ABSTRACT
The clinical resistance of Candida species to antifungal medications, particularly fluconazole, is rising. A major mechanism responsible for this resistance is the alteration in the nucleotide sequence of the gene that codes for the ERG11 protein, a key enzyme in the ergosterol synthesis pathway, which remains the target of fluconazole. This study investigated the distribution of different species of Candida in human and dog vaginal swabs, the susceptibility of the Candida species to fluconazole, the profile of the ERG11 gene, and the phylogenetic relationship of the Candida species based on the nucleotide sequence of the ERG11 gene. A total of 57 human samples and 7 dog samples were screened for the presence of Candida species. Twenty-eight (28) of the human samples were positive (+ve) to yeast growth. A total of 37 Candida isolates were obtained from the 28 human specimens that were positive to yeast growth. Of the 37 isolates, 13 (35%) were C. albicans, 4 (9%) C. glabrata, 4 (9%) C. krusei, 2 (6%) C. tropicalis, and 14 (38%) other Candida species. Of the 28 human specimens that were positive to Candida growth, 21 had single species, 5 had two different species and 2 had three different species. Four different species of Candida, including C. albicans, C. tropicalis, C. krusei, and C. glabrata were identified. The antifungal susceptibility test revealed that 33 (89.2%) of the Candida species were susceptible (≥19mm) to 25 μg fluconazole. The most fluconazole-resistant isolate was C. glabrata, while the most fluconazole-susceptible isolate was a C. albicans. Both isolates were obtained from humans. The four (4) isolates whose ERG11 genes were sequenced, were the most resistant, C. glabrata, the susceptible dose-dependent, C. albicans, the most susceptible, C. albicans and the dog isolate, C. krusei. The nucleotide sequence lengths of the ERG11 gene of these isolates varied from 1431 bases (Can Iso-001) to 1668 bases (Can Iso-029). Similarly, the molecular weight varied from 56183.10Da (Can Iso-001) to 65139.25Da (Can Iso-029), while the isoelectric point varied from 8.88 pI (Can Iso-029) to 9.60 pI (Can Iso-001). The predicted half-life (t1/2) of these proteins in mammalian cells was 100 hours, and the instability coefficients were 35.70, 36.98, 44.50 and 37.69, for Can Iso-001, Can Iso-017, Can Iso-028 and Can Iso-029, respectively. The grand average of hydrophobicity (GRAVY) of these Candida ERG11 proteins were 0.195, 0.478, 0.195 and 0.576, respectively. The four Candida ERG11 proteins were predicted to be localized in the plasma membrane. The first 12 amino acids in the multiple sequence alignment (MSA) make-up the first major conserved domain, while the second major conserved regions are in positions 331- 337, 332-338, 330-336 and 330-336, for Can Iso-001, Can Iso-017, Can Iso-028 and Can Iso-029, respectively. The total residues of alpha helix, beta pleated sheets and turns for these proteins varied. The most fluconazole-resistant isolate (Can Iso-001) had the highest percentage of α-helix in its ERG11 protein, while the most fluconazole-susceptible isolate (Can Iso-028) had the lowest percentage of α-helix in its ERG11 protein. There was a low-level similarity between the Can Iso-017 and Can Iso-028 ERG11 tertiary structural models. The Tertiary protein structure of the Can Iso-001 ERG11 was not similar to the other isolates. Can Iso-001 ERG11 protein had no single fluconazole-binding site, while Can Iso-017, Can Iso-028 and Can Iso-029 ERG11 proteins had unique binding sites to which the drug can effectively bind. Similarly, Can Iso-001 ERG11 protein possesses 10 antigenicity sites, while the Can Iso-017, Can Iso-028 and Can Iso-029 ERG11 proteins, possess 16, 21 and 15 antigenicity sites, respectively. The amino acids; 2-8: ETVIDGI was identified as a disease-causing region common to all, in addition to other disease-causing regions that were peculiar to each of the isolates. The phylogenetic analysis based on the ERG11 gene showed that, the four isolates were closely related. However, the most resistant isolate (Can Iso-001) and the dog isolate (Can Iso-029), seemed to have originated from a common ancestor, implying an even closer evolutionary relatedness.
