3.1. Susceptibility of Planktonic Cells of the C. haemulonii Species Complex to Echinocandins
According to the breakpoints suggested in the M27S3 document published by CLSI, the planktonic cells of all clinical isolates of the
C. haemulonii complex tested herein were considered susceptible to echinocandins, with MIC values ranging from 0.125 to 0.5 mg/L for caspofungin and 0.25–0.5 mg/L for micafungin (
Table 1). For instance, a recent report described the successful use of caspofungin (MIC of ≤0.125 mg/L) in the treatment of a case of catheter-related candidemia caused by
C. haemulonii in a pediatric patient in Mexico [
25], whose fungal isolate exhibited in vitro high MICs for azoles (fluconazole MIC ≥256 mg/L, posaconazole ≥8 mg/L, itraconazole, ketoconazole and voriconazole ≥16 mg/L) and amphotericin B (MIC 1–2 mg/L). Some years before, a catheter-related candidemia in an adult patient hospitalized for a long period was only resolved when fluconazole treatment was replaced by caspofungin [
4].
In general, echinocandins are highly active in vitro against species comprising the
C. haemulonii complex [
7,
26,
27,
28,
29], but the existence of isolates resistant to this class of antifungals has already been reported [
4,
5,
30]. Herein, we conducted a careful review of the literature regarding the susceptibility of the
C. haemulonii species complex to the three clinically available echinocandins, including only papers published after the species reclassification and the creation of the
C. haemulonii complex [
5]. Using the keyword “Candida haemulonii” in the search section, 148, 63, 46 and 5 publications were located from the Web of Science, PubMed, Google Scholar and Scielo databases, respectively (
Table 2). However, only a small fraction of these published papers (varying from 12.2%–28.3%) cited the in vitro susceptibility profile of the
C. haemulonii species complex against echinocandins. In this sense, we recovered a total of 21 distinct papers that fitted our established criteria and, for these reasons, they were selected for data extraction as follows: 5 (23.8%) papers studied the three members forming the
C. haemulonii complex, 6 (28.6%) studied only two species (
C. haemulonii and
C. duobushaemulonii) and 10 (47.6%) studied only one species (
C. haemulonii,
n = 6,
C. duobushaemulonii,
n = 3,
C. haemulonii var.
vulnera,
n = 1). Furthermore, 13 (61.9%) papers detailed the MIC value for each isolate investigated, while the remaining studies (
n = 8; 38.1%) only presented the geometric mean (GM)-MIC and/or the range of MIC values for the fungal isolates against the test echinocandins. Finally, 12 (57.1%) papers tested the three echinocandins, 5 (23.8%) used two and 4 (19.1%) tested only one echinocandin, with caspofungin being the most frequently evaluated.
The results emanating from this literature review revealed that micafungin and anidulafungin appeared to be more effective than caspofungin against the three species forming the
C. haemulonii complex (
Table 3) [
5,
7,
25,
29,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47]. In this respect, 89.8% of the isolates of
C. haemulonii exhibited susceptibility to caspofungin, while 96.3% and 98.4% were susceptible to micafungin and anidulafungin, respectively. Regarding
C. duboushaemulonii, 95.5% of the isolates were susceptible to caspofungin, 99.1% to anidulafungin and 100.0% to micafungin. Finally, considering the clinical isolates of
C. haemulonii var.
vulnera, 85.0% were susceptible to caspofungin, 91.7% to micafungin and 97.1% to anidulafungin. Indeed, the MIC frequency distribution demonstrated that the modal MIC of echinocandins against the
C. haemulonii complex was ≤0.12 mg/L in almost all cases (
Table 4).
Comparing the GM-MIC values of our clinical isolates (
Table 1) with those compiled from the literature reports (for these comparisons, we used the arithmetic mean of the GM-MIC values of the selected works, as summarized in
Table 5), we observed that the GM-MIC values of caspofungin for our isolates of
C. haemulonii,
C. duobushaemulonii and
C. haemulonii var.
vulnera were higher than those reported in the literature (0.33 mg/L versus 0.18 mg/L for
C. haemulonii, 0.18 mg/L versus 0.11 mg/L, for
C. duobushaemulonii and 0.32 mg/L versus 0.21 mg/L for
C. haemulonii var.
vulnera). Similarly, GM-MIC values for micafungin calculated from the literature reports were lower than ours (0.18 mg/L versus 0.33 mg/L for
C. haemulonii, 0.17 mg/L versus 0.30 mg/L for
C. duobushaemulonii, and 0.13 mg/L versus 0.25 mg/L for
C. haemulonii var.
vulnera). Finally, based on the analysis of the literature data, anidulafungin also produced low GM-MIC values for the three fungal species of the
C. haemulonii complex (GM-MICs of 0.16, 0.32 and 0.06 mg/L for
C. haemulonii,
C. duobuhaemulonii and
C. haemulonii var.
vulnera, respectively).
