Candida Biofilms: Environmental and Clinical Aspects

Candida spp. strains are characterized by their ability to form a biofilm structure on biotic and abiotic surfaces, causing significant problems in many industrial branches and threatening human health. Candida biofilm is a heterogeneous, spatially well-organized structure consisting of planktonic and mycelial yeast forms which are interdependent in the quorum sensing system and surrounded by an extracellular polysaccharide substance. Biofilm-forming microorganisms are characterized by high invasiveness, the ability to cause dangerous and difficult to treat infections. Furthermore, the cells in the biofilm, compared to planktonic forms, show reduced sensitivity to chemical compounds with antifungal activity and increased survival under unfavorable environmental conditions. The chapter focuses on the emergence of antifungal resistance with the development of biofilms. The work presents the examples of antibiotic resistance of a variety of Candida, showing that a group of strains expressing intermediate sensitivity or resistance to the tested antibiotics include both clinical and food-borne isolates. Similarities in enzymatic and biochemical profiles of different origin isolates are discussed. A substantial heterogeneity within Candida albicans group is also underlined. Simultaneously, the incidents of biochemical profiles conformity of some clinical and food-borne isolates are presented, which may be a result of Candida transmission via food.


Introduction
Unicellular forms of yeast are rarely found in nature as single, scattered cells, in the form of plankton but they are rather adsorpt at the solid-liquid, liquid-gas, or liquid-liquid interface. Generally, they form organized, settled structures taking the form of multicellular clusters forming biofilm. Biofilm, also called as the biological membrane, is a complex, multicellular, dangerous, if the development of infection is accompanied by a dysfunction of the device colonized by biofilm.

Biofilm definition
Biofilm is defined as a well-organized, three-dimensional social structure surrounded by extracellular matrix and irreversibly bound to the surface, built by microorganisms with altered, with respect to planktonic form, genotype properties [5,6,[8][9][10]. Biofilm enables microorganisms to survive in a changing and unfavorable environment, and therefore is the dominant form of their existence in the nature. It is characterized by structural heterogeneity, genetic diversity, complexity of interaction, and the presence of extracellular substances. It can be either mono-or multilayer, produced by one species or many different species. The biofilm structure depends on many factors such as hydrodynamic conditions, surface type, pH of the environment, microbial mobility, intercellular communication, nutrient content, exopolysaccharides, proteins, or oxygen. Colonization of various surfaces by microorganisms is possible due to their adhesive properties and extracellular polymeric substances (EPS) stabilizing the biofilm structure. Adjacent microorganisms, in a spatially organized structure, produce a common layer of polymeric substances called extracellular matrix, the complex compounds playing an important role in the formation and functioning of the biofilm. Most EPS polysaccharides are the organic compounds with long linear or branched molecules of 106 Da. The amount of polymers depends on the quantitative and qualitative composition of nutrients. The percentage of water in the biofilm matrix is up to 97%. Polymers ability to cyclical accumulation simultaneously with donation of water gives the matrix hydrogel features with exceptional viscoelastic properties [2,[11][12][13][14]. Matrix hydrogel nature effectively protects biofilm microorganisms from desiccation and provides the cells with protection against environmental stress factors such as UV radiation, temperature shifts, pH fluctuations, or toxic substances [2,5,7]. The matrix serves also as a communication system between biofilm cells, where chemical and physical signals are transmitted through a branched open channel system separating individual microcolonies. Thanks to the channel network, oxygen and nutrients are delivered through the channels and the excreted waste products are discharged. Cells in biofilms are present in various metabolic states. On the periphery of the biofilm, where the channel network system is more developed, the cells are large, metabolically active, and its reproducing increases the biofilm thickness. While, microorganisms located inside the biofilm are partially cut off from the water system, which results in their growth rate decreasing. They may also fall in an anabiosis with possible activation in a case of destruction of the outer cell layer, which, no matter how long the biofilm works, uses the features of young biofilm cells [2,12,13]. The biofilm cell has different characteristics than the planktonic cells. An important determinant of biofilm properties is quorum sensing, a specific communication system, strictly controlled by specific genes in response to the abundance of cells in the biofilm-the sense of the piston. The ability of cells to communicate makes the biofilm able to function in a way that resembles an almost one multicellular organism consisting of physiologically diverse subpopulations of microbial cells [2,4,5,12,13,15].

