2. Pathogenic yeast infections: a serious health problem

In the last two decades, fungal infections have shown a significant increment. In addition to the increase in the number of patients with compromised immune system, factors such as increasing number of patients using catheters, the use of broad-spectrum antibiotics, the rising number of patients requiring organ transplantations, as well as those with hematological malignancies and diabetes also contribute to this phenomenon [26, 27].

Even though fungal infections cause significant amount of human morbidity and mortality, the impact of these "opportunistic" diseases on human health is not widely highlighted [2]. Due to this, the research into the pathophysiology of human fungal infections is slow in comparison to other disease-causing pathogens. Recently, an editorial published in the journal Nature Microbiology [28] ratified the importance of not neglecting fungi. The call proposed a reflection on fungi and how these microorganisms have been neglected, even with studies already consolidated showing their medical relevance.

The most frequent fungal diseases affecting populations in the world are candidiasis [29–34] and cryptococcosis [8, 20, 25]. There are several types of candidiasis as mucosal candidiasis, cutaneous candidiasis, onychomycosis, systemic candidiasis [35, 36], and pulmonary candidiasis. An important fact is that candidiasis is an infection that can affect both immunocompromised and healthy people [37, 38]. Candidemia is the most relevant and prevalent nosocomial fungal infection associated with a high mortality rate (up to 49%) in patients with a compromised immune system [39, 40]. The association of Candida with bloodstream infections depends on patient's condition, age, and geographic region. Candidemia is such an important infection that in 10–40% of cases, it is associated with sepsis or septic shock [41].

Candida albicans continues to be the most prevalent species isolated from fungal infections [27, 42–44]. However, the prevalence of other Candida species has increase substantially. These species are C. parapsilosis, C. tropicalis, C. krusei, C. glabrata, C. guilliermondii, C. orthopsilosis, C. metapsilosis, C. famata, and C. lusitaniae [44–46].

Candida species presents high degree of flexibility, being able to grow in extremely different environments regarding to the availability of nutrients, temperature variation, pH, osmolarity, and amount of available oxygen [47]. This fact associated with the high resistance capacity of species to antifungals, their virulent features, and capability of forming biofilms with other species [48, 49] makes the genus Candida a serious risk to human health [50]. Thus, Candida species are highly adaptable and possess numerous strategies to survive in conditions that can affect their overgrowth and alter their susceptibility profiles.

Cryptococcus spp. may remain latent in the lungs, leading to asymptomatic infection, or may cause multifocal lung disease. The latency period of Cryptococcus can range from 6 weeks to more than 1 year after inhalation [51]. The fungus

adherence to host surface, followed by releasing of various hydrolytic enzymes and

Candida species can cause a variety of infections from the mildest to the most severe being candidemia the most frequent hospital infection accounting for up to 15% of bloodstream infections. Candida species are the main causative agents in

Cryptococcus species are other yeasts of medical importance, with more than 39 species, among which Cryptococcus gattii and Cryptococcus neoformans are the most clinically relevant [6–8]. However, other species such as Cryptococcus albidus and Cryptococcus laurentii are emerging pathogens involved in several types of

These yeasts are present in several environmental niches, such as woody sites (decomposing tree trunks, mainly eucalyptus, and soil), vegetable remains, domestic dust, and bird excrement, more precisely in Columba livia [12–14]. The source of the infection is exogenous and occurs primarily by inhalation or by direct inoculation into the tissue after trauma of desiccated spores or yeasts. It is believed that the only source of infection is environmental, since there are no reports of transmission

The main virulence factors of Cryptococcus species are growth capacity at 37°C, polysaccharide capsule, melanin synthesis, and production of urease and antioxidant enzymes, causing primary or opportunistic cryptococcosis, such as pulmonary, cutaneous, and meningitis diseases [6, 8, 13, 16–19]. Cryptococcosis is the third

In addition to delays in yeast diagnosis, there is currently a limited antifungal armamentarium in use against yeast diseases including only four chemical classes: polyenes, triazoles, echinocandins, and flucytosine. Antifungals act by binding specific components of fungal plasma membrane or its biosynthetic pathways or even cell wall components [21]. However, most of the antifungal agents used in the clinic is fungistatic and often led to the development of resistance by fungal species. Modern early antifungal treatment strategies, such as prophylaxis and empirical and preemptive therapy, result in long-term exposure to antifungal agents, which is a

