisms of filamentous fungi.

Mechanism of resistance Effect References

Altering the physiological state of cells Biofilm architecture modification (Baillie and Douglas 1998; Dumitru et al. 2004;

Kuchar



ıkov

a et al. 2011; Beauvais and Latg

2015; Brand~

ao et al. 2018; Kowalski et al.

2019,2020)

Cell density Quorum sensing (Lawrence et al. 1991; Beer et al. 1994; Lass-Fl€

orl

et al. 2003; Perumal et al. 2007; Mowat et al.

2008; Costa-Orlandi et al. 2017)

Extracellular matrix (ECM) Preventing penetration of antifungals into the

biofilm and reaching the targets

(Beauvais et al. 2007; Fontaine et al. 2010;

Loussert et al. 2010; Rajendran et al. 2013;

Bom et al. 2015; Sheppard and Howell 2016)

Up-regulation efflux pumps and ATP-binding-

cassette transporter proteins

Reducing the intracellular performance of

antifungals through pumping out

(Ferreira et al. 2006; Bueid et al. 2010; Rajendran

et al. 2011; Gibbons et al. 2012; Ramage et al.

2012; Muszkieta et al. 2013)

Heat shock proteins 90 (HSP90) Increased drug resistance through possible

enhanced reactive oxygen species capacity

(Robbins et al. 2011; Delattin et al. 2014;Tuet

al. 2020)

Drug tolerance and persister cells Inactivation antifungal drug targets by entering

the dormancy phase

(Schumacher et al. 2009; Lafleur et al. 2010;

Lewis 2010; Wuyts et al. 2018)

Table 3. Summary of filamentous fungal biofilm and their clinical relevance.

Biofilm former Biofilm morphology Matrix component

Devices associated

with biofilm References

Aspergillus spp. Hyphal cells embedded

in ECM

Galactomannan, a-1,3-glucans,

monosaccharides, polyols,

melanins, proteins such as

hydrophobins and DNA

Catheters, cardiac

pacemakers, joint

replacements, heart

valves, and breast

augmentation implants

(Beauvais et al. 2007; Loussert et

al. 2010; Escande et al. 2011;

Jeloka et al. 2011; Sardi et al.

2014; Chatterjee and Das 2020)

Zygomycetes spp. Compact hyphal structures

encased in ECM

ECM contained

N-acetylglucosamine and

glucosamine

Catheters (Singh et al. 2011; Almeida et al.

2018; Ezzat et al. 2020)

Black yeast-like fungi Species-depending high

biomass biofilm

–Water distribution and

supplies systems

(Hilmarsdottir et al. 2005;

Heinrichs et al., 2013a,2013b;

Gao et al. 2016; Kirchhoff et

al. 2017)

Histoplasma capsulatum Dense biofilm of yeast, as

an active biofilm

structural contributor

ECM with

polysaccharide content

Probably healthcare-

associated

biomaterial devices

(Pitangui et al. 2012; Carreto-

Binaghi et al. 2015; Gonc¸alves

et al. 2020)

Scedosporium spp. Dense compact of the

mycelial biomass with

internal channels

enclosed in ECM

ECM consisted of carbohydrate-

rich molecules and

extracellular DNA

Central venous catheter (Mello et al. 2016; Kitisin et al.

2020; Mello et al. 2020)

Fusarium spp. The compacted architecture

of hyphal surrounded

by ECM

ECM composed of extracellular

DNA, RNA, polysaccharides,

and proteins

Contact lenses (Mukherjee et al. 2012; Sav et al.

2018;C



ordova-Alc

antara et al.

2019; Kischkel et al. 2020)

Dermatophyte The dense and thick

network of hyphae,

embedded in EPS

–Water channel and

host tissue(hair)

(Costa-Orlandi et al. 2014; Danielli

et al. 2017; Brilhante et al.

2019; Costa-Orlandi et al. 2020)

Sporothrix schenckii A Network of hyphae,

embedded in EPS

EPS composed of glycoprotein

(mannose rich),

carbohydrates, lipids, and

nucleic acid

–(S

anchez-Herrera et al. 2021)

in A. fumigatus

12 M. ROUDBARY ET AL.

suggesting a possible role in resistance to antifungal

agents of biofilm.

Cell density is also considered as one of the resist-

ance mechanisms within the complex fungal biofilms.

Previous investigations support the idea that the high

density of the cells, which can be regulated by QS, pro-

duces resistance to antifungal agents (Lass-Fl€

orl et al.

2003; Perumal et al. 2007; Mowat et al. 2008). Relatively,

the low cell density (10

cells/mL) of both planktonic

and resuspended biofilm cells showed susceptibility to

azoles. However, ten folds increased cell density leads

to the enhanced resistance to azoles (Perumal et

al. 2007).

6.3. ECM

ECM is an essential component of all fungal biofilms,

rendering physical barrier function against environ-

mental insults, like host defences and antimicrobial

therapies (Mitchell et al. 2016; Pierce et al. 2017;Le

Mauff 2020). The antifungal drug permeation into

the biofilm or binding to the target can be ham-

pered by the presence of a dense ECM (Nett and

Andes 2017; Rodrigues et al. 2017; Singh et al.

