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. 2021 Apr 22;11:645812. doi: 10.3389/fcimb.2021.645812

Trichosporon asahii and Trichosporon inkin Biofilms Produce Antifungal-Tolerant Persister Cells

Rossana de Aguiar Cordeiro 1,*,†, Ana Luiza Ribeiro Aguiar 1,†, Bruno Nascimento da Silva 1, Lívia Maria Galdino Pereira 1, Fernando Victor Monteiro Portela 1, Zoilo Pires de Camargo 2, Reginaldo Gonçalves de Lima-Neto 3, Débora de Souza Collares Maia Castelo-Branco 1, Marcos Fábio Gadelha Rocha 1,4, José Júlio Costa Sidrim 1
PMCID: PMC8100310  PMID: 33968802

Abstract

Persister cells are metabolically inactive dormant cells that lie within microbial biofilms. They are phenotypic variants highly tolerant to antimicrobials and, therefore, associated with recalcitrant infections. In the present study, we investigated if Trichosporon asahii and T. inkin are able to produce persister cells. Trichosporon spp. are ubiquitous fungi, commonly found as commensals of the human skin and gut microbiota, and have been increasingly reported as agents of fungemia in immunocompromised patients. Biofilms derived from clinical strains of T asahii (n=5) and T. inkin (n=7) were formed in flat-bottomed microtiter plates and incubated at 35°C for 48 h, treated with 100 μg/ml amphotericin B (AMB) and incubated at 35°C for additional 24 h. Biofilms were scraped from the wells and persister cells were assayed for susceptibility to AMB. Additionally, we investigated if these persister cells were able to generate new biofilms and studied their ultrastructure and AMB susceptibility. Persister cells were detected in both T asahii and T. inkin biofilms and showed tolerance to high doses of AMB (up to 256 times higher than the minimum inhibitory concentration). Persister cells were able to generate biofilms, however they presented reduced biomass and metabolic activity, and reduced tolerance to AMB, in comparison to biofilm growth control. The present study describes the occurrence of persister cells in Trichosporon spp. and suggests their role in the reduced AMB susceptibility of T. asahii and T. inkin biofilms.

Keywords: antifungal susceptibility, dormant cells, Trichosporon spp., amphotericin B, trichosporonosis

Introduction

Trichosporon spp. are ubiquitous fungi, commonly found as commensals of the human skin and gastrointestinal tract (Duarte-Oliveira et al., 2017). Trichosporon species are emerging opportunistic fungi that have been increasingly reported as agents of fungemia in the last years, especially in immunocompromised patients (Challapilla et al., 2019; Sah et al., 2019; Alp et al., 2020). Furthermore, Trichosporon species have been reported as the second leading cause of invasive yeast infection in patients with malignant hematological diseases (Almeida and Hennequin, 2016).

The ability of Trichosporon species to cause systemic infections is likely associated with the expression of virulence factors, such as extracellular lytic compounds, and, most importantly, biofilms (Montoya et al., 2018), microbial communities surrounded by a polymeric extracellular matrix (Zhao et al., 2017). This structure expresses several mechanisms to escape the action of antimicrobials, as well as to resist physical stress, desiccation, UV radiation, and host immune system (Martinez and Casadevall, 2007; Polke et al., 2015). Previous studies have shown that T. asahii and T. inkin biofilms produce extracellular protease (Cordeiro et al., 2015) and also show increased tolerance to antifungals (Cordeiro et al., 2015; Almeida and Hennequin, 2016; Montoya et al., 2018; Cordeiro et al., 2019). Indeed, T. asahii biofilms may be up to 16,000 times more resistant to voriconazole, the most active antifungal against Trichosporon planktonic cells (Di Bonaventura et al., 2006).

Fungal biofilms tolerate high antifungal concentrations by many simultaneous mechanisms (Taff et al., 2013). Previous studies conducted with Candida albicans have shown that antifungal tolerance may be related to the development of a subpopulation of dormant cells within biofilms, known as persister cells (LaFleur et al., 2006). Such persister cells have been related to relapsing infections, as they tolerate antifungal exposure and can revert to an actively growing state, repopulating the biofilm, once treatment is ceased (Del Pozo, 2018).

Despite their relevance as emergent opportunistic species, few studies have addressed mechanisms of antifungal resistance of Trichosporon species in planktonic or sessile forms (Kushima et al., 2012; Kushima et al., 2015; Kushima et al., 2017; Li et al., 2017; Padovan et al., 2019). The present study aimed to evaluate if T. asahii and T. inkin are prone to develop dormant persister cells within biofilms and to suggest their importance in antifungal tolerance.