Among the available antifungal agents, azoles are the preferred and most frequently used drugs for treatment of Candida and Cryptococcus infections. Fluconazole (FLZ), a type of azole, is often preferred in treatments of Candida infections because of its low cost and toxicity, in addition to availability in varied formulations [22]. However, there are many reports that described resistance development

Considering the limited availability of antifungals in use and the emergence of resistance, the control of Candida and Cryptococcus infections is a challenge in the modern clinic. In this way there is a continuous need for the search for new substances with new mechanisms of action with the aim of developing novel broad

Infectious Diseases Society of America recommends the treatment of cryptococcosis through FLZ and amphotericin B (AMB) with or without combination with 5-flucytosine (5-FC), followed by prolonged maintenance with fluconazole. Other azole compounds such as itraconazole (ITC), voriconazole, and posaconazole may be used as an alternative to FLZ in cases of contraindication or inefficacy of the latter [23, 24]. However, there has been a progressive increase in isolates of Cryptococcus spp. resistant to FLZ, which complicates the management of cryptococcal meningitis [25]. On the other hand, AMB and 5-FC are not available in all countries and are, respectively, nephrotoxic and hepatotoxic, limiting the

other virulence factors for invasion and damage of the host tissues [4].

50–70% of systemic fungal infections [5].

Phytochemicals in Human Health

between animals and humans or between humans [15].

opportunistic infection associated with AIDS [20].

major driving force for the development of resistance.

among Candida species, especially in relation to azoles.

anti-cryptococcal therapeutic [24].

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spectrum antifungal drugs with better efficacy.

infections [6, 9–11].

presents neurotrophism and can migrate to the central nervous system (CNS) through hematogenous dissemination and, when crossing the blood-brain barrier, can cause meningoencephalitis [13, 18]. Episodes of mental confusion in patients with cryptococcosis have been described [52, 53]. Neurocryptococcosis is the most severe form of the disease with high mortality rates in the absence of adequate treatment [18, 23]. The mortality due to cryptococcosis is higher than the mortality caused by tuberculosis and similar to that caused by malaria [54].

Another clinical manifestation is cutaneous cryptococcosis, which is rare and usually secondary to hematogenous dissemination. Cutaneous lesions are characterized by an infiltrative plaque of a solid tumor mass that can present ulcerative and necrotic lesion [17]. Pulmonary and cutaneous lesions due to nodular features may be misdiagnosed as tumor lesions [55]. In addition to the respiratory tract, CNS and skin, other sites may be affected: prostate, eyes, adrenal glands, lymph nodes, bone marrow, and liver [51].

Until now, there are three proposals to explain fungal neurotropism. The first is that neuronal substrates present in the basal ganglia promote cryptococcal growth and survival, and, thus, perivascular spaces may serve as a niche for Cryptococcus, as described by [56] in a healthy female patient who had evidence of Cryptococcus infection within the perivascular spaces of the parenchyma. The second proposal describes that it is possible that there are specific neuronal receptors that can attract Cryptococcus to the CNS [57]. The third hypothesis, one of the most widespread, is that the fungus uses neurotransmitters such as dopamine that aids in the synthesis of melanin [19, 57, 58].

Figure 1.

Figure 2.

235

β-D-glucan synthase, thereby preventing glucan synthesis.

Specific point of action of antifungal drugs in the ergosterol biosynthesis pathway.

Mechanisms of action of some traditional antifungal agents on cellular targets. Azoles inhibit the ergosterol synthesis in the endoplasmic reticulum of the fungal cell by interfering with the enzyme lanosterol 14-αdemethylase. Polyenes act by binding to ergosterol present at the cell membrane. Echinocandins inhibit (1,3)

Phytochemicals and Their Antifungal Potential against Pathogenic Yeasts

DOI: http://dx.doi.org/10.5772/intechopen.87302

Besides the clinical importance of fungal infections caused by theses pathogenic yeasts, interestingly, climatic abnormalities due to phenomena such as La Niña and El Niño have recently been described as important in the distribution and occurrence of mycoses in countries influenced by them [59].