2018), suggesting the possible role of the chemical

composition of the ECM and its regulation in the

resistance mechanisms. A typical ECM consists of

polysaccharides (e.g. galactomannan, a1,3-glucans,

monosaccharides), polyols, melanin, eDNA, lipids (e.g.

ergosterol) and proteins (including antigens and

hydrophobins), however, its composition varies sig-

nificantly based on the growth conditions and the

species (Beauvais et al. 2007; Loussert et al. 2010;

Fiori et al. 2011). In A. fumigatus biofilms, ECM is a

heterogeneous substance that consists of extracellu-

lar DNA, proteins, lipids and polyols, and exopolysac-

charides (e.g. a-glucans, galactomannan, and the

glycan galactosaminogalactan (GAG)) (Beauvais et al.

2007; Fontaine et al. 2010; Loussert et al. 2010;

Rajendran et al. 2013; Sheppard and Howell 2016).

GAG is a partially deacetylated heteropolymer of

a-1,4-linked galactose and N-acetyl galactosamine

(Lee et al. 2014) and is absent in A. fumigatus

spores but exists in growing hyphae. It mediates

protection against host immune defences and also

accelerates the adhesion between both fungi and to

other surfaces (Fontaine et al. 2011; Gravelat et al.

2013; Lee et al. 2016). Deficiency in GAG production

in A. fumigatus fails to produce ECM (the basis of

biofilm formations), indicating a critical role of GAG

in the maintenance of the ECM of A. fumigatus bio-

films (Gravelat et al. 2013; Bamford et al. 2015; Lee

et al. 2015). Therefore, the attenuated virulence of

GAG deficient A. fumigatus strains in the invasive

aspergillosis mouse model, explains the functional

role of this component in evading host defences

(Gravelat et al. 2013; Lee et al. 2015).

Galactofuranose-deficient mutants showed a higher

ability to form biofilms, as a result of enhanced pro-

duction of GAG, confirming the insignificance role of

galactomannan in biofilm formation (Lamarre et al.

2009; Gravelat et al. 2013; Lee et al. 2014). GAG pro-

duction is regulated by the developmental regulatory

proteins MedA (Gravelat et al. 2010; Al Abdallah et

al. 2012), StuA (Gravelat et al. 2013), and SomA (Lin

et al. 2015; Chen et al. 2020). The deletion of the

phosphatases SitA or PtcB through the activation of

cell wall integrity has been linked to the destruction

of biofilm formation and extracellular matrix produc-

tion (Bom et al. 2015; Winkelstr€

oter et al. 2015).

Additionally, A. fumigatus biofilm cells attached to

epithelial cells exhibited raised levels of ECM, with a

coincidental decreased sensitivity to antifungal drugs

(Seidler et al. 2008). Whereas the precise role of the

ECM is not completely understood, it is thought that

the matrix of Aspergillus biofilms contributes to the

higher resistance, by either slowing drug penetration

or direct drug sequestration (Beauvais et al. 2007;

Bugli et al. 2013; Rajendran et al. 2013; Rodrigues et

al. 2017). Moreover, the increased ECM in matured

A. fumigatus biofilm suggests it as a drug resistance

promoter during the later phases of biofilm growth

(Beauvais et al. 2007). Biofilm of A. niger has also

shown an increased thickness of ECM at the matur-

ation stage, proving ECM as the principal constituent

(Bandara et al. 2020).

Apart from exopolysaccharides, eDNA, which is

another key component of the ECM, also appears to

have an impact on drug resistance through providing

structural integrity (Rajendran et al. 2013). The

increased content of eDNA in matured A. fumigatus

biofilms (8 to 48 h) has been confirmed as a genomic

DNA (Rajendran et al. 2013). The association between

eDNA and chitinase activity is a probable conse-

quence of autolysis, conferring the structural support

for the development and maintenance of the com-

plex biofilm architecture (Rajendran et al. 2013).

Furthermore, degradation of eDNA by DNase destabil-

ises the biomass and the integrity of the biofilm and

renders biofilms to become more susceptible to both

amphotericin B and caspofungin (Shopova et al.

2013). However, the mechanisms responsible for the

secretion of DNA are unknown but the integrated

CRITICAL REVIEWS IN MICROBIOLOGY 13

DNA in the ECM of biofilms may develop a more

resistant structural biofilm.

6.4. Efflux pumps and ATP-binding-cassette

transporter proteins

The reduced susceptibility of fungal biofilm to drugs

could be the result of the increased activity of multi-

drug resistance (MDR) pumps, including ATP-binding-

cassette (ABC) and the major facilitator superfamily

(MFS). Frequent exposure to antifungal drugs in the

clinical settings, such as aspergilloma cases, may

enhance the levels of efflux pump expression, thereby

either contributing to or inducing clinical resistance

(Bueid et al. 2010). Genome analysis of A. fumigatus

revealed MDR pumps have been associated with

increased resistance to azoles (Ramage et al. 2012;

Muszkieta et al. 2013). In biofilm conditions, 140 efflux

pump genes, including the ABC transporters MDR1,

MDR2, and MDR4 were upregulated (Ramage et al.

2012; Muszkieta et al. 2013). The highest increase in

resistance in A. fumigatus mature biofilms correlated

with the maximum expression of the MDR4 family

(Ramage et al. et al. 2012; Muszkieta et al. 2013). In a

subcutaneous A. fumigatus biofilm-related mouse

model, the in vivo expression of MDR4 was upregulated

in response to voriconazole treatment (Rajendran et al.

2011). The MFS, azole resistance-related genes, and the

members of the MDR1 family have been significantly

upregulated in A. fumigatus biofilms (Ferreira et

al. 2006).