Biofilm structure
The process of biofilm formation is multistage and depends on the properties of the microorganisms, the construction, and properties of the colonized materials or the host. There are four basic phases: (I) reversible adhesion, (II) irreversible adhesion, (III) biofilm maturation, and (IV) dispersion (Figure 1).
Biofilm formation begins with the adhesion of free floating microorganisms to the biotic or abiotic surface. Reversible adhesion is the result of relatively weak physical interactions causing the first cells to attach to a solid surface such as gravitational interaction, electromagnetic surface charge, van der Waals forces, electrostatic, and hydro-and thermodynamic forces (Brownian motion). These forces play a crucial role when the distance between cells and the surface is relatively large. Biofilm is unstable and can easily be removed by both chemical and physical methods. When the cell distance from the surface is less than 1.5 nm, there is irreversible adhesion due to the formation of specific bonds. First microbial cells attached to the surface help attaching another one by the formation of hydrophobic, non-specific or specific hydrogen bonds, and pairs and ionic complexes (carbon-carbon covalent bonds) [2,5,12,13]. An important place in the biofilm-building process is the interaction of specific receptors, adhesives, and ligands on the cell surface of the microorganism or the target host cell extracellular ligand. Initially, the surfaces are covered by a single layer of microbial cells. In the construction of the basic EPS matrix, which gives the biofilm a defined shape and structure, the increased synthesis and secretion of extracellular biopolymers is important. Biofilms expand by increasing the intensity of cell proliferation. While, glycocalyx, a shell composed of polysaccharide residues of glycolipids and glycoproteins, the components of the cell membrane, is produced up to the total surroundings of the microcolonies. At this stage, biofilm, in addition to living microorganisms, also includes dead cells, mineral substances, and organic compounds. These elements are joined by further microbial cells. Irreversible adhesion allows the formation of microcolonies and biofilm maturation [5,12,13]. Biofilm maturation is followed by the microorganisms' reproduction, their gradual differentiation and the activation or inhibition of expression of certain genes. Biofilm cells acquire features that are not expressed by planktonic cells and can transmit them to adjacent and progeny cells. When reaching the critical thickness of the biofilm membrane, cells migrate from peripheral parts of the mature biofilm to the surrounding environment and the process of colonization begins. Disconnecting cells from biofilm and its dispersion is an intentional separation resulting from a reaction to adverse environmental conditions. Biofilm adapts to environmental stresses and the detached cells begin the process of colonization of new surfaces [2,3,5,8,12,13].
Both bacterial and fungal biofilms, in medicine and in industry, were first described in 1978 [7]. Since then, it has been the subject of numerous studies that aim to understand the molecular mechanisms of its origins and the role it plays in infections and drug resistance [5]. Candida albicans often occurs in the form of biofilm, which is the etiological factor of approximately 90% candidiasis. Among the clinical strains of the genus Candida, biofilm formation depends on the type of a strain [16], and Candida albicans, even of the same genotype, may differ in biofilm features [1].

Candida dimorphism and the biofilm formation
Compared to planktonic forms, biofilm cells lead settled lifestyles and have characteristic gene expression associated with the growth rate and synthesis of some of the adhesion and enzyme proteins. Fungal biofilms with cells differing phenotypically and functionally usually are of much more complex structure than the bacterial biofilm. Polymorphism is a characteristic feature of Candida yeast. The planktonic Candida are usually in a form of blastospores (budding cells), while the biofilm structure is formed by both blastospores and mycelial forms. During biofilm formation, morphological transformation takes place: from blastospores through the germ tubes to the filamentous forms (mycelium or pseudomycelium). The plasticity of Candida biofilm indicates that its cell composition may also change depending on the location and characteristics of the biomaterial surface. Individual cell types exhibit differences in antigenic structure and its adhesion and invasive properties, enzymatic activity, and phagocytosis resistance. Blastospores are responsible for the adhesion and spreading of the biofilm, initiating its production by adhering to biotic and abiotic surfaces and its colonization [5]. Adhesion is a signal that induces germination (morphogenesis) of blastospores and the formation of invasive forms, mycelium, or pseudomycelium, that enter epithelial or endothelial cells via endocytosis or active penetration and because of enzymatic activity they contribute to the destruction of colonized tissues. The presence of mycelial forms is not a prerequisite condition for the biofilm formation; however, it seems indispensable in the process of maturation. In the mature Candida biofilm, the inner layer is composed of blastospores, and the outer multilayer is mycelium and pseudomycelium. Biofilms created by Candida sp. yeast can reach a thickness of 25-450 μm [1,5,10,11,17]. There are three [6,11] or four [1,18] phases of Candida biofilm formation. Sometimes the last dispersion phase is included in the third phase, maturation of the biofilm.

