A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Summary

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Abstract

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.

It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.

The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Introduction

Systemic candidiasis is the fourth major cause of nosocomial infections, which are associated with high morbidity and mortality rates worldwide. Globally, systemic candidiasis affects approximately 700,000 individuals¹. Candida species, namely C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. auris, are the most common cause of invasive Candida infections². Candida species are opportunistic pathogens that produce biofilms³. Biofilms are predominantly associated with Candida virulence, and Candida can withstand oxidative and osmotic stress conditions by inducing biofilm formation⁴. Biofilms further modulate the expression of virulence factors and cell wall components and form an exopolymeric protective matrix, helping Candida to adapt to different host niches⁴. Biofilms contribute to yeast adherence on host tissues and medical instruments⁵. As such, biofilm formation is associated with an advantage to yeasts, as yeast cells within the biofilms can evade the host immune response⁶. Biofilm formation also protects the pathogenic yeasts from the action of antifungal drugs⁵. Decreased susceptibility of C. albicans biofilms to amphotericin B has been demonstrated by Pierce et al.^7,8. Furthermore, biofilms demonstrate antifungal drug resistance to fluconazole, which impairs effective management of systemic candidiasis^9,10.

Microbes have an intrinsic tendency to adhere to various biotic and abiotic surfaces, which results in biofilm formation. Candida albicans, which is a dimorphic fungus, exists in yeast and hyphal forms, and its biofilm formation has been characterized in various in vitro and in vivo model systems¹¹. The steps of biofilm formation include the adhesion of Candida cells to the substrate, filamentation, proliferation, and biofilm maturation¹¹. Initially, the yeast form of C. albicans adheres to substrates, including medical devices and human tissue, followed by filamentation and proliferation of C. albicans into hyphal and pseudohyphal forms, and finally maturation of biofilms embedded in extracellular matrix¹¹. Biofilm formation largely contributes to C. albicans pathogenesis mechanisms¹². Candida species form drug-resistant biofilms, which makes their eradication challenging¹³. A small subset of the C. albicans biofilm-producing population has been described as being highly resistant to the antifungal drugs amphotericin B and chlorhexidine¹⁴. Of note, yeast cells in biofilms exhibit high resistance to multidrug therapy compared to yeast cells in the planktonic phase and proliferation phase¹⁴. It has been suggested that yeast cells existing in biofilms are highly tolerant to antifungal drugs, which contributes to C. albicans survival in biofilms¹⁴. These existing cells were reported to be phenotypic variants of C. albicans and not mutants¹⁴. Furthermore, cells of Candida biofilms known as "persister cells" are tolerant to high doses of amphotericin-B treatment and contribute to Candida survival, thereby posing a great burden of recurring systemic Candida infections in high-risk individuals¹⁵.

The increase in antifungal drug resistance in Candida strains necessitates research for new antifungal agents and immunotherapies. As evident from the abovementioned studies, Candida biofilms show decreased susceptibility to antifungal drugs. Therefore, there is a need for improved immunotherapies to control Candida biofilm formation. Earlier studies have shown that CAGTA can provide effective protection against systemic Candida infections by inhibiting C. albicans biofilm formation in vitro¹⁶. Another study reported that immunization of mice with C. albicans rAls3-N protein induces high antibody titers that interfere with C. albicans biofilm formation in vitro¹⁷. Anti-Als3-N antibodies also exerted an inhibitory effect on C. albicans dispersal from biofilms¹⁷. NDV-3A vaccine based on C. albicans is currently under clinical trial and anti-NDV-3A sera were also found to reduce C. auris biofilm formation¹⁸. A recent study identified inhibition of biofilm formation by Sap2-antibodies as a protection mechanism in a murine model of systemic candidiasis¹⁹.

This paper outlines a detailed in vitro protocol for evaluating the effect of antigen-specific antibodies present in polyclonal serum obtained from different groups of Sap2 vaccinated mice on preformed Candida tropicalis biofilms. To achieve this, a method based on an XTT reduction assay was optimized and developed in the laboratory, which can measure biofilm viability in a fast, sensitive, and high-throughput manner, in the presence or absence of antibodies.

The XTT assay is used to measure cellular metabolic activity as an indicator of cell viability, cellular proliferation, and cytotoxicity²⁰. This colorimetric assay is based on the reduction of a yellow tetrazolium salt, sodium 3´-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) to an orange formazan dye by metabolically active cells. Since only viable cells can reduce XTT, the amount of reduced XTT formazan is proportional to the intensity of color and cell viability. The formazan dye formed is water-soluble and is directly quantified using a plate reader. Due to its water-soluble nature, the XTT assay allows the study of intact biofilms, as well as the examination of biofilm drug susceptibility, without disruption of biofilm structure²¹. Additionally, this method is implemented in Candida fungal viability assessments due to its ease of use, speed, accuracy, high throughput, and high degree of reproducibility^7,22.