CHAPTER ONE
INTRODUCTION
The incidence and prevalence of invasive fungal infections have increased since the 1980s, especially in the large population of immunocompromised patients (Espinel-Ingroff et al., 2009). Candida species are important human fungal pathogens that cause both mucosal and deep tissue infections. Candida species belong to the normal microbiota of an individual’s mucosal oral cavity, gastrointestinal tract, the vagina and other endo-mucosal surfaces (Shao et al., 2007), and are responsible for various clinical manifestations from mucocutaneous overgrowth to bloodstream infections (Eggimann et al., 2003). These yeasts are commensal in healthy humans, and, possibly could cause systemic infections in immunocompromised situations. More than 17 different Candida species are known to be aetiological agents of human infection. However, more than 90% of invasive Candida infections are caused by Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei (Ortega et al., 2011; Pfaller et al., 2015). Candida albicans and other non-albicans Candida (NAC) species such, as C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei are capable of causing superficial oral, vaginal mucosa, disseminated bloodstream and deep-tissue infections. However, species involvement varies by infection site and by geographical location (Sharifzadeh et al., 2013; Cleveland et al., 2015; Klingspor et al., 2015). C. glabrata is the most common NAC species found to be the causative agent in vulvovaginal candidiasis (VVC) (Vermitsky et al., 2008; Mahmoudi Rad et al., 2012). C. parapsilosis is well known for its threat to the pediatric population, as it is responsible for 17–50% of all fungemia in infants and neonates (Krcmery et al., 1999). C. parapsilosis is also second to C. albicans in incidence as a cause of Candida endocarditis with mortality rates between 42% and
65% (Garzoni et al., 2007). C. tropicalis infections are commonly associated with malignancy, with some studies reporting higher prevalence among patients with hematologic diseases such as acute myeloid leukemia (Tang et al., 2015; Cornely et al., 2015). The mortality rate ranges from
30% to 70%, with the highest rates commonly observed among the elderly (Cornely et al., 2015; Wang et al., 2015). C. krusei is the fourth most common NAC species associated with invasive candidiasis and candidemia, accounting for approximately 2.7% of NAC species isolated in clinical studies (Pfaller et al., 2014). The pathogenicity of Candida species is attributed to certain virulence factors, such as adherence to mucosal surfaces, ability to evade host defenses, biofilm
formation (on host tissue and on medical devices) and the production of tissue-damaging hydrolytic enzymes such as proteases, phospholipases and haemolysin (Verstrepen and Klis,
2006). Biofilms are biological communities of Candida with a high degree of organization residing in carbohydrate polymers, in which microorganisms form structured, coordinated and functional colonies. These biological communities are embedded in a highly organized self-created extracellular matrix, primarily composed of structural carbohydrate polymers. Currently, an increase in the number of Candida species that are resistant to antifungal drugs is recognized worldwide (Ingham et al., 2012). The increase in resistant strains necessitates the search for new molecular targets in the organism, as well as new antifungal agents to replace existing ones. There are numerous classes of compounds used to treat Candida infections. The polyenes, azoles, echinocandins, nucleoside analogs, and allylamines are used with varying efficacy, depending on the type and site of infection, as well as the susceptibility of the Candida species to the agent (Pfaller et al., 2015; Pappas et al., 2015). The most commonly prescribed antifungal agents used for most C. albicans infections is fluconazole; a member of the azole class of antifungals (Pfaller et al., 2010). Azoles inhibit lanosterol 14 alpha-demethylase, encoded by the ERG11 gene, which is an enzyme involved in the biosynthesis of the fungal-specific membrane sterol; ergosterol (Lortholary et al., 2011; Fothergill et al., 2014). Azole antifungals have long provided effective treatment for Candida infections, however, recent epidemiological studies indicated that intrinsic azole resistance in some Candida species, including the onset of high-level azole resistance is a problem of critical importance in clinical settings (Pfaller et al., 2015; Shields et al., 2015). While extensive studies to elucidate the molecular machineries of high-level azole resistance in C. albicans has uncovered the role of ergosterol biosynthesis gene (ERG11 gene) mutation and drug efflux pump upregulation as key mediators of azole resistance, there are other factors at play that contribute significantly to such resistance. From previous studies, there exist clear mutations in the ERG11 gene that are found to influence azole resistance in clinical isolates among Candida species (Pfaller et al., 2015; Shields et al., 2015). As azole resistance continues to emerge in these species, a better understanding of the important differences among resistance machineries employed by these species is needed in order to circumvent this crucial clinical problem. Azole antifungals such as fluconazole are often the preferred treatment for many Candida infections due to their efficiency, lower costs, limited toxicity and availability in oral administration. There is an extensive documentation of intrinsic and developed resistance to azole antifungals among
numerous Candida species at different degrees, and in different geographical locations (Vermitsky et al., 2008; Sharifzadeh et al., 2013; Cleveland et al., 2015; Klingspor et al., 2015). As the frequency of azole resistant Candida isolates in the clinical setting increases, it is essential to elucidate the machineries of such resistance in order to both preserve and improve upon the azole class of antifungals for the treatment of Candida infections. Many studies have documented the ability of Candida to develop high-level resistance to azole antifungals (Oxman et al., 2010; Lortholary et al., 2011), therefore, a clear understanding of molecular machineries driving the intrinsic and onset of high-level azole resistance is necessary.
