To cause disease, the infectious agent makes use of both invasiveness factors—the pathogen virulence factors—and the ability to resist and evade the host immune system. The success of the infection process is the result of a complex equation involving pathogen interaction with the host, wherein the expression of several virulence factors (and not just one or the other) will favor the establishment of the pathogen in the host. Fungal pathogens are frequently associated with biofilm formation.
- Fungal biofilms
- Extracellular matrix
- Fungal Infection
Since the seventeenth century, biofilms have been described in multiple systems. Most bacteria preferentially grow as biofilms, in all self-sustaining aquatic ecosystems, and these sessile bacterial cells differ deeply from their planktonic counterparts (cells in suspension) . The definitions of biofilm have evolved over the years, in parallel to the advances of the biology area and research studies on the subject. The definition used today was proposed by Donlan and Costerton in 2002, and it describes a biofilm as a microbial community in which the cells are connected to a substrate, or to each other, embedded in a extracellular matrix of polymeric substances (produced by themselves) and exhibit an altered phenotype regarding the rate of growth and transcription of genes .
In fungi, the ability to colonize surfaces and to form biofilms was initially demonstrated for
Until now, several superficial reports about the ability of a wide range of fungal species to form biofilms
Candidemia and other forms of invasive candidiasis (i.e., infection involving normally sterile sites) are the most prevalent invasive mycoses worldwide [20, 26] with mortality rates close to 40% [27, 28].
The mature biofilm consists of a dense network of cells in the form of yeasts, hyphae and pseudohyphae (Figure 2A) soaked by polymeric extracellular matrix and with water channels between the cells, which facilitate the diffusion of nutrients from the environment through the biomass to the lower layers and which also allow the elimination of waste [43–45].
Biofilms of CNA species are less complex in structure because true-hyphae is not present, culminating in a biofilm formed predominantly by yeasts (
Following the first report in 2007, several studies demonstrated that
Despite the lack of clinical studies substantiating
4. Biofilms of other medically important fungi
Cryptococcosis, caused by yeasts of the genus
The main pathogenic species to humans are
Invasive infections caused by
5. Biofilm resistance behavior
Currently, antifungal therapy is based on four major classes of antifungal drugs: the polyene agents, azoles, allylamines and echinocandins. However, the therapeutic arsenal is limited by several problems, including selectivity, toxicity and development of resistance. Considering invasive mycoses, options are even more restricting, comprising amphotericin B, fluconazole (with several restrictions), itraconazole and voriconazole being the most suitable drugs. Although amphotericin B is considered to be the gold standard drug for these infections, its high degree of hepatotoxicity and nephrotoxicity  may turn it unacceptable for most patients predisposed to invasive fungal infections. Furthermore, some
Fungal infections associated with biofilm formation are often poorly susceptible or even refractory to conventional antifungal therapies, which implies the need for higher dosages—not always possible, as discussed above—or antifungal combination therapy for better penetration of drugs in biofilms. The ineffectiveness of the azole antifungals and classical formulations of amphotericin B (deoxycholate) against biofilms of
6. Biofilm mechanisms of resistance
According to the definition of a biofilm, the cells that compose this structure have an altered phenotype and differ from the planktonic cells (free-floating cells) in the expression of genes, rate of growth and also in its susceptibility to antifungal agents. The increased resistance to antifungals in
7. Cell density
The biofilm architecture is highly ordered to allow the infusion of nutrients and waste expulsion. Mature biofilms, even having high cell density, exhibit spatial heterogeneity with microcolonies and water channels, common feature of both biofilm bacteria and fungi . It has been shown that both planktonic cells and cells resuspended from biofilms exhibit sensitivity to azoles when the cell density is low (103 cells/ml) and became more resistant when cell density is increased ten-fold . It is believed, therefore, that the cell density is an important resistance factor within complex biofilms, particularly to azoles.