Candida adhesion and the ability to the biofilm formation
The biofilm structure depends on the specific gene expression resulting from yeast contact with biotic or abiotic surface. Candida albicans yeast contact with a specific surface and activate the mitogen-activated protein kinase (MAPK) signaling cascade, which carries the extracellular contact signal to eukaryotic cells, then activating transcription factors and expressing a specific set of genes responsible for adherence. MAPK gene mkc1 activity level is higher in cells growing on different surfaces than in planktonic cells. After contacting Candida albicans with a polystyrene surface, the transcriptional level of the gene coding for methionine and cysteine, and the cdr1 and mdr1 codes for the mechanism of active ejection of the drug by efflux are surprisingly increased. Some examples of Candida sp. adhesion to polystyrene are presented in Figure 2.
Genes encoding sulfur amino acids are responsible for the amount of biofilm biomass produced, while the expression of the cdr1 and mdr1 genes is associated with the acquisition of drug resistance by the biofilm phenotype. What is more, the activity of adh1 alcohol dehydrogenase is higher in plankton cells than in biofilm cells, which influences the biofilm formation [1]. This enzyme is probably responsible for inhibition of biofilm formation and inactivation or mutation of the adh1 gene results in stronger biofilm formation in vitro and in vivo [1,5]. Adhesion to both biotic and abiotic surfaces related to recognition of host cell surface receptors, is a precondition for colonization, biofilm formation, initiation, and development of infection. Lack of adhesion prevents yeast cells from biofilm formation. Phenotypic variability and plasticity of cells in relation to changing environmental conditions allow for the settlement of new surfaces. Numerous Candida albicans gene products important for biofilm development have been identified [1,19]. Adhesive genes can be activated by different environmental signals. The potential adhesives involved in biofilm formation are cell wall surface proteins. The input of surface proteins with GPI (glycosylphosphatidylinositol) module is decisive in the formation of fungal biofilm. Ability of Candida albicans to adhesion is an important virulence factor associated with glycoproteins encoded primarily by genes such as hwp1 and als. Such an effect leads to changes in the expression of genes encoding cell wall proteins glycosylphosphatidylinositol dependent. Several key adhesins: als1, als2, als3, als4, als5, eap1, hwp1, hydrolases, lipases, phospholipases, and transcriptional factors bcr1 regulating protein expression are responsible for biofilm formation [1,5,19,20]. Ywp1, mannoprotein with a GPI module, is both the regulator of adhesion and a marker of Candida albicans cells. Blastospores are characterized by weaker intercellular adherence than mycelial forms. In addition, they are probably equipped with mechanisms to regulate the activity of their own adhesins [1]. The ywp1 protein found on yeast cells, a known adherence regulator, can function as an antihistamine and inhibit adherence. The discovery of the ywp1 protein is a scientific proof for the presence of the cell diffusion phase in the biofilm's life cycle. The deletion of the ywp1 gene leads to the enhancement of the blastospor adherence to the various surfaces [1]. On the other hand, in the biofilm layer composed of pseudomycelium Candida albicans identified adhesins with GPI, HWP1, EAP1 module, and the ALS adhesin family. Some studies indicate that the genes als and eap1 within the Candida genus show similarity to the genes regulating adhesion in Saccharomyces cerevisiae [1]. While, the hwp1 gene, known as the gene coding for the main Candida albicans protein, is involved in many functions such as cell wall building, intracellular signaling, and the development of hyphae. In addition, it appears that it is involved in the adhesion of yeast to epithelial cells, which is so important in the initial colonization stage. There is also an evidence for the involvement of the hwp1 gene in systemic candidiasis pathogenesis on mouse model in vivo. It has been shown that strains having the hwp1 and hwp1-null heterologous genes showed, respectively, reduced and no virulence compared to control wild strains. Hwp1 is the first exposed adhesive, required for biofilm formation in vivo, which is not present on cells in the form of yeast and plays no role in the formation of microcolonies. However, it expresses during morphogenesis blastospores to pseudomycelium [1].