6.5. Heat shock proteins 90 (HSP90)

The molecular chaperone heat shock proteins 90

(Hsp90) has been implicated as a principal regulator of

biofilm dispersion and drug resistance (Robbins et al.

2011). Inhibition of Hsp90 by geldanamycin, Hsp90

inhibitor, led to the reduction of resistance of A. fumiga-

tus biofilms to echinocandins (Robbins et al. 2011).

Moreover, vorinostat (suberoylanilide hydroxamic acid,

SAHA), a novel histone deacetylase inhibitor, showed

synergistic effects with azoles, including itraconazole,

voriconazole, and posaconazole, against both biofilm

and planktonic cells of Aspergillus spp. (Tu et al. 2020).

The synergistic effect might be partly associated with

dampened expression of the efflux pump genes and

largely via the inhibition of Hsp90 (Tu et al. 2020).

Given that biofilm formation in C. albicans is associated

with an increased capacity against oxidative stress

(Seneviratne et al. 2008; Delattin et al. 2014), the

increased ROS scavenging capacity might be respon-

sible for increased resistance of biofilm to ROS inducer

antifungal agents. Therefore, more investigation on ROS

scavenging capacity in filamentous fungi and targeting

the oxidative defense system could be a good strategy

to combat resistant fungal biofilms.

6.6. Drug tolerance and persister cells

Persister cells or dormant variants are a subpopulation

of general cells inside a microbial population that are

highly tolerant to drugs (Lewis 2008,2010; Ramage et

al. 2012; Rosenberg et al. 2018). These cells are an

important mechanism of tolerance particularly in

chronic infections. The key feature of a persister cell is

to tolerate the disruptive effect of antifungal agents by

reducing its metabolism and cell division (Lewis 2010).

Since antifungal agents interfere with active metabolic

processes, dormant cells, which are not metabolising

substrates and dividing, are no longer active targets for

the antifungals (Lewis 2010). Within fungal biofilms,

Box 1. An overview of the substitute therapeutic strategies against resistant biofilms.

14 M. ROUDBARY ET AL.

persister cells can resist high concentrations of antifun-

gals by a phenotypic variation that enter dormancy

(Schumacher et al. 2009; Lafleur et al. 2010). As a result,

they are most likely contribute to the resistance and

recalcitrance of biofilm infections (Wuyts et al. 2018).

Candida albicans biofilms have been shown to produce

multidrug tolerant persisters (LaFleur et al. 2006).

Relative to wild-types, persisters are not mutants but

rather phenotypic variants (LaFleur et al. 2006).

Remarkably, C. albicans persisters have only been

observed in the biofilm but not in a planktonic state

(LaFleur et al. 2006). Identification of possible genes

and mechanisms involved in persister, particularly in

filamentous fungi, may uncover a way to design an

effective anti-biofilm therapy, like the conventional anti-

fungal combination with a compound inhibiting per-

sister formation or maintenance.

7. Concepts in anti-biofilm approaches

Given the resistance of fungal biofilm-related infections

towards conventional antifungals, alternative anti-bio-

film agents and strategies are urgently needed. Since

the MICs of sessile cells are often notably higher than

the MICs of planktonic cells (Mowat et al. 2007; Seidler

et al. 2008), the higher values of agents are required to

reach the MIC in vivo, which is not possible due to the

toxicity and side effects. Consequently, the treatment

with conventional drugs that are currently used may

only decrease the biofilms, but might not eliminate the

entire biofilm, resulting in chronic and persistent infec-

tions (Wuyts et al. 2018). Therefore, to overcome the

drug resistance of biofilm communities, alternative

approaches are required (Box 1). Biofilm-associated

infections can be treated in two manners; either by

inhibition of biofilm formation or by disruption of

established biofilms (Ahmad Khan et al. 2020; Mishra et

al. 2020).

Combination therapy is a novel and relevant antibio-

film strategy. It could be based on either antifungal

drug combinations or an antifungal with a non-antifun-

gal or a potentiator to accelerate the antifungal efficacy

(Livengood et al. 2020; Tits et al. 2020). Combination of

caspofungin with amphotericin B or voriconazole

against Aspergillus biofilms demonstrated a synergistic

effect with a significant reduction of the metabolic

activity of A. fumigatus biofilm, in comparison to caspo-

fungin, amphotericin B, or voriconazole alone at the

same concentrations (Liu et al. 2012). Furthermore, the

combination of vorinostat as a potentiator with azoles,

revealed a synergistic effect against both biofilm and

planktonic cells of Aspergillus spp., including A.

fumigatus,A. flavus,A. terreus (Tu et al. 2020). Besides,

some enzymes also showed potential roles in combin-

ation with conventional antifungal agents (Bugli et al.

2013; Lohse et al. 2020). For instance, A. fumigatus bio-

films exposed to alginate lyase, an enzyme degrading

the EPSs of biofilm, combined with amphotericin B,

indicated more susceptibility, proposing EPS-degrading

enzymes as a promising combination therapy to

improve the management of biofilm-associated infec-

tions (Bugli et al. 2013).

Apart from conventional antifungal agents, many

novel antifungals have already been described (Vahedi-

Shahandashti and Lass-Fl€

orl 2020), which could be

promising either alone or in combination. The antibio-

film activity of olorofim and voriconazole against

Lomentospora prolificans showed promising efficacy,

while amphotericin B and micafungin did not exhibit a

desirable antibiofilm activity (Kirchhoff et al. 2020),

which emphasises the importance of more investigation

on antibiofilm activity of novel agents.

