The Threat Called Candida haemulonii Species Complex in Rio de Janeiro State, Brazil: Focus on Antifungal Resistance and Virulence Attributes

Abstract

Although considered rare, the emergent Candida haemulonii species complex, formed by C. haemulonii sensu stricto (Ch), C. duobushaemulonii (Cd) and C. haemulonii var. vulnera (Chv), is highlighted due to its profile of increased resistance to the available antifungal drugs. In the present work, 25 clinical isolates, recovered from human infections during 2011-2020 and biochemically identified by automated system as C. haemulonii, were initially assessed by molecular methods (amplification and sequencing of ITS1-5.8S-ITS2 gene) for precise species identification. Subsequently, the antifungal susceptibility of planktonic cells, biofilm formation and susceptibility of biofilms to antifungal drugs and the secretion of key molecules, such as hydrolytic enzymes, hemolysins and siderophores, were evaluated by classical methodologies. Our results revealed that 7 (28%) isolates were molecularly identified as Ch, 7 (28%) as Chv and 11 (44%) as Cd. Sixteen (64%) fungal isolates were recovered from blood. Regarding the antifungal susceptibility test, the planktonic cells were resistant to (i) fluconazole (100% of Ch and Chv, and 72.7% of Cd isolates), itraconazole and voriconazole (85.7% of Ch and Chv, and 72.7% of Cd isolates); (ii) no breakpoints were defined for posaconazole, but high MICs were observed for 85.7% of Ch and Chv, and 72.7% of Cd isolates; (iii) all isolates were resistant to amphotericin B; and (iv) all isolates were susceptible to echinocandins (except for one isolate of Cd) and to flucytosine (except for two isolates of Cd). Biofilm is a well-known virulence and resistant structure in Candida species, including the C. haemulonii complex. Herein, we showed that all isolates were able to form viable biofilms over a polystyrene surface. Moreover, the mature biofilms formed by the C. haemulonii species complex presented a higher antifungal-resistant profile than their planktonic counterparts. Secreted molecules associated with virulence were also detected in our fungal collection: 100% of the isolates yielded aspartic proteases, hemolysins and siderophores as well as phospholipase (92%), esterase (80%), phytase (80%), and caseinase (76%) activities. Our results reinforce the multidrug resistance profile of the C. haemulonii species complex, including Brazilian clinical isolates, as well as their ability to produce important virulence attributes such as biofilms and different classes of hydrolytic enzymes, hemolysins and siderophores, which typically present a strain-dependent profile.

Keywords: Candida haemulonii complex; antifungal resistance; biofilm formation; hemolysins; hydrolytic enzymes; siderophores.

Figure 1

Distribution of species comprising the C. haemulonii complex obtained from Brazilian hospitals (Rio de Janeiro State). (A) General species distribution considering the total number of clinical isolates (n = 25). (B) Percentage of clinical isolates obtained from each anatomical site considering the total number of fungal isolates, followed by the anatomical sites’ distribution per species forming the C. haemulonii complex. (C) Timeline comprising the distribution of species of the C. haemulonii complex used in the present work recovered per year. The numbers inside the black dots represent the number of total isolates obtained in the corresponding year.

Figure 2

Biofilm formation by C. haemulonii species complex on a polystyrene surface. Biofilms were formed for 48 h at 37 °C and then processed to detect the fungal biomass (590 nm) and cell viability (492 nm). The results are expressed as absorbance (ABS) values per clinical isolate studied (A,C) and mean per fungal species (B,D). The results represent means ± standard deviation of three independent experiments. The numbers on the X-axis of the graph represent each of the 25 isolates of the C. haemulonii complex used, in which Ch means C. haemulonii, Cd means C. duobushaemulonii and Chv means C. haemulonii var. vulnera.

Figure 3

Hydrolytic enzymes produced by C. haemulonii species complex. The distribution of aspartic protease, caseinase, phospholipase, esterase, phytase, hemolysin and siderophores production in each clinical isolate was shown (left side). Note that the gray lines divide the graph according to the intensity of enzymatic activity as follows: below the thin gray line are found the excellent producing fungal isolates, between the gray lines are found the good producing isolates and above the gray thick line are found the weak producing strains. In parallel, the mean ± standard deviation regarding the aspartic protease, caseinase, phospholipase, esterase, phytase, hemolysin and siderophores production in each species forming the C. haemulonii complex was plotted (right side). Note that the dotted line represents the overall average for the production of each enzyme in all studied strains (n = 25). Ch, C. haemulonii; Cd, C. duobushaemulonii; Chv, C. haemulonii var. vulnera.