1.1 Epidemiology of Candida Infection
Numerous Candida species are commensals and colonize the skin and mucosal surfaces of humans. Critically ill or otherwise immunocompromised patients are more predisposed to developing both superficial and life-threatening Candida infections (Hasan et al., 2009). C. albicans is the predominant cause of invasive fungal infections (Horn et al., 2009), and represents a serious public health challenge with increasing medical and economic importance. This is due to the high mortality rates and increased costs of care (Lai et al., 2012). Although C. albicans is the most prevalent species involved in invasive fungal infections, the incidence of infections due to non-albicans species is increasing. In a recent study, it was found that 28.3% of patients exhibited invasive fungal infection, with C. albicans as the most frequently isolated (58%), followed by C. tropicalis (17%) and C. glabrata (15%) (Yapar, 2014). In Europe, an analysis showed that more than half of the cases of Candidaemia were caused by C. albicans, and the incidence rates for non- albicans Candidaemia infections were 14% each for C. glabrata and C. parapsilosis, 7% for C. tropicalis and 2% for C. krusei (Tortorano et al., 2006). Changes in the epidemiology have also been observed in Latin America. For instance, in Chile, the prevalence of C. albicans has changed, and a progressive increase of non-albicans infection has been observed; C. parapsilosis is the most frequent species, followed by C. tropicalis and C. glabrata. According to the Brazilian Network Candidaemia study, C. albicans accounted for 40.9% of cases in Brazil, followed by C. tropicalis (20.9%), C. parapsilosis (20.5%) and C. glabrata (4.9%) (Nucci et al., 2010). For Nigeria, however, such a cohort of study is yet to be executed and reported.
1.2 Pathogenicity of Candida Species
Candida species are considered important pathogens due to their versatility and ability to survive in various anatomical sites. Candida species are commensal eukaryotic opportunistic pathogens that reside on the mucosa of the gastrointestinal tract, mouth, oesophagus, vagina and other mucosal linings in an asymptomatic manner. While Candida species can infect different anatomical sites of the human host, there are indications that immune protection is site-specific. Cutaneous candidiasis and vaginal candidiasis are more likely to be connected with a phagocytic response involving neutrophils and mononuclear phagocytes (Vidigal and Svidzinski, 2009). It can however, become one of the most significant causes of death if not treated effectively (Wisplinghoff et al., 2006; Vincent et al., 2009). Infection caused by Candida is called candidiasis or candidosis, with a wide spectrum of clinical manifestations. It can be classified as superficial (as with cutaneous and mucosal infections), deep, widespread and of high severity (as is the case of invasive candidiasis). In years past, fungi emerged as major causes of nosocomial infections, mainly affecting immunocompromised patients or those who were hospitalized for long periods as a result of serious underlying diseases (Vidigal and Svidzinski, 2009). Most people usually have a single strain of Candida in different places in the body for a long period, while a comparatively lower number of individuals have more than one strain or species at the same time, as frequently observed among hospitalized patients (Kojic and Darouiche, 2004; Klotz et al., 2007). Virulence in C. albicans comprises of host recognition, which enables the pathogen to bind to host cells and proteins, with degradative enzymes playing special roles in their virulence. Extracellular hydrolytic enzymes appear to play an important part in adherence, tissue penetration, invasion and the damage of host tissues (Silva et al., 2009). Candida pathogenicity is aided by a number of virulence factors, the most important of which are those for adherence to host tissues and medical devices, biofilm formation and secretion of hydrolytic enzymes (e.g. proteases, phospholipases and haemolysins). The primary mechanism in the fungal colonization of human tissues is adherence to host surfaces; a process controlled and induced by numerous cell-signaling cascades in both the fungus and its immediate surrounding environment. The initial attachment of Candida cells is facilitated by non-specific factors (electrostatic forces and hydrophobicity) and promoted by specific adhesins that are present on the surface of fungal cells, and can identify ligands such as proteins (including fibrinogen and fibronectin). Adhesins can unambiguously bind to amino acids and sugars on the surface of other cells (Verstrepen and Klis, 2006). The presence of biofilm