Many studies have been focused on inhibiting fungal

adhesion to surfaces through a range of surface coating

manners, including coating with metal nanoparticles,

antimicrobials, and lock solutions (Cateau et al. 2008;

Redding et al. 2009; Barad et al. 2017; Paulone et al.

2017; Lagree et al. 2018; Naderi et al. 2019; Kitisin et al.

2020; Vera-Gonz

alez and Shukla 2020; Vladkova et al.

2020). For instance, in Candida spp., nanoparticles such

as silver, zinc oxide, and titanium dioxide, graphen

oxide (Asadi Shahi et al. 2019), and gold nanoparticles

(Rahimi et al. 2019) showed potent activity against fun-

gal biofilm by different mechanisms. These included

ROS production, lipid peroxidation, cell wall disruption,

and decreased gene expression profiles mainly involved

in biofilm formation (Haghighi et al. 2013; Lara et al.

2015; Barad et al. 2017; Nikoomanesh et al. 2019).

Additionally, synthesis, or function inhibition of GAG, as

adhesive features, in A. fumigatus, has been introduced

as a potential therapeutic target (Lee et al. 2016). This

adhesion activity demands deacetylation by the carbo-

hydrate esterase type 4 Agd3, which has a crucial role

in virulence and extracellular localisation, proposing

Agd3 to be a promising therapeutic target (Lee et

al. 2016).

Some research has highlighted the anti-biofilm effi-

cacy of natural products (Khan and Ahmad 2012; Sardi

et al. 2013; Roemer and Krysan 2014) and metabolites

(Klausmeyer et al. 2005; Estrela and Abraham 2016),

that could lead to the development of unique alterna-

tive therapeutics. Several structural parts of the biofilm

can be potential targets for the development of

enzyme-based therapies, such as matrix and eDNA

CRITICAL REVIEWS IN MICROBIOLOGY 15

(Martins et al. 2012; Rajendran et al. 2013). This is the

case of glycoside hydrolases Sph3 and Ega3, enzymes

involved in the GAG biosynthetic pathway, that have

been repurposed as anti-GAG therapeutics which can

disrupt biofilm (Bamford et al. 2019; Le Mauff et

al. 2019).

Furthermore, interrupting signalling pathways that

are involved in biofilm formation is also a compelling

strategy, such as QS signalling (Pierce and Lopez-Ribot

2013;Mishraetal.2020). Quorum-quenching molecules

(QQMs) or QS inhibitors (Sharma et al. 2015), are an

interesting alternative therapy. They typically interfere in

the process of QS, using three main tactics: (1) prevent-

ing QSMs production, (2) degrading QSMs, and (3) block-

ing the QSMs receptors. Currently, some QQMs have

been applied against bacterial and fungal (e.g. Candida

spp.), biofilm; for instance, thiazolidinedione-8, QQ-5,

and QQ-7 (Feldman et al. 2014; Weiland-Br€

auer et al.

2019). Further screening, either on new QQMs in natural

products libraries or QSMs secreted by other microor-

ganisms in polymicrobial communities (such as filament-

ous fungi), may play a QQ role against other species and

may provide more therapeutic options.

Additionally, probiotics with different features have

revealed a distinct horizon to combat biofilms. They pos-

sess less cytotoxicity than QS-inhibitors and less induc-

tion of selective pressure on resistant isolates than

conventional agents (Sadiq et al. 2019; Barzegari et al.

2020). Probiotics, particularly Lactobacillus spp., produce

different anti-biofilm metabolites, which have been

widely studied in bacteria, such as organic acids, perox-

ides, fatty acids, bacteriocins, and biosurfactants

(Shokouhfard et al. 2015; Vahedi Shahandashti et al.

2016; Barzegari et al. 2020). Some studies have shown

the beneficial effects of probiotics, either cells or cell-free

supernatants, on fungal biofilm, mostly C. albicans (Vilela

et al. 2015; Matsubara et al. 2016;Tanetal.2017;



Cern

akov

aetal.2019) and bacterial-fungal polymicrobial

biofilms (Hager et al. 2019). However, the influence of

probiotics on filamentous fungal biofilm remains

unknown, which implies opening new research paths to

provide more treatment or/and prevention strategies.

Another plausible alternative therapy is photo-

dynamic therapy (PDT), which has been applied in bac-

teria and Candida spp.. Antimicrobial PDT uses different

photosensitizers or non-toxic dyes, various light sour-

ces, and wavelengths, which produce ROS and attack

proteins, lipids, and nucleic acids present within the

biofilm matrix (Plaetzer et al. 2009; Hu et al. 2018). In

Candida spp. biofilms, antimicrobial PDT showed prom-

ising results, featuring the potential use of this method

in other fungal biofilm eradication (Lopes et al. 2014).

PDT might also be suitable to be adopted for filament-

ous fungal biofilms eradication (Biel and Gomer 2010;

Hu et al. 2018; Sharma et al. 2019).

Finally, the CRISPR-based tool is one of the most

powerful methods in underlying genetic interactions

determination (Shapiro et al. 2018; Sharma et al. 2019).

