Biofilm Formation and Resistance to Fungicides in Clinically Relevant Members of the Fungal Genus Fusarium

Clinically relevant members of the fungal genus, Fusarium, exhibit an extraordinary genetic diversity and cause a wide spectrum of infections in both healthy individuals and immunocompromised patients. Generally, Fusarium species are intrinsically resistant to all systemic antifungals. We investigated whether the presence or absence of the ability to produce biofilms across and within Fusarium species complexes is linked to higher resistance against antifungals. A collection of 41 Fusarium strains, obtained from 38 patients with superficial and systemic infections, and three infected crops, were tested, including 25 species within the Fusarium fujikuroi species complex, 14 from the Fusarium solani species complex (FSSC), one Fusarium dimerum species complex, and one Fusarium oxysporum species complex isolate. Of all isolates tested, only seven strains from two species of FSSC, five F. petroliphilum and two F. keratoplasticum strains, recovered from blood, nail scrapings, and nasal biopsy samples, could produce biofilms under the tested conditions. In the liquid culture tested, sessile biofilm-forming Fusarium strains exhibited elevated minimum inhibitory concentrations (MICs) for amphotericin B, voriconazole, and posaconazole, compared to their planktonic counterparts, indicating that the ability to form biofilm may significantly increase resistance. Collectively, this suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.


Introduction
Despite being well known as plant pathogens, Fusarium species (order Hypocraeles) cause a broad spectrum of superficial infections, such as keratitis and onychomycosis, as well as locally invasive and disseminated fusarioses in human and animals [1,2]. The genus Fusarium also contains species which may spoil crops by the production of persistent mycotoxins that affectconsumers' health [1].
At present, the genus Fusarium consists of more than 200 species, divided in 22 species complexes, differing by morphology, host association, and molecular characteristics [3,4]. Among them, the Fusarium solani species complex (FSSC) and Fusarium oxysporum species complex (FOSC) are responsible for approximately 60% and 20% of human fusariosis, respectively [5][6][7]. Importantly, clinically relevant members of the genus Fusarium display high levels of resistance to systemic azoles, echinocandins, and polyenes [8][9][10][11][12]. The antifungal susceptibility within each species complex also varies from one species to another, which poses a major challenge in the management of patients with Fusarium infections [6,13].
In human pathogenic fungi, such as Candida and Aspergillus, biofilm formation increases fungal resistance to antifungal compounds [14][15][16][17], while it also plays a role in the colonization of specific surfaces [18,19]. The biofilm-forming ability of Fusarium strains, and its link with reduced antifungal susceptibility, has been reported in keratitis patients [20][21][22][23]. Previous studies also reported the possibility of biofilm-formation on contact lenses in outbreaks of keratitis caused by Fusarium species [21,22,24]. We therefore investigated whether various levels of biofilm formation or absence of this feature, across and within Fusarium species complexes, are linked to higher resistance against systemic antifungals.

Fungal Strains
A collection of 38 clinical Fusarium strains obtained from 38 patients with superficial and systemic infections, and 3 isolates from Fusarium infections in crops, was used. Table 1 describes the reference numbers of the isolates, the species complexes they reside in, sources, geographic origins, and in the case of clinical infections, the underlying disease, for all of the Fusarium strains. All strains were obtained from the reference collection of the CBS-KNAW Fungal Biodiversity Center (housed at Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) and handled under biosafety laboratory regulations. Identity of the organisms was confirmed by sequencing of the internal transcribed spacer regions of rDNA, translation elongation factor 1α (TEF1α) and the RNA polymerase II gene (RPB2), as described previously [6]. Prior to testing, all isolates were subcultured on Sabouraud glucose agar (Merck, Darmstadt, Germany), at 25 • C for 3-5 days.