Candida germ tubes and the ability to the biofilm formation
Pseudomycelium is formed by a germ tube process and as a key component of the biofilm provides its integrity. Both morphological forms of blastospore and pseudomycelium are capable of a biofilm formation, but strains capable of growth only in the form of blastospores produce only residual biofilm. The transcription factor efg1 plays a key role in regulating the morphology and virulence of yeast Candida albicans. It was first identified as an inducer of the development of pseudomycelium in Saccharomyces cerevisiae and then as a necessary for the growth of mycelium Candida albicans. The consequence of deletion of this gene is a loss of ability to transition into mycelial forms in response to majority of stimulation factors, but may occur in hypoxia and abiotic conditions. The efg1 gene fulfills many of the important functions in Candida albicans yeast cells, and most important is the virulence of vast infection models. Cells with efg1 gene deletion do not attack the human epithelium. In addition, efg1 is one of the key regulators of transition from the "white" form to the "opaque" form and essential to keep the default "white" phenotype. Moreover, unlike many other biofilm process regulators, the efg1 gene is essential for biofilm development under hypoxia and oxygenation conditions. Even when the yeast cells have adhered to the abiotic surface it is necessary to produce resistance to antifungal agents. Efg1 is a part of a network of six transcription factors that regulate the expression of at least 1000 genes involved in the development of the Candida albicans biofilm [21]. Several thousands of intergenic regions bound by the transcriptional factor efg1, which binds to promotors at least 53 genes in Candida albicans, including many transcription factors have been identified. The binding of efg1 is closely related to the transition from the basic form of the yeast cell to the pseudomycelium. Mutants with the deletion of efg1, cph1, and tec1 genes encoding transcription factors do not form pseudomycelium, and consequently have no ability to form mature biofilm structures. This indicates that transcriptional factors efg1, cph1, and tec1 play a key regulatory role in the formation of mature Candida albicans biofilm [1,21]. Furthermore, the mutations of the genes suv3, nup85, mds3, and kem1 inhibit the formation of pseudomycelium, which in turn promotes the formation of "immature" biofilm. In addition, mutants with the deletion of the bcr1 gene produce pseudomycelium, but do not produce biofilm, since the inhibition of gene expression for adhesin als and hwp1, involved in biofilm formation and regulated by bcr1 [1]. Separation of the filamentation process and biofilm formation showed that the morphogenesis of blastopores to pseudomycelium and consequently the presence of pseudomycelium did not clearly determine the biofilm formation. Pseudomycelium is only a basis, on which under control of the transcription factor bcr1, the adhesins gene gradually express. It is therefore necessary to provide the proper functionfilamentous adherence, without which mature biofilm will not be formed. Mutants lacking the activity of tec1, bcr1, als3, or hwp1 proteins exhibit large abnormalities in the biofilm production, which may underline the importance of all these proteins in the early stages of biofilm formation. The ability of residual biofilm formation by these mutants may at the same time indicate that these proteins are not directly involved in adherence to the surface, but in adherence between the blastospore, the mycelium forms, or the adherence mixed between both forms [1, 5, 21].

Candida communication and the ability to the biofilm formation
For the proper functioning of biofilm, communication between the cells and density regulation is necessary. These tasks are executed by small signaling particles called autoinducers and by responding to the generated signals within population in the quorum sensing system. In the culture with a high population density, there are signaling particles which, through diffusion, penetrate other cells running different signals. Exchanging signals lead to specific cell effects and coordination of cellular activity like multicellular behavior. Candida yeasts produce several signaling molecules, the accumulation of which determines the development, existence, and breakdown of the biofilm through having a direct influence on the process of mycelial forms creation. The best-known molecule is farnesol (C 15 H 26 O), a terpene alcohol isolated from Candida albicans cells. In the reproduction and maturation phase of the biofilm, the density of cells is relatively small, allowing the formation of mycelium. With the cell concentration increase, the concentration of farnesol, which interacts with the blastospore cell receptors, is increasing, preventing transformation into pseudomycelium and maturation of biofilm. The consequence is the phase of the biofilm dispersion in which individual blastospores and their aggregates are detached [1,2,5,18]. The release of blastospore requires weakening of the adhesive properties, which corresponds to the anti-adhesion ywp1 protein, and the main regulator of the process is the hsp90 protein [5]. Farnesol exogenously inhibits biofilm formation by blocking the expression of many genes responsible for the formation of pseudomycelium and induces expression of the adh gene taking part in inhibition of the biofilm formation. The endogenous accumulation of this signaling molecule in biofilm structures may therefore be a factor initiating the breakdown of the biofilm upon reaching a critical cell concentration. Farnesol also influences the expression of ergosterol metabolism genes. At the time of action of azoles blocking the ergosterol synthesis, the substrate to produce this molecule is increased by Candida albicans, and the amount of farnesol may even rise 45 times. Farnesol also affects many other processes, such as production of chlamydospores, iron transport, and activation of genes responsible for antibiotic resistance and oxidative stress. Unfortunately, it also has an adverse effect on host cells by inhibiting macrophage activity in the mouse model [1,2,5].