In C. albicans, single or double deletions of 12 adhesin

genes by the CRISPR system, led to a combination

adhesin mutant with 144 single- and double-knockout

(Shapiro et al. 2018). A combined adhesin mutant dem-

onstrated a set of adhesins that can be targeted in bio-

film impairment, highlighting the contribution of

CRISPR in the feasibility of generating a combination of

mutations. The potential applications of CRISPR-based

methods look promising in uncovering the distinctive

pathways and genes involved in filamentous fun-

gal biofilm.

8. Perspective

The biofilm formation of saprophytic and pathogenic

fungi is a worldwide health concern. Invasive fungal

infections directly linked to biofilms are associated with

severe clinical conditions, and their treatment remains

incredibly challenging due to the high resistance to the

available antifungals. Despite recent progress in the

understanding of biofilms formed by bacteria and

yeasts, a comprehensive understanding of the molecu-

lar mechanisms and crucial components involved in

establishing filamentous fungal biofilm infections is still

required. Because of the distinctive biofilm growth and

planktonic growth, developing biofilm-based treatment

alternatives to manage or eradicate these microbial

communities seems an urgent necessity. Moreover,

future studies are needed to understand the scale of

biofilm formation and its clinical impact by patho-

genic fungi.

Acknowledgments

C.F.R. would like to acknowledge the UID/EQU/00511/2020

Project—Laboratory of Process Engineering, Environment,

Biotechnology and Energy (LEPABE), financed by national

funds through FCT/MCTES (PIDDAC). The authors warmly

thank Azin Kheirkhah for the scientific linguistic revision.

Author contributions

Maryam Roudbary contributed to data curation, the review

design and writing. Roya Vahedi-Shahandashti contributed to

review design, writing, Figure preparation and editing. Andr

Luis Souza dos Santos contributed to interpretation/selection

and editing. Shahla Roudbar Mohammadi and Peyman Aslani

contributed to review draft and revision. Cornelia Lass-Fl€

orl

16 M. ROUDBARY ET AL.

and C

elia F. Rodrigues contributed to the review design,

editing and supervision. They are all accountable for their

own contributions and ensure the accuracy of any part of

the work.

Disclosure statement

The authors declare no conflict of interest.

ORCID

C

elia F. Rodrigues http://orcid.org/0000-0001-8633-2230

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CRITICAL REVIEWS IN MICROBIOLOGY 25

Biofilm formation in Zymoseptoria tritici

Preprint

Full-text available

Jul 2023

Zymoseptoria tritici is an economically damaging fungal pathogen of wheat which is able to survive for long periods on the leaf surface. In this environment, the fungus is exposed to many stresses, including fluctuations in temperature, UV radiation, drying, and foliar fungicide applications. We observed biofilm-like cell aggregations on the surface of wheat leaves infected with Z. tritici in both compatible and non-compatible interactions. A literature search revealed few examples of plant pathogenic fungi forming biofilms, but demonstrated that such biofilms have a number of key characteristics, which are shared with other biofilm forming microbes. These include adhesion of cells to the substrate, production of extracellular matrix, altered gene expression and increased tolerance to various stresses. We used a combination of microscopy, qRT-PCR and stress tolerance assays to determine whether putative Z. tritici biofilms possessed these diagnostic characteristics. We show that Z. tritici biofilms resemble in form and function those formed by other filamentous and dimorphic fungi, producing extra-cellular matrix in which cells become embedded, creating layers of hyphal bundles and blastospores. These biofilms show increased tolerance to drying and high temperature. There is also isolate-dependent resistance to reactive oxygen stress and the fungicide carboxin. Taken together, these findings demonstrate that Z. tritici is indeed able to produce biofilms.

Effects of Coleus amboinicus L. Essential Oil and Ethanolic Extracts on Planktonic Cells and Biofilm Formation of Microsporum canis Isolated from Feline Dermatophytosis

Article

Full-text available

Dec 2022

Microsporum canis is an important zoonotic fungus that causes dermatophytosis in domestic animals and their owners. Domestic cats are the primary reservoir for M. canis. Antifungal drugs frequently produce adverse effects on the host animal, increasing the demand for novel alternative treatments derived from nature. We evaluated the antifungal activity of Coleus amboinicus essential oil (CEO) and ethanolic extracts (CEE) against M. canis in planktonic and biofilm growth. Twelve clinical isolates of M. canis were identified in feline dermatophyte samples. Using GC-MS, 18 compounds were identified in CEO, with carvacrol being the major constituent. HPLC analysis of CEE revealed that it contained rosmarinic acid, apigenin, and caffeic acid. The planktonic growth of all M. canis isolates was inhibited by C. amboinicus extracts. The minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC50) was 128 µg/mL (32–256 µg/mL) for both CEO and CEE. The MIC90 values of CEO and CEE were 128 and 256 µg/mL, respectively. CEO at MIC (128 µg/mL) and 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of weak, moderate, and strong biofilm-producing M. canis. CEE at 2× MIC (256 µg/mL) significantly inhibited the biofilm formation of all isolates. Overall, C. amboinicus extracts inhibited planktonic growth and exhibited a significant antibiofilm effect against M. canis. Thus, C. amboinicus is a potential source of natural antifungal compounds.