Material and Methods

Microorganisms

A total of 12 strains of Trichosporon spp. were evaluated in this study ( Table 1 ). Strain identification was based on analysis of micromorphological features on malt agar (De Hoog et al., 2000) and sequencing of intergenic spacer region IG1 of rDNA (Silvestre Junior et al., 2010). Strains were recovered from storage and maintained on potato dextrose agar (PDA; Himedia, India), at 35°C, for 48 h. The isolates belong to the culture collection of the Specialized Medical Mycology Center of Federal University of Ceará, Brazil. The chosen T. asahii strains do not have mechanisms of acquired resistance to amphotericin B (wild-type), according to the epidemiological cutoff values (ECVs), proposed by Francisco et al. (2019) for the species.

Table 1.

Trichosporon strains used in this study.

Species Strain GenBank Source
T. asahii CEMM 05-6-072 JX124945 urine
CEMM 05-6-073 JX124961 catheter
CEMM 03-1-072 MW291565 blood
CEMM 80 MW291562 tracheal aspirate
CEMM 81 MW291563 blood
T. inkin CEMM 01-1-143 JX125002.1 skin lesion
CEMM 01-1-144 JX124989.1 skin lesion
CEMM 01-1-145 JX124958.1 urine
CEMM 05-6-057 HM46988.1 white piedra
CEMM 05-6-074 JX124953 urine
CEMM 05-6-075 JX124985 perigenital area
CEMM 03-1-073 MW291566 nails
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C. albicans ATCC 10231 was included as internal control for persister cell isolation; moreover, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality controls for susceptibility assays (CLSI, 2008).

Drugs

Stock solution of amphotericin B (AMB; Sigma Chemical Co., St. Louis, MO, United States) was prepared as recommended by the document M27-A3 (CLSI, 2008). Serial two-fold dilutions of AMB were performed in RPMI 1640 medium (Sigma-Aldrich, MO, United States), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, MO, USA).

Biofilm Formation

Biofilm growth was induced as described by Cordeiro et al. (2015). Strains of T. asahii (n=5) and T. inkin (n=7) were grown on potato dextrose agar (PDA, Himedia, Mumbai, India) at 35°C for 48 h. Aliquots of 200 μl of fungal suspension adjusted to 2 x 106 cells/ml in RPMI 1640 medium were added to flat-bottomed 96-well polystyrene microplates and incubated at 35°C for 6 h (adhesion period) at 80 rpm. After incubation, non-adherent cells were removed by washing with sterile phosphate buffer saline with 0.05% Tween 20 (PBS-Tween 20). Afterwards, the wells were filled with RPMI medium and incubated at 35°C for 48 h (maturation period) at 80 rpm.

Detection of Persister Cells

The presence of persister cells in Trichosporon spp. biofilms was evaluated according to the methodology described by LaFleur et al. (2006) for C. albicans biofilms. Mature biofilms were washed with sterile PBS-Tween 20 and then treated with 100 μg/ml AMB and incubated at 35°C for additional 24 h (La Fleur et al., 2006). Thereafter, the wells were washed with sterile PBS-Tween 20, scraped with a pipette tip and the cells were resuspended in 100 μl in sterile PBS. Suspensions were serially diluted, plated on PDA agar and incubated at 35°C for 24 h for viable cell counting. Cells that survived exposure to 100 μg/mL AMB treatment were considered persister cells (LaFleur et al., 2006) Biofilms formed in RPMI 1640 medium without antifungal drug were included as biofilm growth control. C. albicans ATCC 10231 was included as control for validation of the methodology described by LaFleur et al. (2006), as it has been shown to produce dormant cells in the presence of AMB (Boucherit et al., 2007). All assays were performed in triplicate, at two independent experiments.

Regeneration of Biofilms by Persister Cells

After observing the presence of persister cells in Trichosporon biofilms, we tested their ability to regenerate biofilms on an abiotic surface. Persister cells were induced and isolated as described above. Thereafter, cell suspensions were adjusted in RPMI medium, transferred to flat-bottomed 96-well polystyrene plates and incubated at 35°C for 48 h at 80 rpm order to form progeny biofilms. Results were compared with the following paired controls: (1) mature biofilms produced by planktonic cells; and (2) progeny biofilms produced by cells detached from mature biofilms. Biofilms were evaluated for metabolic activity and biomass, by the XTT reduction (Cordeiro et al., 2015) and crystal violet staining assays (Pierce et al., 2015), respectively.