#### 3. Traditional antifungal agents against yeasts

In the last two decades, there has been an increasing, but limited, discovery of antifungal agents [47]. These include azoles, such as fluconazole, itraconazole, ketoconazole (KTC), miconazole, and clotrimazole, polyenes (amphotericin B [AMB] and nystatin), allylamines, thiocarbamates, morpholines, 5-fluorocytosine, and echinocandins (for instance, caspofungins) [21]. However, fungal cells and human cells are eukaryotic, so antifungal compounds target both cell types, resulting in considerable side effects in patients and fewer available targets for drug action. Antifungals target three cellular components of fungi (Figure 1). Azoles inhibit ergosterol biosynthesis by interfering with the enzyme lanosterol 14-αdemethylase in endoplasmic reticulum of the fungal cell. This enzyme is involved in the transformation of lanosterol into ergosterol, a component that is part of the plasma membrane structure of the fungus (Figures 1 and 2). Thus, as the concentration of ergosterol is reduced, the cell membrane structure is altered, thereby inhibiting fungal growth [60].

Azoles comprise a five-member azole ring containing two (imidazole) or three nitrogen atoms (triazole) attached to a complex side chain [61, 62]. Imidazoles include KTC, miconazole, econazole, and clotrimazole, and triazoles include FLZ, ITC, voriconazole (synthetic triazole derivative of FLZ of second generation), and posaconazole (hydroxylated analog of itraconazole) [63].

AMB and nystatin bind to ergosterol causing the disruption of the membrane structure and promoting extravasation of intracellular constituents such as ions and sugars and, consequently, cell death [21] (Figure 1).

Phytochemicals and Their Antifungal Potential against Pathogenic Yeasts DOI: http://dx.doi.org/10.5772/intechopen.87302

#### Figure 1.

presents neurotrophism and can migrate to the central nervous system (CNS) through hematogenous dissemination and, when crossing the blood-brain barrier, can cause meningoencephalitis [13, 18]. Episodes of mental confusion in patients with cryptococcosis have been described [52, 53]. Neurocryptococcosis is the most severe form of the disease with high mortality rates in the absence of adequate treatment [18, 23]. The mortality due to cryptococcosis is higher than the mortality

Another clinical manifestation is cutaneous cryptococcosis, which is rare and usually secondary to hematogenous dissemination. Cutaneous lesions are characterized by an infiltrative plaque of a solid tumor mass that can present ulcerative and necrotic lesion [17]. Pulmonary and cutaneous lesions due to nodular features may be misdiagnosed as tumor lesions [55]. In addition to the respiratory tract, CNS and skin, other sites may be affected: prostate, eyes, adrenal glands, lymph nodes,

Until now, there are three proposals to explain fungal neurotropism. The first is that neuronal substrates present in the basal ganglia promote cryptococcal growth and survival, and, thus, perivascular spaces may serve as a niche for Cryptococcus, as described by [56] in a healthy female patient who had evidence of Cryptococcus infection within the perivascular spaces of the parenchyma. The second proposal describes that it is possible that there are specific neuronal receptors that can attract Cryptococcus to the CNS [57]. The third hypothesis, one of the most widespread, is that the fungus uses neurotransmitters such as dopamine that aids in the synthesis

Besides the clinical importance of fungal infections caused by theses pathogenic yeasts, interestingly, climatic abnormalities due to phenomena such as La Niña and El Niño have recently been described as important in the distribution and occur-

In the last two decades, there has been an increasing, but limited, discovery of antifungal agents [47]. These include azoles, such as fluconazole, itraconazole, ketoconazole (KTC), miconazole, and clotrimazole, polyenes (amphotericin B [AMB] and nystatin), allylamines, thiocarbamates, morpholines, 5-fluorocytosine, and echinocandins (for instance, caspofungins) [21]. However, fungal cells and human cells are eukaryotic, so antifungal compounds target both cell types,

resulting in considerable side effects in patients and fewer available targets for drug action. Antifungals target three cellular components of fungi (Figure 1). Azoles inhibit ergosterol biosynthesis by interfering with the enzyme lanosterol 14-αdemethylase in endoplasmic reticulum of the fungal cell. This enzyme is involved in the transformation of lanosterol into ergosterol, a component that is part of the plasma membrane structure of the fungus (Figures 1 and 2). Thus, as the concentration of ergosterol is reduced, the cell membrane structure is altered, thereby

Azoles comprise a five-member azole ring containing two (imidazole) or three nitrogen atoms (triazole) attached to a complex side chain [61, 62]. Imidazoles include KTC, miconazole, econazole, and clotrimazole, and triazoles include FLZ, ITC, voriconazole (synthetic triazole derivative of FLZ of second generation), and

AMB and nystatin bind to ergosterol causing the disruption of the membrane structure and promoting extravasation of intracellular constituents such as ions and

caused by tuberculosis and similar to that caused by malaria [54].

rence of mycoses in countries influenced by them [59].