Mixed Fungal Biofilms: From Mycobiota to Devices, a New Challenge on Clinical Practice

Article

Full-text available

Aug 2022

Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that allow the evaluation of fungal morphology and the identification of the etiologic agent of mycosis. Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that enable the examination of the fungi for further identification of the etiological agent of the mycosis. The isolation of fungi from pure cultures is typically recommended, as when more than one species is identified, the second agent is considered a contaminant. Fungi mostly survive in highly organized communities that provoke changes in phenotypic profile, increase resistance to antifungals and environmental stresses, and facilitate evasion from the immune system. Mixed fungal biofilms (MFB) harbor more than one fungal species, wherein exchange can occur that potentialize the effects of these virulence factors. However, little is known about MFB and their role in infectious processes, particularly in terms of how each species may synergistically contribute to the pathogenesis. Here, we review fungi present in MFB that are commensals of the human body, forming the mycobiota, and how their participation in MFB affects the maintenance of homeostasis. In addition, we discuss how MFB are formed on both biotic and abiotic surfaces, thus being a significant reservoir of microorganisms that have already been associated in infectious processes of high morbidity and mortality.

Exploring the Potential of Farnesol as a Novel Antifungal Drug and Related Challenges

Article

Full-text available

Feb 2024
Curr Infect Dis Rep

Purpose of Review Farnesol (FOH) is a quorum-sensing molecule with potential as an antifungal drug. Given the growing concern about fungal drug resistance, exploring new solutions is crucial. Therefore, summarizing the antifungal activity of FOH is expected to be the basis for further FOH research and application. Herein, we reviewed the in vitro and in vivo antifungal efficacy of FOH alone and in combination with conventional antifungal drugs, as well as its mechanisms of action. Furthermore, we discussed the prospects and challenges of the FOH application in detail. Recent Findings Recent studies have revealed that FOH can target various aspects, such as reactive oxygen species production, induction of apoptosis, and modulation of virulence factors, to inhibit fungal growth and reduce fungal pathogenicity, thereby exerting its antifungal activity. Furthermore, FOH can suppress resistance-associated genes, such as those of biofilm and ergosterol, so as to enhance the fungicidal effectiveness of conventional antifungal drugs. However, the action mechanism of FOH on drug efflux pumps remains unclear and warrants further investigation. Summary FOH can prevent and treat fungal infections. It exerts significant antimicrobial effects on fungal planktonic and biofilm cells, enhances the antimicrobial efficacy of conventional antifungal drugs, and reverses and reduces fungal drug resistance. However, further in vitro and in vivo studies are needed to assess the safety of FOH due to potential adverse effects on immune cells and other factors.

Antifungal Activity of Chitosan against Histoplasma capsulatum in Planktonic and Biofilm Forms: A Therapeutic Strategy in the Future?

Article

Full-text available

Dec 2023
J. Fungi

Histoplasmosis is a respiratory disease caused by Histoplasma capsulatum, a dimorphic fungus, with high mortality and morbidity rates, especially in immunocompromised patients. Considering the small existing therapeutic arsenal, new treatment approaches are still required. Chitosan, a linear polysaccharide obtained from partial chitin deacetylation, has anti-inflammatory, antimicrobial, biocompatibility, biodegradability, and non-toxicity properties. Chitosan with different deacetylation degrees and molecular weights has been explored as a potential agent against fungal pathogens. In this study, the chitosan antifungal activity against H. capsulatum was evaluated using the broth microdilution assay, obtaining minimum inhibitory concentrations (MIC) ranging from 32 to 128 µg/mL in the filamentous phase and 8 to 64 µg/mL in the yeast phase. Chitosan combined with classical antifungal drugs showed a synergic effect, reducing chitosan’s MICs by 32 times, demonstrating that there were no antagonistic interactions relating to any of the strains tested. A synergism between chitosan and amphotericin B or itraconazole was detected in the yeast-like form for all strains tested. For H. capsulatum biofilms, chitosan reduced biomass and metabolic activity by about 40% at 512 µg/mL. In conclusion, studying chitosan as a therapeutic strategy against Histoplasma capsulatum is promising, mainly considering its numerous possible applications, including its combination with other compounds.

Berberine inhibits Candida albicans growth by disrupting mitochondrial function through the reduction of iron absorption

Article

Nov 2023
J APPL MICROBIOL

Aims This study aimed to investigate whether berberine (BBR) can inhibit the iron reduction mechanism of Candida albicans, lowering the iron uptake of the yeast and perhaps having antimicrobial effects. Methods and results We determined that BBR may cause extensive transcriptional remodeling in C. albicans and that iron permease Ftr1 played a crucial role in this process through eukaryotic transcriptome sequencing. Mechanistic research showed that BBR might selectively inhibit the iron reduction pathway to lower the uptake of exogenous iron ions, inhibiting C. albicans from growing and metabolizing. Subsequent research revealed that BBR caused significant mitochondrial dysfunction, which triggered the process of mitochondrial autophagy. Moreover, we discovered that C. albicans redox homeostasis, susceptibility to antifungal drugs, and hyphal growth are all impacted by the suppression of this mechanism by BBR. Conclusions The iron reduction mechanism in C. albicans is disrupted by BBR, which disrupts mitochondrial function and inhibits fungal growth. These findings highlight the potential promise of BBR in antifungal applications.