matrix restricts the penetration of drugs, through the formation of a diffusion barrier, causing clinical problems of concern, by increasing resistance to antifungal therapy, since only the superficial layers are in contact with lethal doses of the drug (Kojic and Darouiche, 2004). Recent evidence suggests that many of the diseases produced by C. albicans are associated with biofilm growth (Ramage and Lo´pez-Ribot, 2005). Biofilms can thrive on any moist biotic or abiotic surface as a form of protection for their proliferation and defense against antifungal treatment, as well as to withstanding competitive pressure from other organisms. This strategy also encourages symbiotic relationships, and allows survival in hostile environments (Davey and O’toole, 2000; Ramage and Lopez-Rib, 2005). In lung infections, the association between C. albicans and Pseudomonas aeruginosa is an instance of an antagonistic interaction between bacteria and fungi, where P. aeruginosa kills yeast hyphae and biofilms of C. albicans (Morales et al., 2010). Generally, the biofilm matrix comprises carbohydrates, proteins, phosphorus and hexosamines; though, environmental circumstances such as medium composition, pH, oxygen concentration and the strain can affect biofilm formation along with matrix composition. For instance, C. parapsilosis biofilms contain large amounts of carbohydrates, and the protein content is lower in comparison with the biofilms of C. glabrata and C. tropicalis (Silva et al., 2009). According to the US National Institutes of Health (NIH), biofilms are the most common form of microbial growth in nature, and cause the majority of infections in humans (Nett et al., 2010). Several studies have shown that a relationship exists between an increase in the activity of extracellular hydrolytic enzymes and an increase in the pathogenic capacity of the yeasts, leading to clinical signs of severe candidiasis (Ingham et al., 2012). The roles of these fungal extracellular lipases include the digestion of lipids for nutrient acquisition, adhesion to host cells and tissues, unspecific initiation of inflammatory processes and self-defense through lysing of any competing microflora (Verstrepen and Klis,
2006). Iron, an inorganic element, is also essential for the survival of microorganisms, including yeasts (Candida), and the capacity to obtain this element is contributory to the on-set of an infectious process (Dongari-Bagtzoglou et al., 2009). Biofilms are hard to diagnose and treat, and have the possibility to serve as infectious reservoirs for a variety of microorganisms that include bacteria (such as Staphylococcus epidermidis, Staphylococcus aureus and Enterococcus species) and fungi (Klotz et al., 2007; Harriott and Noverr, 2011). Biofilms however, thus far, have not been demonstrated in the gastrointestinal tract (Harriott and Noverr, 2011). C. albicans reversibly transforms from unicellular yeast cells to either pseudohyphae or hyphae (filamentous growth
form); a morphogenesis phenomenon. This phenomenon has been observed in C. albicans and C. dubliniensis (Bruder-Nascimento et al., 2010). The growth of hyphae, a virulence mechanism, plays an important role in tissue invasion and resistance to phagocytosis (Jayatilake et al., 2006). The morphological transformation from the yeast to the mycelial form (dimorphic switching) is induced by many environmental factors, such as serum, high temperatures (370C) and neutral pH (Yi et al., 2011). Genetic analyses show that both yeast cells and hyphae are crucial for biofilm formation, suggesting that each cell type has a unique role in this process (Douglas, 2003).