Antifungal Susceptibility Testing of Planktonic Cells
The planktonic cells of each Fusarium isolate were tested for in vitro susceptibility to amphotericin B  [25]. Final concentrations of the following antifungal agents ranged from 0.016 to 16 µg/mL: amphotericin B, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole. Fluconazole and flucytosine, were assessed over a two-fold concentration range, from 0.064 to 64 µg/mL.
The minimum inhibitory concentrations (MICs) of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and posaconazole were determined visually; an inverted mirror was used for comparing the growth in wells containing the drugs with that in the drug-free control well. The minimum effective concentrations (MECs) of anidulafungin and caspofungin were read using a plate microscope (Olympus SZX9; Olympus Nederland, Zoeterwoude, The Netherlands), at 25× to 50× magnification. Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019), and C. krusei (ATCC 6258) were used for quality controls in all experiments. All experiments on each strain were performed using three independent replicates on different days. The geometric means (GMs) MICs and MECs of three independent replicates were determined for each species and drug, after 48 h of incubation. If no growth was observed, or the growth was not adequate, the incubation was extended to 72 h.

In Vitro Biofilm Formation Assay
The ability of the Fusarium strains to form biofilms was tested using the Crystal violet staining method in three independent replicates, as described previously [26]. Briefly, conidial suspension of each strain was adjusted to a final concentration of 1 × 10 6 conidia/mL in phosphate-buffered saline (PBS). One hundred µL of this suspension was placed in a tube containing 2 mL of brain-heart infusion broth (BHIB) with glucose (0.25%). The tubes were incubated at 37 • C for 24 h, and the suspensions were diluted in a ratio of 1:20 in freshly prepared BHIB with glucose. A 200-µL aliquot of this suspension was added to each well of a flat-bottom 96-well polystyrene microtiter plate (Corning Inc., Corning, NY, USA). After incubation for 24 h at 37 • C, the microplate was rinsed three times with PBS, and then inverted to drain, and 200-µL of 1% crystal violet was added to each well. After incubation for 15 min at room temperature, the microplate was again rinsed three times with PBS. Next, 200-µL of an ethanol:acetone mixture (80:20 w/v) was added to each well. The plates were read at 450 nm using a plate reader (Biotek EL × 808, Winooski, VT, USA). The percent transmittance (%T) value of each test sample was subtracted from the %T value of the reagent blank to obtain a measure of the relative amount of light blocked by the sample (%T bloc ). The biofilm production of each isolate was considered negative (%T bloc , <5), + (%T bloc , 5-20), ++ (%T bloc , 20-50), or +++ (%T bloc , >50). The biofilm activity of C. albicans ATCC 92228 (%T bloc , 5-20) was considered the positive quality control.

Antifungals Susceptibility of Pre-Formed Biofilms
To analyze the effects of antifungals on pre-formed biofilms, Fusarium biofilms were first established on the surface of 96-well, flat-bottomed microtiter plates, as described previously [27]. Briefly, the planktonic cell suspensions of 1 × 10 6 cells/mL in PBS, containing 0.025% (v/v) Tween-20, were prepared, and 200-µL was added to select wells and the suspensions were incubated at 37 • C for 24 h. Then, non-adherent cells were removed by washing with PBS, and a 200-µL RPMI 1640 medium, containing various antifungal concentrations, was added to the selected wells and incubated at 37 • C for an additional 24 h. Negative-control wells received 200-µL RPMI 1640 alone. The effects of antifungals on the pre-formed biofilms were then estimated using a semi-quantitative viability based XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) reduction assay, within 2 h of incubation, at 35 • C to 37 • C, as described previously [27,28]. Briefly, XTT (Sigma-Aldrich, St Louis, MO, USA) was prepared as a saturated solution (0.5 g/liter) in PBS. The solution was filter sterilized through a 0.22 µm pore-size filter, aliquoted, and stored at −70 • C. Prior to use, an aliquot of stock XTT was thawed, and 10 mM menadione (Sigma-Aldrich, St Louis, MO, USA), prepared in acetone, was added to the XTT, to make a final concentration of 1 µM. Subsequently, 100 µL of the above-mentioned XTT-menadione solution was added to each pre-washed biofilm, and to the control wells, to measure background XTT levels. The plates were further incubated at 37 • C for 2 h, in order to allow conversion of XTT to its formazan derivative. XTT conversion, as a direct correlation of the metabolic activity of the biofilm, was then measured as optical density (OD), with a microtitration plate spectrophotometric reader (Biotek EL × 808, USA) at 450 nm/630 nm. For each well, XTT conversion was calculated after subtraction of the background OD, which was the OD of a simultaneously incubated well with 100 µL of XTT-menadione solution, but no biofilm. Percentages of fungal growth were calculated for each well by dividing the XTT conversion of each well by the XTT conversion of the drug-free growth control well.