AMB Susceptibility of Persister Cells, Biofilm Cells, and Planktonic Cells

Susceptibility of persister cells to AMB was accessed by the broth microdilution method (CLSI, 2008). For isolation of persister cells, 48-h mature biofilms grown in RPMI-medium without antifungals were incubated with 100 µg/ml AMB for 24 h. Progeny biofilms were formed by inoculating persister cells into drug-free RPMI medium and repeating the biofilm growth procedure described above. Results were compared with the following paired controls: (1) planktonic cells; (2) mature biofilms produced by planktonic cells; (3) progeny biofilms produced by cells detached from mature biofilms; and (4) progeny biofilms produced by persister cells. Planktonic cells were obtained from 48-h culture on PDA at 35°C. Cell suspensions from biofilms were obtained after scraping of the wells and centrifugation at 9,167 xg for 10 min. Inocula were prepared in sterile saline solution and adjusted to a final concentration of 0.5–2.5 × 103 cells/ml in RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS. AMB was tested in concentrations ranging from 0.25 to 128 µg/ml (Cordeiro et al., 2015). Plates were incubated at 35°C for 48 h and fungal growth was visually determined analyzed. The minimum inhibitory concentration (MIC) was defined as the lowest concentration able of inhibiting 100% of visual fungal growth. Isolates were tested in triplicate, at two independent experiments. Controls were grown in RPMI medium without AMB. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included as quality controls.

Biofilm Structure and Morphology

Structural analysis of Trichosporon biofilms (T. asahii, CEMM 05-6-072, urine; T. inkin, CEMM 05-6-074, urine) was performed by scanning electron microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM), as described elsewhere (Di Bonaventura et al., 2006; Cordeiro et al., 2017). Biofilms were formed on Thermanox® slides (Thermo Fisher Scientific, NY, USA) in 12-well polystyrene plates with RPMI medium as described above and incubated at 35°C for 48 h at 80 rpm. Paired controls were grown in RPMI medium without antimicrobials. For SEM analysis, biofilms were fixed with 2.5% glutaraldehyde in 0.15M sodium cacodylate buffer and incubated overnight at 4°C. Biofilms were washed twice with 0.15M cacodylate buffer for 5 min and dehydrated in ethanol. Slides were dried with hexamethyldisilazane (Polysciences Europe, Germany) for 30 min, coated with 10nm gold (Emitech Q150T, Lewes, UK) and observed in a SEM (FEI Inspect S50, Thermo Fisher Scientific, Hillsboro, Oregon, USA) in the high vacuum mode at 15 kV. For CLSM, biofilms were stained with the Live/DeadTM (Invitrogen, Molecular Probes, Carlsbad, CA, USA) and evaluated with a confocal Nikon C2 C microscope (Nikon, Melville, NY, USA), at 488 nm, for the detection of SYTO 9 (live cells) and at 561 nm, for the detection of propidium iodide (dead/damaged cells). Images were processed using ImageJ Software (Collins, 2007). The software COMSTAT was used for quantitative analysis of the image stacks produced by CLSM.

Statistical Analysis

Parametric data were analyzed using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. For data with asymmetry, Wilcoxon’s or Friedman’s nonparametric tests followed by Dunn’s post-hoc test were applied. P-values < 0.05 were considered statistically significant. Statistical Analysis was performed using the software GraphPad Prism 7.0 (GraphPad Software, CA, USA).

Results

Presence of Persister Cells in Trichosporon Biofilms

The colony-forming unit (CFU) counts showed the presence of viable cells in Trichosporon biofilms after exposure to 100 μg/mL AMB. Counts ranged from 1.1 x 105 to 2.26 x 105 CFU/mL for T. asahii and 2.6 x 104 to 2.5 x 105 T. inkin strains ( Figure 1A ). Biofilm growth controls ranged from 3.53 x 106 to 7.7 x 106 CFU/mL and 1.12 x 106 to 5.75 x 106, for T. asahii and T. inkin, respectively. Persister cells consisted approximately 4% of viable cells for both T. asahii and T. inkin biofilms ( Figure 1B ). Statistically significant differences were observed when compared to drug-free growth control (P<0.05). The number of persister cells of C. albicans (2 x 104 CFU/ml) corresponds to approximately 1.45% of the total number of cells within biofilm growth control (1.38 x 106 CFU/ml).

Figure 1.

Figure 1

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Detection of persister cells in T. asahii and T. inkin biofilms (grey bars). (A) Mature biofilms formed in RPMI medium were treated with 100 µg/mL AMB. (B) Survival of T. asahii and T. inkin biofilms challenged with 100 µg/mL AMB. Biofilm controls were grown in drug-free RPMI medium (black bars). Values are shown as mean ± SD. *Statistically significant differences when compared to the respective drug-free control (P < 0.05).

Persister Cells of Trichosporon Can Form New Biofilms

Mature Trichosporon biofilms were challenged with AMB (100 µg/ml), then, progeny biofilms were formed by inoculating persister cells into RPMI medium, as previously described. Persister cells from each strain were able to form progeny biofilms. In general, a significant reduction in metabolic activity and biomass was observed for both species, when compared to progeny biofilms produced by cells detached from mature biofilms (P<0.05), as shown in Figure 2 .

Figure 2.

Figure 2

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Regeneration of T. asahii and T inkin biofilms. Biofilms were formed in RPMI medium and produced by planktonic cells (black bars), cells detached from mature biofilms (grey bars) or persister cells (white bars). Metabolic activity and total biomass were expressed as relative percentages of absorbance by the XTT reduction (A) and crystal violet staining (B) assays. *Statistically significant differences when compared to the biofilm growth control produced by planktonic cells (P < 0.05).