1.3.1 Candida Biofilms and Conventional Antifungals
Different antifungal classes utilize a different means to inhibit the growth of fungal pathogens (Pfaller et al., 2012). The molecular mechanisms of antifungal resistance are categorized as either primary or secondary, and are connected to intrinsic or acquired qualities of the fungal pathogen. This encompasses either interference with the antifungal machineries of the corresponding drug or a reduction in the drug levels. Resistance also surfaces when environmental influences lead to the colonization or replacement of a susceptible species with a resistant species. The antifungal properties of polyene and azole antifungals could be attributed to their actions on the fungal cell membrane, whereas echinocandins act by disrupting the fungal cell wall (Pfaller et al., 2012). The ability of Candida to form drug-resistant biofilms is a vital factor in its influence in human disease. The development of biofilms causes clinical complications of concern because they elevate the resistance to antifungal therapies, and the mechanism of biofilm resistance to antimicrobial agents is currently not completely known. A notable hypothesis to account for this resistance is that, the formation of a diffusion barrier, through the presence of the matrix, restricts the infiltration of drugs (Nett et al., 2011); therefore, only the most superficial layers are in direct contact with lethal doses of antimicrobials. Numerous molecular tools of resistance to antifungal agents in C. albicans have been described. In particular, these include the increased efflux of antifungal agents as a result of the overexpression of the efflux genes, CDR1, CDR2 and MDR1. The CDR1 and CDR2 are in the family of ABC (ATP-binding cassette) membrane transport proteins (Sardi et al., 2011). CDR1, CDR2 and other genes are often co-regulated, and are overexpressed at the same time (Staib et al.,
1999). Amino acid substitutions in the enzyme ERG11p (lanosterol 14-α-demethylase), encoded by the gene ERG11 is also another possible factor that could be responsible (Flowers et al., 2015). Due to the resulting increased fungal infections, two triazoles (voriconazole and posaconazole) and three echinocandins (anidulafungin, caspofungin and micafungin) have been developed and
approved to treat and prevent these infections (Mattiuzzi and Giles, 2005) (Fig. 1). Among these three classes of antifungal agents currently in clinical use, only amphotericin B and the echinocandins, e.g. caspofungin, have validated reliable in vitro activity against C. albicans biofilms (Kuhn et al., 2002). Despite the success of these two agents, Candida biofilm-related infections are awfully difficult to eradicate. The combined use of echinocandins with other drugs that have antifungal activity is becoming an important alternative form of therapy in mycoses resulting from fungi that are resistant to standard antifungal monotherapy in biofilm-associated diseases. In C. albicans biofilms, only a small subcategory of yeast cells are described to be highly resistant to amphotericin B (LaFleur et al., 2006).
1.3.2 Candida Biofilm and New Antifungal Strategies
The rising occurrence of drug-resistant pathogens and the side-effects associated with existing antifungal substances has attracted attention in the direction of the antimicrobial action of natural products. The small number of medications available for fungal treatment, most of which are fungistatic, and the developing resistance to antifungal agents encourage the search for more suitable substitute treatments (Sardi et al., 2011). Plants are found to be good options for obtaining a wide diversity of medications used in medicine due to their easy accessibility and application to various pathologies (Sardi et al., 2011). Plants therefore have proven to be an excellent source for substances that are useable in the formulation of new antifungal agents (Holetz et al., 2002). The extracts of some Romanian medicinal plants such as Artemisia absinthium, Arnica montana and Urtica dioica, have significant antimicrobial activities that are preferentially directed against fungi (C. albicans) and bacteria (S. aureus) (Stanciuc et al., 2011). Humans have also been a source of antifungal agents. In a study by Rossignol et al. (2011), the 18-amino acid cationic tryptophan- rich ApoEdpL-W peptide, derived from human ApoE apolipoprotein was studied, and showed antifungal activity against pathogenic yeasts of the Candida genus, with the exception of C. glabrata. ApoEdpL-W proved to be active against planktonic cells and early-stage biofilms but with less activity against mature biofilms, possibly because of its attraction for extracellular matrix b-glucans (Stanciuc et al., 2011).
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MOLECULAR PROFILING AND PHYLOGENETIC ANALYSIS OF THE ERG11 GENE OF FLUCONAZOLE RESISTANT STRAINS OF CANDIDA SPECIES ISOLATED FROM HUMANS AND DOGS OF REPRODUCTIVE AGE
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