Data Analysis
Data were analyzed using GraphPad Prism, version 5.0, for Windows (GraphPad Software, San Diego, CA, USA). MIC/MEC distributions between the groups were compared using Student's t test and the Mann-Whitney-Wilcoxon test; differences were considered statistically significant at p values of ≤0.05 (two-tailed).

Antifungal Susceptibility Profile of Planktonic Cells
As shown in Table 1, amphotericin B had the highest in vitro activity against the planktonic form of all Fusarium species tested, with the MIC ranging from 0.125 to 8 µg/mL. Both voriconazole and posaconazole showed interspecies variability, across and within Fusarium species complexes, with the MIC ranging from 1 to 16 µg/mL and 0.125 to >16 µg/mL, respectively. However, all the species indiscriminately showed high MIC/MEC values for flucytosine, fluconazole, itraconazole, anidulafungin, and caspofungin. The MICs/MECs were identical between replicates.

Biofilm Formation
Of all isolates tested, only seven strains from FSSC, including five F. petroliphilum strains and two F. keratoplasticum strains, scored a %T bloc 20-50, displaying the capacity to form biofilms. The F. petroliphilum strains were identified in blood (n = 4), and nasal biopsy (n = 1) samples of patients with underlying acute lymphoblastic leukemia and myelodysplastic syndrome, respectively, while the F. keratoplasticum strains (n = 2) were recovered from nail scrapings of onychomycosis patients. The ability to produce biofilms was not detected in the remaining 34 strains.

Sessile Susceptibilities of Fusarium Strains
Amphotericin B showed the lowest MIC values against planktonic cells of all biofilm-positive species, with the MIC ranging from 0.25 to 2 µg/mL, but did not significantly differ from most other species (p > 0.05). Biofilm-forming strains of the two species showed higher MIC to azoles and echinocandins, whereas non-biofilm forming species had more variability in their susceptibility to these compounds. Of note, intraspecies variation exhibited within F. keratoplasticum and F. proliferatum species, with the MIC ranging 8 to 16 µg/mL, and 0.125 to >16 µg/mL for voriconazole and posaconazole, respectively.