Another Candida albicans signaling molecule is an autoinducer thiamazole that stimulates the production of mycelium during the intermediate phase of biofilm growth. Comparing to planktonic cells, biofilm cells produce higher amounts of thiamazole [2,18]. It protects the cells from a decrease in the expression of DNA replication genes, chromosome segregation, and a cell cycle control [1,2].
The active regulation of the process of detachment from biofilm surface layers, in the state of achieving critical concentration of cells inside, is a crucial role of signaling molecules [1].

Candida antibiotic resistance
Candida albicans, like most pathogens, developed a number of mechanisms that regulate their virulence. It has developed different strategies to colonize host tissues and break down and weaken its barriers and defense mechanisms. One of the most important virulence factors of Candida sp. is the ability to produce mycelial forms that allow a host tissue invasion, at the same time repelling an effective phagocytes attack. The virulence of Candida sp. is strongly related to proteins determining of cell integrity, adherence, colonization, or change of phenotypic forms. These proteins are also an effective weapon in the fight against host defense. Most of them are characterized by the presence of anchored glycosylphosphatidylinositol and represent 88% of all covalently bound Candida albicans cell membrane proteins. Increasing clinical drug resistance because of abuse of antimicrobial agents is an important phenomenon hindering the fight against these yeasts. Candida albicans drug resistance is closely related to the antifungal activity of the drugs used.
Most drug resistance mechanisms to antifungal agents are the results of gene mutations. Usually, these are point mutations of genes encoding drug-binding molecules, enzymes of metabolic pathways, or transcription factors [22]. Such mutations are stable and their acquisition takes time. It is believed that they are the expression of a cell response to chronic stress, for example, resistance-inducing azoles [23] or genetic aneuploidy [24], which changes the expression of multi-drug pump points or transcription factors. Antifungal drugs can also activate a classic, immediate response to a stress. Resistance acquired on this path does not involve the change of genetic material and is reversible, for example, Candida sp. phenotype form change or biofilm formation. This reversible change allows us to obtain the time necessary to induce permanent resistance dependent on genetic mechanisms. For the resistance of one of the oldest antifungal agents, 5-FC, the most responsible is uracil phosphoribosyltransferase mutation preventing conversion of 5-fluorouracil to fluorouradine 5-monophosphate [25].
Resistance to polyene, which is still relatively rare today, is obtained by decreasing ergosterol content in the cell membrane, inter alia by erg3 gene mutation. Lowering ergosterol content in the cell membrane also leads to azole resistance by the increased expression of the erg11p molecule, the azoles binding point. Point mutations of this molecule are responsible for replacing the toxic ergosterol precursors accumulated in the yeast cell by non-toxic ones [25]. In vitro studies show different patterns of drug resistance to azoles, frequently overlapping with clinical trials. In vitro, the role of the hsp90 molecule chaperone for calcineurin in promoting the rapid acquisition of Candida albicans resistance to fluconazole has been identified [26,27]. Interestingly, the ability to maintain azole resistance even after treatment has ended [28]. Another effective mechanism of azoles resistance is the high expression of multilayer membrane pumps (MFS Mdr1p drug pump or ATP binding cartridge (ABC) of the Cdr1p or Cdr2p pump). These pumps beside azoles are active against a variety of other drugs, apart from echinocandins [25,29]. Limited resistance to echinocandin is most likely related to their relatively rare use. Although, in recent years, there have been reports both in vitro and in vivo on Candida sp. resistance to echinocandins. The best-known mechanism of resistance to these antibiotic agents is the mutation of the β-1,3-d-glucan synthase Gsc1p subunit [25,29].