Amino sugars influence Aspergillus fumigatus cell wall polysaccharide biosynthesis, and biofilm formation through interfering galactosaminogalactan deacetylation

Article

Oct 2023

Antifungal therapy: Novel drug delivery strategies driven by new targets

Article

Jun 2023
ADV DRUG DELIVER REV

In patients with compromised immunity, invasive fungal infections represent a significant cause of mortality. Given the limited availability and drawbacks of existing first-line antifungal drugs, there is a growing interest in exploring novel targets that could facilitate the development of new antifungal agents or enhance the effectiveness of conventional ones. While previous studies have extensively summarized new antifungal targets inherent in fungi for drug development purposes, the exploration of potential targets for novel antifungal drug delivery strategies has received less attention. In this review, we provide an overview of recent advancements in new antifungal drug delivery strategies that leverage novel targets, including those located in the physio-pathological barrier at the site of infection, the infection microenvironment, fungal-host interactions, and the fungal pathogen itself. The objective is to enhance therapeutic efficacy and mitigate toxic effects in fungal infections, particularly in challenging cases such as refractory, recurrent, and drug-resistant invasive fungal infections. We also discuss the current challenges and future prospects associated with target-driven antifungal drug delivery strategies, offering important insights into the clinical implementation of these innovative approaches.

Detection of microscopic filamentous fungal biofilms – Choosing the suitable methodology

Article

Jan 2023
J MICROBIOL METH

Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi.

Biofilm Formation by Chromoblastomycosis Fungi Fonsecaea pedrosoi and Phialophora verrucosa: Involvement with Antifungal Resistance

Article

Full-text available

Sep 2022
J. Fungi

Patients with chromoblastomycosis (CBM) suffer chronic tissue lesions that are hard to treat. Considering that biofilm is the main growth lifestyle of several pathogens and it is involved with both virulence and resistance to antimicrobial drugs, we have investigated the ability of CBM fungi to produce this complex, organized and multicellular structure. Fonsecaea pedrosoi and Phialophora verrucosa conidial cells were able to adhere on a polystyrene abiotic substrate, differentiate into hyphae and produce a robust viable biomass containing extracellular matrix. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed the tridimensional architecture of the mature biofilms, revealing a dense network of interconnected hyphae, inner channels and amorphous extracellular polymeric material. Interestingly, the co-culture of each fungus with THP-1 macrophage cells, used as a biotic substrate, induced the formation of a mycelial trap covering and damaging the macrophages. In addition, the biofilm-forming cells of F. pedrosoi and P. verrucosa were more resistant to the conventional antifungal drugs than the planktonic-growing conidial cells. The efflux pump activities of P. verrucosa and F. pedrosoi biofilms were significantly higher than those measured in conidia. Taken together, the data pointed out the biofilm formation by CBM fungi and brought up a discussion of the relevance of studies about their antifungal resistance mechanisms.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms

Article

Full-text available

May 2021

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008–0.03 mg/L), and fluconazole (FLZ) (1–512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2–7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

Combination Therapy to Treat Fungal Biofilm-Based Infections

Article

Full-text available

Nov 2020
INT J MOL SCI

An increasing number of people is affected by fungal biofilm-based infections, which are resistant to the majority of currently-used antifungal drugs. Such infections are often caused by species from the genera Candida, Aspergillus or Cryptococcus. Only a few antifungal drugs, including echinocandins and liposomal formulations of amphotericin B, are available to treat such biofilm-based fungal infections. This review discusses combination therapy as a novel antibiofilm strategy. More specifically, in vitro methods to discover new antibiofilm combinations will be discussed. Furthermore, an overview of the main modes of action of promising antibiofilm combination treatments will be provided as this knowledge may facilitate the optimization of existing antibiofilm combinations or the development of new ones with a similar mode of action.

The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus

Article

Full-text available

Nov 2020

The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking β-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus . We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

Drug-Resistant Fungi: An Emerging Challenge Threatening Our Limited Antifungal Armamentarium

Article

Full-text available

Dec 2020

The high clinical mortality and economic burden posed by invasive fungal infections (IFIs), along with significant agricultural crop loss caused by various fungal species, has resulted in the widespread use of antifungal agents. Selective drug pressure, fungal attributes, and host- and drug-related factors have counteracted the efficacy of the limited systemic antifungal drugs and changed the epidemiological landscape of IFIs. Species belonging to Candida, Aspergillus, Cryptococcus, and Pneumocystis are among the fungal species showing notable rates of antifungal resistance. Drug-resistant fungi from the environment are increasingly identified in clinical settings. Furthermore, we have a limited understanding about drug class-specific resistance mechanisms in emerging Candida species. Establishment of antifungal stewardship programs in both clinical and agricultural fields and inclusion of species identification, antifungal susceptibility testing, and therapeutic drug monitoring practices in the clinic can minimize the emergence of drug-resistant fungi. New antifungal drugs featuring promising therapeutic profiles have great promise to treat drug-resistant fungi in the clinical setting. Mitigating antifungal tolerance, a prelude to the emergence of resistance, also requires development of effective and fungal-specific adjuvants to be used in combination with systemic antifungals.

Natural Anti-biofilm Agents:strategies to control Biofilm forming pathogens.

Article

Full-text available

Oct 2020

Pathogenic microorganisms and their chronic pathogenicity are significant concerns in biomedical research. Biofilm-linked persistent infections are not easy to treat due to resident multidrug-resistant microbes. Low efficiency of various treatments and in vivo toxicity of available antibiotics drive the researchers toward the discovery of many effective natural anti-biofilm agents. Natural extracts and natural product-based anti-biofilm agents are more efficient than the chemically synthesized counterparts with lesser side effects. The present review primarily focuses on various natural anti-biofilm agents, i.e., phytochemicals, biosurfactants, antimicrobial peptides, and microbial enzymes along with their sources, mechanism of action via interfering in the quorum-sensing pathways, disruption of extracellular polymeric substance, adhesion mechanism, and their inhibitory concentrations existing in literature so far. This study provides a better understanding that a particular natural anti-biofilm molecule exhibits a different mode of actions and biofilm inhibitory activity against more than one pathogenic species. This information can be exploited further to improve the therapeutic strategy by a combination of more than one natural anti-biofilm compounds from diverse sources.