Discussion
Our study showed that the seven tested isolates of two species, F. petroliphilum and F. keratoplasticum, both from the FSSC, could produce biofilms. These strains were recovered from blood, nails, and nasal biopsies of superficial and systemic fusariosis, but so were many of the non-biofilm producing strains, indicating that the biofilm formation trait is not the main contributing factor that causes these infections in the genus, Fusarium. The biofilm formation has been shown a major virulence attribute of pathogenicity in medically important fungi, such as Candidia, Aspergillus, and Pseudallescheria/Scedosporium species [17,19,[29][30][31]. In Fusarium, the ability to form biofilms was suggested as a pathogenicity determinant in an outbreak of fusarial keratitis, irrespective of the thickness of these biofilms [21].
The planktonic forms of biofilm-forming isolates all showed high resistance to tested azoles and echinocandins, whereas non-biofilm producers showed more variation and some of these were less resistant to these compounds. In addition, in the liquid culture tested, sessile Fusarium biofilms exhibited elevated MICs, compared to their planktonic counterparts, for amphotericin B, voriconazole, and posaconazole, indicating that the ability to form a biofilm may significantly (p ≤ 0.05) increase resistance, as shown in Table 2. This suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.
Similarly, Zhang et al. [20] reported that Fusarium species producing mature biofilms were intrinsically resistant to azole antifungal compounds. In another study, Imamura et al. [22] also observed that Fusarium biofilms in contact lenses may reduce susceptibility to lens care solutions in a time-dependent manner, suggesting that this extracellular matrix prevents antifungal penetration or that the biofilm increases the expression of a drug efflux pump system [14,15,32]. In contrast, however, Mukherjee et al. [21] reported that biofilms had no apparent effect on the natamycin susceptibility of FSSC and FOSC; voriconazole was active against biofilms formed by FSSC, and amphotericin B was active against FOSC.
In our study, we only tested one F. oxysporum strain, which did not form detectable biofilms. However, Mukherjee et al. showed that members of FOSC were able to produce (lower) levels of biofilms [21]. F. oxysporum is reported from localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients. Studies have also shown that clinically important lineages of F. oxysporum are linked with water systems in hospitals [33], supporting the possibility of nosocomial F. oxysporum infections. In addition to F. oxysporum, none of the tested F. solani strains originating from superficial and systemic infections had the ability to form biofilms, which is in contrast with previous reports suggesting that the ability of F. solani to form biofilms on contact lenses may have had a role in the keratitis outbreak [20][21][22]24]. Importantly, F. petroliphilum and F. keratoplasticum, the two biofilm-forming species in our study, were abundantly found in sinks and drains-man-made environments typically inhabited by biofilm-forming microorganisms [34]. Collectively, this suggests that the biofilm formation may also be a trait that also enables a species to establish itself in common human environments where people, including immunocompromised patients vulnerable to infection, may encounter them.
Fusarium strains generally show high intrinsic levels of resistance to the tested antifungal drugs. The underlying mechanisms leading to antifungal resistance in Fusarium are not yet understood, and a complex of involved regulatory proteins, enzymes, and transporter genes is suggested [35]. The observed mechanism of increased resistance in Fusarium species includes specific transcription regulators, such as CCG-8 [36], up-regulated ABC-transporters [35,37], and in the case of azole resistance, the presence of three lanosterol 14 alpha-demethylase paralogues (CYP51A, B, and C) [38]. Some of these mechanisms may also be involved in the antifungal resistance in Fusarium biofilms. Several studies have demonstrated that in Candida species, biofilm formation leads to dramatically increased levels of resistance to the most commonly used antifungal agents [39], and that the reason is multifactorial (mechanical, biochemical, and genetical factors); one mechanism of the increased resistance proves to be the up-regulation of efflux pumps and other resistance genes [15], as well as increased metabolically activity [18], during the development of biofilms. The three-dimensional architecture of the biofilm with increased cell densities and the formed extracellular "exopolymeric substance" (EPS) matrix have been found to be important factors [40]. Furthermore, nutrient limitation in biofilms may influence growth rates, and phenotypically altered "persister" cells are typically formed in the biofilms [41]. Also, in Aspergillus fumigatus, biofilm formation has been shown to increase antifungal drug resistance [16], with multifactorial principles, including the formation of extra cellular DNA (eDNA), to stabilize the EPS matrix [42].

Conclusions
Fusarium species are emerging in human infections. Biofilm formation is a relatively common feature in fungal etiological agents that renders biofilm-producing Fusaria even more refractory to treatment, while non-biofilm producing strains already possess a high level of innate resistance to most antifungal drugs available. The fact that biofilm production also allows the fungi to establish themselves well in human-made environments, like sinks and bathrooms, where they can act as reservoirs for nosocomial infections, makes them even a more serious threat to humans. Further studies, however, are warranted, to explore this association in greater detail, and to determine the mechanisms of virulence and antifungal resistance in biofilm-producing Fusarium species.