In summary, the majority of literature reported GM-MIC concentration values of <0.5 mg/L for the three echinocandins against the
C. haemulonii species complex. Nevertheless, two works warranted specific attention: Cendejas-Bueno et al. [
5], in which the GM-MIC values for caspofungin for the three members of the
C. haemulonii complex were disproportionately high in comparison to our present results and those given in the other literature publications; and Isla et al. [
36], in which the GM-MIC value obtained for caspofungin against the
C. duobushaemulonii isolates was considerably higher (
Table 5). A possible explanation for the high MIC values found in the aforementioned papers is the possible occurrence of paradoxical growth effect (also known as the Eagle effect), that is characterized by reduced activity of the antifungal agents at high concentrations. In fact, Cendejas-Bueno et al. [
5] stressed this discussion in their study, but in a superficial way. A recent study conducted with 106 clinical isolates of
C. auris demonstrated that the vast majority of isolates were susceptible to the echinocandins; however, they exhibited different intensities of paradoxical growth effect in the presence of caspofungin, whilst four isolates were resistant to echinocandins and had a mutation in hot spot region 1 of the
FKS gene [
48]. Interestingly, those isolates presenting paradoxical growth effect were susceptible to caspofungin at doses used in human treatment, while those with
FKS1 mutation were still resistant in a murine model of invasive candidiasis, demonstrating that only the isolates with the mutations display in vivo echinocandin resistance [
48].
3.2. Effects of Echinocandins on the Biofilm Formed by C. haemulonii Species Complex
In order to evaluate the effects of echinocandins (caspofungin and micafungin) on the viability and biomass of the biofilms formed by the clinical isolates of the
C. haemulonii complex, the mature biofilms were firstly incubated with different concentrations of the antifungals and then analyzed. The metabolic activity of viable fungal cells was assessed by their ability to reduce XTT to formazan, whilst the decrease in biofilm biomass was measured spectroscopically by looking at the incorporation of crystal violet into methanol-fixed, non-viable cells (
Figure 1 and
Figure 2). In general, the test echinocandins were found to be more efficient at reducing cell viability than decreasing the biomass of the
C. haemulonii complex biofilms.
The decrease of both viability and biomass parameters by caspofungin was isolate-dependent. At the lowest concentration used (0.25 mg/L) this echinocandin caused a statistically significant reduction in the viability of all of the fungal cells tested (
p < 0.05; One-way ANOVA analysis of variance, Dunnett’s multiple comparison test), varying from 30–80% among the different isolates (
Figure 1). However, caspofungin was unable to reduce the biomass of some of the
C. haemulonii isolates (LIP
Ch2, LIP
Ch3 and LIP
Ch4) even at the highest concentration used. Nevertheless, for the remaining fungal isolates the drug caused a biomass reduction of up to 60% (mainly against the
C. duobushaemulonii isolates) (
Figure 2). The isolates LIP
Ch2 (
C. haemulonii), LIP
Ch1 (
C. duobushaemulonii) and LIP
Ch5 (
C. haemulonii var.
vulnera) were less susceptible to caspofungin at the higher concentrations (
Figure 1).
Micafungin proved to be more effective than caspofungin at disturbing both biofilm viability and biomass. A decrease in biofilm viability of up to 60% was seen among most of the clinical isolates, especially against
C. duobushaemulonii and
C. haemulonii var.
vulnera (
Figure 1). Unlike caspofungin, micafungin showed a decrease of up to 60% on the biofilm biomass of
C. haemulonii isolates, with the exception of isolate LIP
Ch4, which forms a very dense and robust biofilm (
Figure 2). For the
C. duobushaemulonii and
C. haemulonii var.
vulnera isolates, micafungin reduced biomass in the range 20–60% (
Figure 2). In summary, the lowest concentration of micafungin used was able to significantly reduce the cell viability and the biomasses of biofilms formed by all of the test isolates, expect for the biomass of one isolate.