Natural Anti-biofilm Agents: Strategies to Control Biofilm-Forming Pathogens

Article

Full-text available

Oct 2020

Satpal Singh Bisht

Fungal oxylipins direct programmed developmental switches in filamentous fungi

Article

Full-text available

Oct 2020

Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

Dynamics of Mono-and Dual-Species Biofilm Formation and Interactions Between Paracoccidioides brasiliensis and Candida albicans

Article

Full-text available

Oct 2020

The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono-and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic-and confocal laser scanning-microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans coexistence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections.

Inhibitory Effects of Antifungal Drugs on Biofilm Producing Aspergillus spp. Recovered from Drinking Water System

Article

May 2020

Aims: Biofilms formed in drinking water distribution systems serve as a continuous source of fungal infections. Biofilms are thick aggregates of adherent microorganisms including pathogenic species of fungi. Respiratory diseases and skin allergy reactions are caused by drinking water containing biofilm forming fungus and bacteria. One of the main causes of nosocomial infections and respiratory diseases in hospitals is due to the fungal biofilm formation in machines, catheters and other surgical instruments. There are some antifungal drugs which are used to control biofilm formation to minimize the infection rate. Methodology and results: The present study was conducted to isolate and identify Aspergillus species which are the main fungal spp. responsible for the biofilm formation in drinking water and to check their antifungal susceptibility against antifungal drugs. The isolated fungal samples from drinking water were cultivated on Potato dextrose agar for the isolation of Aspergillus species. Isolated Aspergillus species were identified on the basis of cultural, morphological and microscopic examination. Then in-vitro ability of biofilm produced by isolated Aspergillus species was estimated using microtitre plate method and quantification by crystal violet assay. Antifungal susceptibility testing against isolated fungal spp. was done by antifungal drug Amphotericin B. Results: From results, it is concluded that drinking water of labs, hospitals and common water chillers were more prevelant by Aspergillus species whereas water from reverse osmosis plants showed negative results. From microtitre plate method and crystal violet assay, it was concluded that Aspergillus spp. are Susceptible against Amphotericin B drug as compared to miconazole. Keywords: Aspergillus spp; Biofilm; Drinking water; Disk diffusion method; Amphotericin B

Surface engineered biomaterials and ureteral stents inhibiting biofilm formation and encrustation

Article

Dec 2020
SURF COAT TECH

Protection of ureteral stents against infections is a significant current challenge raised by increasing the number of stentings and infections, microbial resistance to conventional antibiotics and multi-drugs treatments, side effects for the patients like damage of surrounding tissue and high morbidity. Surface engineered biomaterials and ureteral stents, reducing biofilm formation and encrustation would contribute to mitigation of the problem. The aim of this review is to present the progress during the last 5 years in the development of surface engineered antimicrobial biomaterials and stents with expectation to raise some new fruitful ideas in this direction. Strategies aimed at preventing, disrupting, or removing adherent microbes and biofilms from biomaterials and ureteral stents are its main subject. Various antibacterial agents, modifications, and coatings as well as preparation, deposition and characterization techniques are among the topics covered. A brief market analysis is also included covering the significance of ureteral stents associated infections and a mode of development of a biofilm. The review of the progress during the last 5 years shows a continuing interest in surface modification and coating employing three principal anti-biofilm strategies: 1) mechanical detachment; 2) killing microbial cells and 3) creation of low-adhesive surfaces. The known surface engineering solutions report a reduced, to some extent, biofilm formation and encrustation of biomaterials and ureteral stents, but none of them is able to totally stop their development. Some new trends are observed, such as complementary antimicrobial protection by coating and flow dynamic; biodegradable coatings releasing antimicrobial agent; quasi irremovable surface coatings, delivering drug or antimicrobial agent as well as new carbon and biodegradable materials; bacteriophages and phage cocktails, etc. Almost all of them are under intense in vitro studies; only few of them were studied in vivo animal models and none in humans. Some of the liquid infused coatings, already tested in animal models, seem to be the closest to clinical application but so far no one has been applied to ureteral stents. This review outlines some future research directions and major challenges in the surface engineered biomaterials and ureteral stents. It gives ideas how, by surface engineering, to approach more closely the “clean” ureteral stent to not allow microbial adhesion and encrustation and thus sharply reduce the ureteral stents associated infections and stent dysfunction. The most important prerequisite of the non-toxic “clean” surface is to be low adhesive. Its anti-biofilm performance could be improved by including bio-surfactant and/or inhibitor of quorum sensing (QS) and/or inhibitor of the crosslinking of the exopolymeric substances (EPSs). The key to identifying a “universal” surface that would repel/release all microbial cells is maybe hidden in the in-depth understanding of the mechanism of the initial reversible adsorption of the EPSs secreted by microbial cells, since it initiates the whole biological cascade of biofilm development.

Biofilm formation in clinically relevant filamentous fungi: a therapeutic challenge

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