Persister Cells Showed Tolerance to AMB

Persister cells presented increased tolerance to AMB, compared to planktonic cells and biofilm-detached cells ( Table 2 ). MICs against persister cells from T. asahii and T. inkin biofilms ranged from 64 to >128 µg/mL (geometric mean: 111.43 µg/mL and 115.93 µg/mL, respectively). MICs against planktonic cells ranged from 0.25 to 2.0 µg/mL for T. asahii (geometric mean: 1.41 µg/mL) and 0.5 µg/mL to 1.0 µg/mL for T. inkin (geometric mean: 1.0 µg/mL). MICs against mature biofilm-detached cells ranged from 16 to 64 µg/mL (geometric mean: 36.75 µg/mL and 28.98 µg/mL, for T. asahii and T. inkin, respectively). MICs against progeny biofilm-detached cells ranged from 8 to 32 µg/mL (geometric mean: 21.11 µg/mL and 14.5 µg/mL, for T. asahii and T. inkin, respectively). Cells from progeny biofilms from persister cells showed increased susceptibility to AMB, when compared to other biofilm-derived cells, displaying MICs ranging from 2 to 8 µg/mL (geometric mean: 5 µg/mL for both species). AMB MIC of control strains C. krusei ATCC and C. parapsilosis ATCC 22019 were according CLSI guidelines (CLSI, 2008).

Table 2.

Minimum inhibitory concentration (MIC) of amphotericin B (AMB) against persister cells, biofilm cells and planktonic cells of T. asahii and T. inkin strains.

Species/Strains AMB MIC (µg/mL)*
Planktonic cells a Biofilm cells b Progeny biofilm c Persister cells d Progeny biofilm-persister cells e
T. asahii CEMM 05-6-072 0.5 32 16 128 8
CEMM 05-6-073 1 16 16 64 8
CEMM 03-1-072 2 32 32 128 8
CEMM 80 0.25 64 16 >128 2
CEMM 81 0.5 64 32 >128 4
Geometric mean 1.41 36.75 21.11 111.43 5
T. inkin CEMM 01-1-143 0.5 16 8 >128 8
CEMM 01-1-144 0.5 32 16 128 4
CEMM 01-1-145 0.5 32 16 128 4
CEMM 05-6-057 1 32 16 128 8
CEMM 05-6-074 0.5 16 8 64 2
CEMM 05-6-075 1 32 16 128 8
CEMM 03-1-073 1 64 32 128 8
Geometric mean 1 28.98 14.5 115.93 5
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*100% inhibition of visible fungal growth.

a

Obtained from 48-h cultures on PDA at 35°C.

b

Disaggregated cells from mature biofilms (48 h) produced by planktonic cells.

c

Disaggregated cells from progeny mature biofilms (48 h) produced by cells detached from mature biofilms.

d

Persister cells.

e

Disaggregated cells from progeny mature biofilms (48 h) produced by persister cells.

Biofilm Ultrastructure

Persister cells were compared to biofilm growth control by SEM ( Figure 3 ) and CLSM ( Figure 4 ). Structured biofilms with high cell density enclosed by a dense extracellular material were seen in drug-free growth controls ( Figures 3A, B , and 4A, B ). AMB (100 µg/mL) was able to disrupt the three-dimensional structure of mature biofilms, leaving deformed fungal structures ( Figures 3C, D ) and rare live cells ( Figures 4C, D ). Persister cells were able to form progeny biofilms with fewer filaments and less extracellular matrix (Figures 3E, F and 4E, F ) than biofilm growth control. Progeny biofilms produced by cells detached from mature biofilms also showed fewer filaments and less extracellular matrix than the drug-free growth control ( Figures 3G, H and 4G, H ).

Figure 3.

Figure 3

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Ultrastructure of T. asahii CEMM 05-6-072 and T. inkin CEMM 05-6-074 biofilms by SEM. Sessile cells were formed on Thermanox™ coverslips in drug-free RPMI medium as controls (A, B). Mature biofilms (48 h) were challenged with 100 μg/mL AMB and incubated at 35°C for 24 h (C, D). Surviving cells were considered persisters. Biofilms were then scrapped with a pipette tip and inocula were prepared in RPMI medium. Progeny biofilms produced by persister cells (E, F) or mature biofilms-detached cells (G, H) were formed in RPMI medium.

Figure 4.

Figure 4

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Scanning electron microscopy of T. asahii CEMM 05-6-072 and T. inkin CEMM 05-6-074 biofilms. Sessile cells were formed on Thermanox™ coverslips in drug-free RPMI medium as controls (A, B). Mature biofilms (48 h) were challenged with 100 μg/mL AMB (C, D) for detection of persister cells. Progeny biofilms produced by persister cells (E, F) or mature biofilms-detached cells were formed in RPMI medium (G, H). Live cells are shown in green and dead/damaged cells are shown in yellow/red after stain with SYTO9 and propidium iodide, respectively. Magnification: 400x. Bar: 100 μm.