3. Traditional antifungal agents against yeasts

posaconazole (hydroxylated analog of itraconazole) [63].

sugars and, consequently, cell death [21] (Figure 1).

bone marrow, and liver [51].

Phytochemicals in Human Health

of melanin [19, 57, 58].

inhibiting fungal growth [60].

234

Mechanisms of action of some traditional antifungal agents on cellular targets. Azoles inhibit the ergosterol synthesis in the endoplasmic reticulum of the fungal cell by interfering with the enzyme lanosterol 14-αdemethylase. Polyenes act by binding to ergosterol present at the cell membrane. Echinocandins inhibit (1,3) β-D-glucan synthase, thereby preventing glucan synthesis.

Pyrimidine analogs include 5-fluorocytosine and 5-fluorouracil (5FU). The first has fungistatic properties and enters the fungal cell through cytosine permease, inhibiting the thymidylate-synthetase enzyme and interfering with DNA. 5-fluorouracil, which in turn can be phosphorylated to 5-fluorodeoxyuridine monophosphate, can be incorporated into RNA molecules [63]. Due to toxicity [64]; stronger side effects, such as hepatic impairment; interference with bone marrow function; and rapid occurrence of resistance especially among Candida species, the clinical use of 5-FC is preferred in association with AMB [65, 66]. In addition, the nephrotoxicity and hepatotoxicity of AMB and 5-FC, respectively, and the unavailability of these antifungals in many countries have limited their use in cryptococcal therapeutic [24].

Therefore, limitations of treatment and drug resistance associated with pathogenicity of the clinical isolates support the urgent need to identify substances that are more effective, with new mechanisms of action in the fight against Candida and

Most antifungals target sterols or the enzymes that synthesize them. However, the fungistatic nature of many of these antifungals and emergence of clinical drug resistance limits their success. Increased drug resistance in fungi is a problem that cannot be avoided, particularly for FLZ, which is the preferred antifungal for

The number of people at risk for fungal infections has been increasing, resulting

in an increased use of antifungal agents, even as prophylaxis. Thus, besides the existence of some non-albicans Candida (NAC) species presenting inherent resistance to azoles, higher minimum inhibitory concentrations (MICs) for antifungals against C. albicans strains have been observed [76]. The World Health Organization (2014) categorizes antimicrobial resistance as that developed by the microorganism to an antimicrobial drug, which was initially effective in treatment of such infections. Low-dose prophylactic administration of azole derivatives, such as FLZ, for

prolonged periods to prevent the occurrence of opportunistic infections in

zole, itraconazole, and voriconazole in comparison to clinical isolates.

immunosuppressed patients also results in resistant phenotypes [27, 75]. Therapeutic failures and empiric treatment are facts which are likely to collaborate to the

In the last decade, a number of new clinical problems have arisen, requiring new guidelines regarding the treatment of cryptococcosis, mainly because clinical data have suggested that cryptococcal strains have become more resistant to drugs [23, 25]. Some relates say that clinical Cryptococcus isolates are frequently less susceptible to fluconazole than environmental isolates. However, Chowdhary et al. [77] evaluated the susceptibility profile of environmental and clinical strains of C. gattii and observed that environmental samples were less susceptible to flucona-