8. Drug target alteration
The antifungal agents of the azole class, including fluconazole, itraconazole, voriconazole and posaconazole, act by inhibiting sterol 14-α-demethylase enzyme encoded by ERG11 gene. The main target of azoles, Erg11p protein, can develop point mutations or be overexpressed, reducing the drug activity and culminating in an ineffective treatment. Treatment of
9. Drug efflux pumps expression
The primary molecular mechanism leading to resistance to the azoles, in
The increased expression of genes encoding drug efflux pumps has been reported in
10. Role of the extracellular matrix of the biofilm in resistance
In most biofilms, the population of microorganisms corresponds to 10% of the total mass and the extracellular matrix (ECM) corresponds to 90%. The ECM is a key biofilm component, which exerts a physical barrier function, protecting the cells from environmental factors such as host immunity and antifungal agents . In 2004, Al-Fattani and Douglas demonstrated that, although the diffusion of small molecules can be hampered by the presence of a dense ECM, reducing the penetration of antifungal drugs does not play a key role in biofilm resistance . Recent studies have provided new insights suggesting that the chemical composition of the ECM and its regulation may play the central role in resistance.
The overall composition of the ECM of
The contribution of the β-1,3-glucan for the biofilm resistance in
In addition to polysaccharides, the extracellular DNA (eDNA) present in ECM of
Other than physical components, transcription factors that regulate glucan synthesis and hydrolases are also associated with biofilm resistance. The CaZAP1 transcription factor is a negative regulator of the release of soluble β-1,3-glucan for the ECM in
The ability to form
11. Persister cells
Persister cells are an important mechanism of tolerance in chronic infections and recently have received special attention in fungi biofilms . By definition, these cells are “dormant variants of regular cells inside a microbial population that are highly tolerant to antibiotics” . The main disruptive effect of antifungal agents in the cells relates to its interference with metabolic processes (synthesis of cell membrane, cell wall or DNA). The main characteristic of a “dormant” or “persister” cell is the reduction of its metabolism and cell division. So, because they are not metabolizing substrates and not dividing, these cells are no longer a target for the antifungal and become tolerant to its presence . The presence of persister cells has been demonstrated in biofilms of
In summary, the major studies published to date that attempt to elucidate the main factors involved in antifungal resistance of biofilms were performed with
12. Biofilm and pathogenesis
Pathogenesis involves the interaction between the pathogen and the host. To cause disease, the infectious agent makes use of both invasiveness factors—the pathogen virulence factors—and the ability to resist and evade the host immune system. Often these two topics communicate, mainly because the molecules and metabolic adaptations produced by the pathogen to escape the immune response are considered as virulence factors.
The ability to grow as a biofilm cannot be considered a
The relationship between biofilm and pathogenicity relies mainly on two unique features of this community life-style: its increased resistance and the dispersion of infectious cells. Biofilms are a natural survival strategy of microorganisms to resist environmental threats . In the clinical setting, the encased highly dense colony of fungal cells is protected not only from antifungal penetration, as discussed above, but also from the immune system. A single yeast or hyphae cell can be recognized and eliminated by the innate immunological response, either via phagocytosis by macrophages or induction of apoptosis by degranulation of mast cells. However, biofilms are too big to be phagocytosed and, yet, ECM may impair recognition of fungal surface epitopes. Thus, biofilm formation may also contribute to the escape from the host immunological response, favoring the establishment of the infection.
Cells that are released from mature biofilms are called “dispersion cells” and may colonize adjacent surfaces, expanding the biofilm or, in a clinically relevant scenario, use the bloodstream to disseminate the infection and allow the colonization of deep organs . Additionally,
Recently, a prospective analysis of patients with
13. Final considerations
The ability to form biofilms is widespread among pathogenic fungi, but understanding of the mechanisms that govern their formation, physiology and drug resistance is still limited. The continuous development of knowledge of the molecular mechanisms underlying biofilm formation, maintenance and molecular basis of metabolic dormancy of subpopulations of cells, such as persister cells, could lead to a drug-based strategy that could help us solve clinical diseases associated with fungal biofilms.
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