Discussion

Mechanisms associated with antifungal tolerance have already been described in Candida biofilms and include increased efflux pump activity; drug sequestration by extracellular matrix (Cohen et al., 2013; Wuyts et al., 2018); expression of resistance genes (Li et al., 2015); reduction of cell ergosterol concentration (Mukherjee et al., 2003) and production of persistent cells (LaFleur et al., 2006; Al-Dhaheri and Douglas, 2008; Delarze and Sanglard, 2015; Wuyts et al., 2018). Although the antifungal tolerance in Trichosporon biofilms is widely reported (Falk et al., 2003; Di Bonaventura et al., 2006; Iturrieta-González et al., 2014; Cordeiro et al., 2015), the mechanisms underlying this phenotype have not been described.

LaFleur et al. (2006) were the first to describe the occurrence of persister cells in fungal biofilms, presenting evidence that this phenomenon occurs in C. albicans biofilms. Authors challenged mature biofilms with up to 900 µg/mL of AMB and showed that most of the population was killed by AMB at low concentrations but nearly 1% of the cells seemed to be completely invulnerable to higher concentrations of the drug. Since then, persister cells have been described in biofilms of C. parapsilosis, C. krusei (Al-Dhaheri and Douglas, 2008) and Saccharomyces cerevisiae (Bojsen et al., 2014).

Persister cells are phenotypic variants that are maintained in a state of dormancy, being able to survive the oxidative stress induced by high concentrations of antifungals through the activation of energy storage pathways (Wuyts et al., 2018). As these dormant cells have low metabolic activity, they have been detected by counting colony-forming units, instead of using viability reagents (LaFleur et al., 2006; Al-Dhaheri and Douglas, 2008; LaFleur et al., 2010; Li et al., 2015; Sun et al., 2016; Wu et al., 2019; Yee et al., 2019; Galdiero et al., 2020; Wu et al., 2020). In the present study, we observed that clinical strains of T. asahii and T. inkin produced persister cells inside their biofilms. Using the methodology originally proposed by LaFleur for the detection of persister cells in C. albicans biofilms, it was observed that the proportion of persister cells varied from 2 to 5% for T. asahii and from 2 to 6% for T. inkin. These values are higher than those found in C. albicans (LaFleur et al., 2006), C. parapsilosis and C. krusei biofilms (Al-Dhaheri and Douglas, 2008), which usually comprise up to 2% of the total population. We do not know if these results indicate intrinsic properties of Trichosporon biofilms or if they are derived from the small number of strains investigated in the present study.

The concept of persister cells also encompass a biphasic pattern of killing when a sessile population is challenged with an antifungal drug at concentrations well above MIC (Wuyts et al., 2018). In our study, mature biofilms of Trichosporon were challenged with increased concentrations of AMB (10, 50 and 100 µg/mL) and a biphasic pattern of killing was seen: whereas 50 µg/mL AMB suppressed Trichosporon biofilms (data not shown), few cells remained alive after challenge with 100 µg/mL of AMB, as shown by CFU counting and confocal microscopy. Previous studies have shown that MIC values for AMB against Trichosporon biofilm-cells were, in general, below 50 µg/mL (Iturrieta-González et al., 2014; Cordeiro et al., 2015; Montoya et al., 2018).

In the present study, persister cells of T. asahii and T. inkin were able to originate progeny biofilms, however, these biofilms presented less biomass and reduced metabolic activity when compared to the progeny biofilms produced by cells detached from mature biofilms. Such results were corroborated by the ultrastructural analysis, which revealed that the biofilms deriving from persister cells have lower cell density. Detached cells from these progeny biofilms were less tolerant to AMB than persister cells contained in the starter inoculum. These results suggest that Trichosporon persister cells are not mutants but phenotypic variants of regular cells, as previously demonstrated for other fungal species (LaFleur et al., 2006; Al-Dhaheri and Douglas, 2008; Wuyts et al., 2018).

Besides the intracellular accumulation of energy storage molecules (Li et al., 2015; Wuyts et al., 2018), it is supposed that persister cells may have a different cell wall composition (Wuyts et al., 2018) and increased extracellular matrix production (Li et al., 2015). Previous studies have shown that the extracellular matrix actively contributes to the antifungal resistance of biofilms, by inducing drug-sequestration and harboring drug-efflux proteins (Li et al., 2015; Wuyts et al., 2018; Berman and Krysan, 2020). Although we were not able to study the cell wall properties of persister cells of T. asahii and T. inkin, microscopy analyses showed that these cells and their progeny biofilms had less extracellular matrix, resulting in thinner biofilms. The reduced amount of extracellular matrix observed in our results reinforces the hypothesis that the tolerance of Trichosporon biofilms to AMB at 100 µg/mL is derived from the presence of persister cells.