Heteroresistance is also a worrying phenomenon. It consists of the ability of a subpopulation of microorganism to adapt to high concentrations of the drug, resulting in resistant homogenous populations. However, heteroresistant strains return to the initial phenotype when the stimulus with the drug is withdrawn [78]. Some mechanisms for cellular and molecular resistance to FLZ in yeasts are described. In Candida and Cryptococcus, the first is related to the induction of multidrug pumps, which decrease the concentration of drug available in the intracellular compartment of yeast cells. Various genes belonging to the ATP-binding cassette superfamily or to the major facilitator superfamily encode efflux pumps were identified in C. albicans. Overexpression of some transporter genes or of their regulated genes can confer cross-resistance to various azoles [21]. In C. gattii and C. neoformans, AFR1, MDR1, and AFR2 genes encode ABC transporters that expel the azole out of the fungal cell, thereby causing resistance to these drugs [79]. A second mechanism of resistance involves modification of the target enzyme

encoded by the ERG11 gene, also known as cytochrome P450 lanosterol 14-αdemethylase (Cyp51). Mutations in this gene prevent azoles from binding to enzyme sites. Another mechanism of resistance is related to mutations in the ERG3 gene which does not convert 14-α-methylfecosterol into 14-α-methyl-3,6-diol in the ergosterol synthesis pathway. This substitution causes azoles to have no fungistatic

4. Resistance in pathogenic yeasts: a significant problem

Phytochemicals and Their Antifungal Potential against Pathogenic Yeasts

DOI: http://dx.doi.org/10.5772/intechopen.87302

Cryptococcus infections.

treating yeast infections [75].

increased incidence of fungal infections.

effects on the fungal cell membrane [21].

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Host's immunity, type of infection, site of origin of the samples, toxicity, bioavailability of the drug, and the sensitivity/resistance profile of the isolates interfere in the choice of the type of agent to be used [22]. AMB is considered the gold standard drug for most mycoses that affect patients at risk [67], although it has high toxicity. Azoles have fungistatic properties that affect cell growth and proliferation [65]. Among azoles, KTC was one of the firsts to emerge and was the first alternative to AMB [68]. Currently, FLZ is the drug of choice for most Candida and Cryptococcus infections [64] and is the most recommended antifungal agent for use in invasive candidiasis [47, 49].

For cryptococcosis, the choice of treatment depends on the patient's immunological status and mainly on the clinical of the disease, if it is just a pulmonary manifestation or if the infection is systemic. Fluconazole is recommended in cases of lung disease with mild to moderate symptoms. Amphotericin B with or without combination with 5-flucytosine is the recommended therapy for more serious infections such as meningoencephalitis, followed by prolonged maintenance with fluconazole [23, 24].

Although azoles are generally well-tolerated, they have limitations such as hepatotoxicity and the emergence of resistance among fungal isolates [69] which provide motivation for improving this class of antifungal agents [68]. For instance, alterations in triazole molecule gave rise to voriconazole (structurally related to FLZ) and posaconazole (related to ITC), both available for systemic therapy [66].

Echinocandins, which include caspofungin, micafungin, and anidulafungin, are a new class of antifungals and have fungicidal effects in all Candida species [66]. They inhibit (1,3) β-d-glucan synthase, thereby preventing glucan synthesis, which is present in the cell membrane of fungi (Figure 1). As this drug acts on the wall structure of the fungus, it has the advantage of a lower side effect in animal cells [47].

Allylamines (terbinafine and naftifine) and thiocarbamates inhibit the enzyme squalene epoxidase, which participates in the synthesis of ergosterol and is encoded by the ERG1 gene (Figure 2). This activity leads to membrane rupture and accumulation of squalene. Allylamine effects can also prevent the production of other sterol derivatives.

To minimize toxicity and resistance, some pharmacological strategies were developed. The preparation and use of new antifungal formulas (liposomal AMB (Ambisome®), AMB lipid complexes (Abelcet®), AMB colloidal dispersions (Amphocil®/Amphotech®), and AMB lipid nanosphere formulations and β-cyclodextrin itraconazole) are one strategy [68]. Others include combination therapies of antifungal compounds (e.g., AMB + 5-FC, FLZ + 5-FC, AMB + FLZ, caspofungin + liposomal AMB, and caspofungin + FLZ) and nanostructuring of conventional antifungal agents [70–73].

However, all traditional antimycotic drugs have at least one restriction related to their use. Some do not have a broad spectrum of action or are fungistatic. Others have high toxicity and low bioavailability with significant side effects [74].

Therefore, limitations of treatment and drug resistance associated with pathogenicity of the clinical isolates support the urgent need to identify substances that are more effective, with new mechanisms of action in the fight against Candida and Cryptococcus infections.