Candida biofilm and its drug resistance
Particularly dangerous from a clinical point of view is the ability of most clinically important Candida species (Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida tropicalis, and Candida parapsilosis) to biofilm formation. The clinical significance of biofilm is increasing with the increasing introduction of various medical devices into the human body. Almost all the contaminations of these devices are the results of their colonization by microorganisms forming organized biofilm structures.
Biofilm Candida sp. is characterized by high resistance to all antifungal agents currently used: azoles (fluconazole, itraconazole, voriconazole, posaconazole), echinocandins (kaspofungin, mikafungin, anidulafungin), amphotericin B, flucytosine, but the level of this resistance is different for different drugs. Studies have shown that Candida sp. biofilm is resistant to fluconazole at a concentration of 2000 times higher than the MIC value for the planktonic form. The liposomal form of amphotericin B and echinocandin are the most active against Candida sp. biofilm. These antibiotic agents exhibit anti-biofilm activity in concentrations 2-25 times higher than the MIC values against planktonic forms [25,30,31]. Biofilm resistance is a complex, multi-factor phenomenon that uses the different mechanisms generated by planktonic forms at different stages of biofilm formation. There is also the possibility of generating different mechanisms of drug resistance by individual cell in the biofilm. For example, in the early stages of biofilm-building with low cell concentration, the increase in the activity of drug pumps, lowering the intracellular concentration of azoles, is noted. In mature biofilms characterized by greater cell concentration and many extracellular substances, resistance to amphotericin, azoles, and echinocandins is generated [11,25,30]. In addition, it appears that the lower content of ergosterol in mature biofilm is also one of the mechanisms of defense against antifungal agents [22]. The change from planktonic forms to biofilm is a response to unfavorable environmental conditions, which starts a rapid response to stress, which generates, for example, drug resistance. Acquired by acute stress resistance, it is associated with protein kinase activity, calcineurin or hsp90p heat shock protein. Drug-resistant subpopulation protects the pool of cells needed to rebuild the biofilm [25]. An extracellular matrix (ECM) is an important factor in the generation of multi-drug resistance. β-1,3-d-glucan, one of the ECM components, is responsible for drug resistance to fluconazole and amphotericin B. While the role of ECM in generating multi-drug resistance is unquestioned, the mechanisms leading to it remain unexplained.
In the fight against Candida biofilm, there are two main problems: a penetration of the drug into the biofilm structure and to overcome the yeast resistance produced by the cells organized in the biofilm. A method of "lock therapy" is conformed to deliver the antifungals directly into the places colonized by the biofilm. To conquer the growing antibiotic biofilm resistance the following strategies are applied: (i) novel antifungal agents in the forms of conjugates, (ii) a multi-drug therapy, (iii) a combination of antifungal agents with nonsteroidal anti-inflammatory drugs, and (iv) agents interfering the communication of cells in the biofilm.
The use of high drug concentrations, for example, higher echinocandin doses used to treat endocarditis, is one of the proposed methods of fighting against Candida sp. biofilm. "Lock therapy" uses medical devices (e.g., vascular catheters) for treatment, where high drug doses are introduced into the catheter [32,33].
Hudson et al. [34] describe a novel form of amphotericin B, dextran aldehyde conjugate with amphotericin B, preservative gel formulation used in local treatment of infections (ligaments, vascular catheters, bones) caused by Candida sp. biofilm. In vitro, also other compounds: EDTA, ethanol, and high doses of monocycline, are effective in the fight against Candida sp. biofilm as "lock therapy" [32,35].
Another method of fighting infections caused by Candida sp. biofilm is the combination therapy of antifungal agents (fluconazole, echinocandin, and amphotericin B) with calcineurin inhibitors such as cyclosporin A or tacrolimus. Such therapy exhibited good in vivo activity in the treatment of rat-associated venous catheter infections [35]. Other promising preparations used in "lock therapy" in combination with antifungal agents are compounds that target hsp90 heat shock proteins such as geldanamycin [25,35]. However, none of these preparations are suitable for systemic use due to their toxicity or lack of confirmed safety in clinical trials.