Experimental evidences suggest that persister cells are directly related to the occurrence of recalcitrant microbial infections. Unequivocal proof was first presented by Mulcahy et al. (2010) who showed a direct relationship between the presence of persister cells of Pseudomonas aeruginosa in patients with cystic fibrosis pneumonia. Studies performed with persister cells from bacterial biofilms prove that these cells, besides presenting reduced susceptibility to antibiotics, are better suited to survive the host immune system, persist within catheter-associated biofilm infection (Daubert et al., 2020) and cause more severe disease (Yee et al., 2019). Regarding fungal infections, LaFleur et al. (2010) showed that in vivo selection for high-persister mutants occurs in cancer patients that harbor Candida. Recently, it was hypothesized that persister cells inside biotic biofilms formed on vaginal epithelium and underlying tissues are related to recalcitrant vulvovaginal candidiasis (Wu et al., 2019; Wu et al., 2020). Although the virulence of fungal persister cells is not fully understood, the occurrence of dormant cells in biotic biofilms demands the attention of clinicians and researchers, since the available antifungals have low activity against biofilms and persister cells (Galdiero et al., 2020).

In conclusion, the results presented here show that T. asahii and T. inkin produce persister cells in their biofilms. These cells have a high tolerance to AMB and provide an inoculum for cell growth and new biofilm formation. Future studies should be conducted in order to understand the genetic regulation associated with the production of persister cells in Trichosporon biofilms and their phenotypic tolerance to AMB. Physicians should be aware that the production of persister cells within biofilms may be related to therapeutic failure in invasive trichosporonosis.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Author Contributions

RC and AA designed the research, analyzed the data and wrote the manuscript. AA, BS, LP, and FP performed the experiments. DC-B captured CLSM images. DC-B, ZC, RL-N, MR, and JC critically revised the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (Process 430193/2018-1). The authors are grateful to Central Analítica-UFC/CT-INFRA/MCTI-SISNANO/Pró-Equipamentos CAPES and Centro de Tecnologias Estratégicas do Nordeste-MCTIC.