The determination of MBEC, which was defined as the lowest antifungal concentration able to reduce the biofilm viability in 50% [
19], revealed that the biofilms of all isolates remained susceptible to echinocandins, with the exception of the isolate LIP
Ch4 of
C. haemulonii (
Table 6). This fact could be explained by the ability of the isolate LIP
Ch4 to form very robust biofilm on polystyrene in comparison with the other isolates [
18,
21], hampering the action of echinocandins due to the high amount of fungal cells-forming the biofilm architecture as well as due to the high production of extracellular matrix that can block the antifungal penetration into the biofilm structure.
As micafungin was more active than caspofungin against the mature biofilms formed by the
C. haemulonii species complex it was chosen for further studies. In order to verify the 3-D organization of the biofilms following exposure to micafungin two isolates of
C. haemulonii were selected: LIP
Ch3, to represent the isolates having susceptible biofilms, and LIP
Ch4, to represent isolates forming resistant biofilms. CLSM analysis was conducted using Calcofluor white, which binds to the chitin in the fungal cell wall, to evidence the biofilm biomass. The CLSM analysis corroborated the results observed by crystal violet approach, with the lowest antifungal concentration used causing a drastic reduction in the biofilm biomass of LIP
Ch3, whilst even the highest concentration of micafungin failed to affect the biofilm formed by LIP
Ch4 (
Figure 3).
Until now, no information has been available in the literature regarding the activity of conventional antifungal agents against the biofilm formed by the
C. haemulonii species complex. A recent study conducted with
C. auris, which belongs to the
C. haemulonii clade, showed that, despite the susceptibility of planktonic cells to echinocandins and amphotericin B, the biofilms were not vulnerable, exhibiting MBECs which were 512-fold higher than their planktonic MIC counterparts [
19]. Actually, the biofilm formed by
C. auris is not as robust as those arising from
C. albicans and
C. glabrata, but its tolerance to the major classes of antifungal agents is notable, especially for amphotericin B and micafungin, which are the recommended antifungal therapeutics for infections caused by
C. albicans biofilms [
49]. The antifungal tolerance of the
C. auris biofilm has been shown to be phase-dependent, with the mature biofilms resistant to the three available antifungal drug classes [
50]. On the other hand, micafungin has been shown to be effective against both planktonic and biofilm-forming
C. albicans cells, while its effectiveness against
C. parapsilosis was considered to be moderate [
51]. Additionally, micafungin concentrations >2 mg/L prevented the regrowth of
Candida biofilm cells [
51]. Regarding the
C. parapsilosis complex, caspofungin was more active against biofilms of
C. orthopsilosis than
C. parapsilosis sensu strictu, with 20% and 86% of isolates resistant to this antifungal, respectively, suggesting that a treatment of catheter-related candidemia caused by
C. orthopsilosis with caspofungin would be more effective than against
C. parapsilosis sensu strictu [
52]. A study, conducted with five different
Candida species recovered from cases of bloodstream infections demonstrated both species-specific and drug-specific differences in
Candida biofilms regarding their susceptibility to echinocandins [
53]. In this sense, while
C. albicans and
C. krusei biofilms were susceptible to the three clinically available echinocandins,
C. lusitaniae,
C. guilliermondii and
C. parapsilosis were quite resistant to them [
53]. In addition, micafungin seemed to be the most effective echinocandin against
C. parapsilosis biofilms, presenting lower MBECs against this
Candida species in comparison to caspofungin and anidulafungin [
53]. These observations reinforce the need to determine the correct identification of the actual fungal species causing the candidiasis infection and, further, to assess its antifungal susceptibility profile against both planktonic and biofilm-forming cells in order to choose the best therapeutic option for each case.
Furthermore, we observed that one isolate of each species forming the
C. haemulonii complex showed a smaller reduction in cell viability when incubated in the presence of higher concentrations of the echinocandins. This phenomenon is called paradoxical growth, and it corresponds to the decreased sensitivity to echinocandins in the presence of concentrations higher than the MIC values. To date, the evidence strongly suggests that this paradoxical effect is more commonly associated with caspofungin than either micafungin or anidulafungin [
54]. This effect has already been documented for biofilms formed by other
Candida species, such as
C. albicans [
53,
55],
C. parapsilosis [
53],
C. tropicalis [
55] and
C. dubliniensis [
56].
To finalize, we recognize some of the limitations associated with the present study, such as the limited number of isolates used and the exclusion of anidulafungin. The experiments were conducted with only 12 clinical isolates of the C. haemulonii complex due to the difficulties in obtaining more isolates, since it is quite a rare fungal complex. Additionally, we tested only two of the three echinocandins currently in clinical use, and this was because at the time the experiments were conducted anidulafungin was not available for scientific research purposes.