An interesting proposal seems to be the combination of antifungal preparations with widely used nonsteroidal anti-inflammatory drugs (NSAIDs). Their activity by inhibiting cyclooxygenase prevents yeasts filamentation and thus biofilm formation [32].
Its activity in mouse model studies in vitro was comparable to that of azoles. However, in vivo studies on animal models have not been conducted [32,36].
Pulmozyme preparation, comprising recombinant human deoxy ribonuclease (rkDNase), is currently used in inhalation therapy of patients with cystic fibrosis, which targets bacterial biofilm DNA [32].

Probable environmental circulation of Candida strains
Besides the most frequent fungal pathogen Candida albicans, non-albicans Candida strains are isolated from the patients and clinical environments. Among non-albicans, the common clinical isolates are Candida glabrata, Candida lusitaniae, Candida parapsilosis, Candida auris, Candida tropicalis, and Candida dubliniensis. Candida sp. are the widespread yeasts in food products serving as natural flora members or food-contaminants. The examples of food-associated Candida yeasts are Candida lusitaniae, Candida famata, Candida parapsilosis, Candida colliculosa, Candida tropicalis, Candida krusei, Candida boidinii, and Candida pelliculosa. Considering the possibilities of Candida strains' natural circulation between food and clinical environments, a question arises if the food-borne strains can be a threat for specific groups of patients. Our previous work presented the examples of antibiotic resistance of a variety of Candida clinical and food-borne isolates [37]. Within the study, 24 clinical strains of Candida albicans and 1 Candida glabrata strain as well as a Candida lusitaniae strain were compared with 18 nonalbicans food-borne candidas. The set of food-borne isolates consisted of Candida lusitaniae (four strains), Candida famata (two strains), Candida parapsilosis (one strain), Candida colliculosa (one strain), Candida tropicalis (one strain), Candida krusei (four strains), Candida boidinii (three strains), Candida rugosa (one strain), and Candida pelliculosa (one strain). The strains sensitivity to the nystatin (polyenes), fluconazole (triazoles I generation), voriconazole (triazoles II generation), and caspofungin (echinocandins) were checked. It was found that all the tested strains were sensitive to caspofungin but 15 strains differed in sensitivity to nystatin, fluconazole, and voriconazole irrespective of their origin. Interestingly, two of four tested foodborne strains of Candida krusei were not susceptible to fluconazole, and the third one was classified as intermediate. All Candida krusei isolates were sensitive to fluconazole. One clinical isolate of Candida glabrata was not sensitive to fluconazole. Triazoles were the last effective not totally inhibiting the growth of the clinical isolates and five food-borne strains. The results proved that a group of strains expressing intermediate sensitivity or resistance to the tested antibiotics include both clinical and food-borne isolates.
According to the biochemical profiles, the tested strains were classified in two groups: (i) 24 Candida albicans clinical isolates and 1 strain of food-borne yeast Candida tropicalis, which was isolated from pickled cucumbers; (ii) 17 food-borne strains and 2 clinical isolates Candida glabrata and Candida lusitaniae. What is more, Candida albicans isolates expressed vast biochemical heterogeneity. A yeast adaptation to the host organism may explain these differences.
Both Candida albicans and Candida glabrata, typical human pathogens, were not found in food [37]. The noted biochemical profiles conformity of some clinical and food-borne isolates may be a result of Candida transmission via food. The similarity of food-borne Candida tropicalis to the Candida albicans strains isolated from clinical patients implies the possibility of circulating of antibiotic-resistant strains outside the hospital environment and the possible yeast infection caused by yeasts entered into the body with food.

Conclusions
Biofilm-forming microorganisms, including Candida species, are characterized by high invasiveness, the ability to cause dangerous, and difficult to treat infections. Furthermore, the cells in the biofilm, compared to planktonic forms, show reduced sensitivity to chemical compounds with antifungal activity and increased survival under unfavorable environmental conditions. The morphological diversity of the biofilm structures formed by Candida albicans and non-albicans strains allows these yeasts to colonize both biotic and abiotic surfaces. The emergence of antifungal resistance with the development of biofilms is still a problem. The incidences of medical equipment colonization by Candida yeasts are constantly noted. Moreover, the proven biochemical profiles conformity of some clinical and food-borne isolates may be a result of Candida transmission via food.