References

  1. Al-Dhaheri R. S., Douglas L. J. (2008). Absence of amphotericin B-tolerant persister cells in biofilms of some Candida species. Antimicrob. Agents Chemother. 52, 1884–1887.   10.1128/AAC.01473-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Almeida J. N., Jr., Hennequin C. (2016). Invasive Trichosporon Infection: a systematic review on a re-emerging fungal pathogen. Front. Microbiol. 7, 1629.   10.3389/fmicb.2016.01629 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alp S., Gulmez D., Ayaz C. M., Arıkan-Akdaglı S., Akova M. (2020). Fungemia due to rare yeasts in a tertiary care university center within 18 years. Mycoses 63, 488–493.   10.1111/myc.13072 [DOI] [PubMed] [Google Scholar]
  4. Berman J., Krysan D. J. (2020). Drug resistance and tolerance in fungi. Nat. Rev. Microbiol. 18, 319–331.   10.1038/s41579-019-0322-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bojsen R., Regenberg B., Folkesson A. (2014). Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase. BMC Microbiol. 14, 305.   10.1186/s12866-014-0305-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boucherit Z., Seksek O., Bolard J. (2007). Dormancy of Candida albicans cells in the presence of the polyene antibiotic amphotericin B: simple demonstration by flow cytometry. Med. Mycol. 45 (6), 525–533.   10.1080/13693780701487821 [DOI] [PubMed] [Google Scholar]
  7. Challapilla M., Patel K., Patel B., Soman R., Rodrigues C., Shetty A. (2019). Trichosporon-blood stream infection. J. Assoc. Physicians India 67, 19–21. [PubMed] [Google Scholar]
  8. CLSI (2008). “Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard - Third Edition,” in CLSI document M27-A3. Wayne, PA, USA. [Google Scholar]
  9. Cohen N. R., Lobritz M. A., Collins J. J. (2013). Microbial persistence and the road to drug resistance. Cell Host Microbe 13, 632–642.   10.1016/j.chom.2013.05.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collins T. J. (2007). ImageJ for microscopy. Biotechniques 43, 25–30.   10.2144/000112517 [DOI] [PubMed] [Google Scholar]
  11. Cordeiro R., Serpa R., Alexandre C. F. U., Marques F. J. F., Melo C. V. S., Franco J. S., et al. (2015). Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals. J. Med. Microbiol. 64, 1277–1286.   10.1099/jmm.0.000159 [DOI] [PubMed] [Google Scholar]
  12. Cordeiro R. A., Serpa R., Mendes P. B. L., Evangelista A. J. J., Colares A. R. C., Franco J. S., et al. (2017). The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth, biofilm formation and proteolytic activity in Trichosporon spp. Biofouling 33, 640–650.   10.1080/08927014.2017.1350947 [DOI] [PubMed] [Google Scholar]
  13. Cordeiro R. A., Aguiar A. L. R., Pereira V. S., Pereira L. M. G., Portela F. V. M., Brilhante R. S. N., et al. (2019). Sodium butyrate inhibits planktonic and sessile cells of Trichosporon spp. Microb. Pathog. 130, 219–225.   10.1016/j.micpath.2019.03.013 [DOI] [PubMed] [Google Scholar]
  14. Daubert T. A., Kluthe K., Nuxoll A. (2020). S. aureus persister cells have increased survival to components of innate immunity, leading to persistence within a catheter-associated biofilm infection. J. Immunol. 204 (1 Supplement), 156.11. [Google Scholar]
  15. De Hoog G. S., Guarro J., Gene´ J., Figueiras M. J. (2000). Atlas of Clinical Fungi. 2nd ed (Washington: ASM Press; ). [Google Scholar]
  16. Del Pozo J. L. (2018). Biofilm-related disease. Expert. Rev. Anti. Infect. Ther. 16, 51–65.   10.1080/14787210.2018.1417036 [DOI] [PubMed] [Google Scholar]
  17. Delarze E., Sanglard D. (2015). Defining the frontiers between antifungal resistance, tolerance and the concept of persistence. Drug Resist. Update 23, 12–19.   10.1016/j.drup.2015.10.001 [DOI] [PubMed] [Google Scholar]
  18. Di Bonaventura G., Pompilio A., Picciani C., Iezzi M., D’antonio D., Piccolomini R. (2006). Biofilm formation by the emerging fungal pathogen Trichosporon asahii: development, architecture and antifungal resistance. Antimicrob. Agents Chemother. 50, 3269–3276.   10.1128/AAC.00556-06challa [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Duarte-Oliveira C., Rodrigues F., Gonçalves S. M., Goldman G. H., Carvalho A., Cunha C. (2017). The cell biology of the Trichosporon-host interaction. Front. Cell Infect. Microbiol. 7, 118.   10.3389/fcimb.2017.00118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Falk R., Wolf D. G., Shapiro M., Polacheck I. (2003). Multidrug-resistant Trichosporon asahii isolates are susceptible to voriconazole. J. Clin. Microbiol. 41, 911.   10.1128/JCM.41.2.911.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Francisco E. C., de Almeida Junior J. N., de Queiroz Telles F., Aquino V. R., Mendes A. V. A., de Andrade Barberino M. G. M., et al. (2019). Species distribution and antifungal susceptibility of 358 Trichosporon clinical isolates collected in 24 medical centres. Clin. Microbiol. Infect. 25, 909.e1–909.e5.   10.1016/j.cmi.2019.03.026 [DOI] [PubMed] [Google Scholar]
  22. Galdiero E., de Alteriis E., De Natale A., Siciliano A., Guida M., Lombardi L., et al. (2020). Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625. Sci. Rep. 10, 5780.   10.1038/s41598-020-62746-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Iturrieta-González I. A., Padovan A. C. B., Bizerra F. C., Hahn R. C., Colombo A. N. (2014). Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B. PLoS One 9, e109553.   10.1371/journal.pone.0109553 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kushima H., Tokimatsu I., Ishii H., Kawano R., Shirai R., Kishi K., et al. (2012). Cloning of the lanosterol 14-α-demethylase (ERG11) gene in Trichosporon asahii: a possible association between G453R amino acid substitution and azole resistance in T. asahii . FEMS Yeast Res. 12, 662–667.   10.1111/j.1567-1364.2012.00816.x [DOI] [PubMed] [Google Scholar]
  25. Kushima H., Tokimatsu I., Ishii H., Kadota J. (2015). Antifungal susceptibility and drug-resistant mechanism of Trichosporon . Med. Mycol. J. 56, J123–J128.   10.3314/mmj.56.J123 [DOI] [PubMed] [Google Scholar]
  26. Kushima H., Tokimatsu I., Kawano R., Watanabe K., Kadota J.-i. (2017). A new amino acid substitution at G150S in lanosterol 14-α demethylase (Erg11 protein) in multi-azole-resistant Trichosporon asahii . Med. Mycol. J. 58, E23–E28.   10.3314/mmj.16-00027 [DOI] [PubMed] [Google Scholar]
  27. LaFleur M. D., Kumamoto C. A., Lewis K. (2006). Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob. Agents Chemother. 50, 3839–3846.   10.1128/AAC.00684-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LaFleur M. D., Qi Q., Lewis K. (2010). Patients with long-term oral carriage harbor high-persister mutants of Candida albicans. Antimicrob. Agents Chemother. 54, 39–44.   10.1128/AAC.00860-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li H., Wang C., Chen Y., Zhang S., Yang R. (2017). Integrated transcriptomic analysis of Trichosporon Asahii uncovers the core genes and pathways of fluconazole resistance. Sci. Rep. 7, 17847.   10.1038/s41598-017-18072-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li P., Seneviratne C. J., Alpi E., Vizcaino J. A., Jin L. (2015). Delicate metabolic control and coordinated stress response critically determine antifungal tolerance of Candida albicans biofilm persisters. Antimicrob. Agents Chemother. 59, 6101–6112.   10.1128/AAC.00543-15hoog [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martinez L. R., Casadevall A. (2007). Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light. Appl. Environ. Microbiol. 73, 4592–4601.   10.1128/AEM.02506-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Montoya A. M., Elizondo-Zertucge M., Treviño-Rangel R. J., Becerril-Gargia M., González G. M. (2018). Biofilm formation and antifungal susceptibility of Trichosporon asahii isolates from Mexican patients. Rev. Iberoam Micol. 35, 22–26.   10.1016/j.riam.2017.02.008 [DOI] [PubMed] [Google Scholar]
  33. Mukherjee P. K., Chandra J., Kuhn D. M., Ghannoum M. A. (2003). Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect. Immun. 71, 4333–4340.   10.1128/iai.71.8.4333-4340.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mulcahy L. R., Burns J. L., Lory S., Lewis K. (2010). Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J. Bacteriol. 192, 6191–6199.   10.1128/JB.01651-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Padovan A. C. B., Rocha W. P. D. S., Toti A. C. M., Jesus D. F. F., Chaves G. M., Colombo A. L. (2019). Exploring the resistance mechanisms in Trichosporon asahii: Triazoles as the last defense for invasive trichosporonosis. Fungal Genet. Biol. 133, 103267.   10.1016/j.fgb.2019.103267 [DOI] [PubMed] [Google Scholar]
  36. Pierce C. G., Chaturvedi A. K., Lazzell A. L., Powell A. T., Saville S. P., McHardy S. F., et al. (2015). A novel small molecule inhibitor of Candida albicans biofilm formation, filamentation and virulence with low potential for the development of resistance. NPJ Biofilms Microbiomes 1, 15012.   10.1038/npjbiofilms.2015.12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Polke M., Hube B., Jacobsen I. D. (2015). Candida survival strategies. Adv. Appl. Microbiol. 91, 139–235.   10.1016/bs.aambs.2014.12.002 [DOI] [PubMed] [Google Scholar]
  38. Sah R., Soin A. S., Chawla S., Wadhwa T., Gupta N. (2019). Disseminated Trichosporon asahii infection in a combined liver-kidney transplant recipient successfully treated with voriconazole. Immun. Inflamm. Dis. 7, 125–129.   10.1002/iid3.250 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silvestre Junior A. M., Miranda M. A. B. R., Camargo Z. P. (2010). Trichosporon species isolated from the perigenital region, urine and catheters of a Brazilian population. Braz. J. Microbiol. 41, 628–634.   10.1590/S1517-83822010000300013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sun J., Li Z., Chu H., Guo J., Jiang G. (2016). Candida albicans amphotericin B-tolerant persister formation is closely related to surface adhesion. Mycopathologia 181, 41–49.   10.1007/s11046-015-9894-1 [DOI] [PubMed] [Google Scholar]
  41. Taff H. T., Mitchell K. F., Edward J. A., Andes D. R. (2013). Mechanisms of Candida biofilm drug resistance. Future Microbiol. 8, 1325–1337.   10.2217/fmb.13.101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wu X., Zhang S., Xu X., Shen L., Xu B., Qu W., et al. (2019). RAFT-derived polymethacrylates as a superior treatment for recurrent vulvovaginal candidiasis by targeting biotic biofilms and persister cells. Front. Microbiol. 10, 2592.   10.3389/fmicb.2019.02592 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wu X., Zhang S., Li H., Shen L., Dong C., Sun Y., et al. (2020). Biofilm formation of Candida albicans facilitates fungal infiltration and persister cell formation in vaginal candidiasis. Front. Microbiol. 11, 1117.   10.3389/fmicb.2020.01117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wuyts J., Van Dijck P., Holtappels M. (2018). Fungal persister cells: the basis for recalcitrants infections? PLoS Pathog. 14, e1007301.   10.1371/journal.ppat.1007301 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yee R., Yuan Y., Shi W., Brayton C., Tarff A., Feng J., et al. (2019). Infection with persister forms of Staphylococcus aureus causes a persistent skin infection with more severe lesions in mice: failure to clear the infection by the current standard of care treatment. Discov. Med. 28, 7–16. [PubMed] [Google Scholar]
  46. Zhao X., Zhao F., Wang J., Zhong N. (2017). Biofilm formation and control strategies of foodborne pathogens: food safety perspectives. RSC Adv. 7, 36670–36683.   10.1039/C7RA02497E [DOI] [Google Scholar]

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Data Availability Statement

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