In addition to the XTT reduction assay, numerous alternative techniques have also been identified for the measurement of biofilm quantity. Some of these include the use of the MTT reduction assay, crystal violet staining, DNA quantification, quantitative PCR, protein quantification, dry cell weight measurement, and viable colony counting. These procedures vary widely in terms of their time and cost requirements. Taff et al. performed a comparative analysis of seven different Candida biofilm quantitation assays and found that the XTT assay provided the most reproducible, accurate, and efficient method for the quantitative estimation of C. albicans biofilms²³. Staining techniques such as crystal violet have certain limitations; the crystal violet test indirectly determines the amount of biofilm by measuring the optical density of the crystal violet-stained biofilm matrix and cells. Although the crystal violet assay provides a good measure of biofilm mass, it does not give a measure of biofilm viability as it stains both microbial cells and the extracellular matrix²⁴. Dhale et al. further reported that the XTT reduction assay was the most sensitive, reproducible, accurate, efficient, and specific method to detect biofilm production as compared to crystal violet assay²⁵. Literature reports have shown that the XTT assay correlates well with the CFU/mL parameter in the CFU counting method. However, compared to the XTT assay, the CFU method is labor-intensive and slow²⁶. Furthermore, the fraction of detached live cells may not be representative of the initial biofilm population²⁷. Although the XTT reduction assay seems the best available option to quantify viability, there are a few limitations of this technique. While the XTT method is useful for comparisons involving one fungal strain, its use may be limited when comparing different fungal strains and species. Interstrain comparisons may be difficult in the absence of detailed standardization since different strains metabolize substrates with different capabilities²¹.

Protocol

BALB/c mice were housed in the Small Animal Facility at IIT Roorkee. All animals were maintained in a 12 h:12 h light:dark cycle at 25 °C and were provided with a pellet diet and water ad libitum. All animal procedures were approved by the Institutional Animal Ethics Committee (IAEC) of IIT Roorkee.

1. Preparation of C. tropicalis

NOTE: The fungus Candida tropicalis belongs to Risk Group 2 pathogens and is classified as a BSL2 microorganism. Always use certified Class II Biological Safety Cabinets when working with Candida species. Practice aseptic and sterile techniques during work with C. tropicalis and follow the recommended biosafety procedures for proper disposal of this pathogen.

1. Streak C. tropicalis (strain ATCC 750) onto a Sabouraud dextrose (SAB) agar plate.
2. Prepare an overnight grown culture of C. tropicalis by inoculating a single colony from the SAB agar plate into a sterile 50 mL conical tube containing 10 mL of SAB broth medium. Alternatively, use a frozen glycerol stock of C. tropicalis and inoculate 100 µL of the glycerol stock into a sterile 250 mL conical flask containing 50 mL of SAB broth medium.
3. Incubate C. tropicalis culture in an orbital shaker at 180 rpm at 30 °C for 24-48 h.
4. Centrifuge the fungal culture (cells in logarithmic phase) at 2,150 × g for 15 min at 21 °C.
5. Discard the supernatant and add 50 mL of sterile 1x PBS to the pellet. Wash and resuspend the pellet in sterile 1x PBS with gentle vortexing.
6. Centrifuge again at 2,150 × g for 15 min at 21 °C. Discard the supernatant and resuspend the fungal pellet in 10 mL of sterile 1x PBS.
7. Calculate the concentration of cells by counting with a haemocytometer.
8. Prepare fungal stocks at a final density of 1.0 × 10⁶ cells/mL in RPMI 1640 morpholinepropanesulfonic acid (MOPS) medium. Use the cell suspension from step 1.6 immediately.
   ​NOTE: To set up one 96-well plate, the total fungal stock volume needed is 10 mL (100 µL/well). Scale as needed.

2. C. tropicalis biofilm formation

1. Prepare Candida biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1)^28,29.
2. Add 100 µL of C. tropicalis culture (from 10⁶ cells/mL stock, prepared as above) to a 96-well microtiter plate using a multichannel pipette (Figure 2A). Keep the last two columns (11 and 12) as 'no fungus plus serum' and 'no fungus and no serum' negative controls by not adding fungal cells. Fill columns 11 and 12 with 100 µL of RPMI 1640 MOPS medium alone.
3. Cover the microtiter plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 °C under stationary conditions.
4. The next day, aspirate the medium carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual medium.
5. Wash the plate with 200 µL of 1x PBS (per well) using a multichannel pipette. Add PBS very gently along the side walls of the well to avoid disrupting biofilms. Aspirate the PBS carefully using a multichannel pipette. Repeat the PBS wash 2x (a total of three washes).
6. To remove excess PBS, air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet.

3. Treatment of biofilm with antibodies

NOTE: Biofilms can now be processed for assessing the inhibition of biofilm maturation by antibodies. Murine serum was used as the source of polyclonal antibodies. Different groups of Sap2-immunized (Sap2-albicans, Sap2-tropicalis, and Sap2-parapsilosis) along with sham-immunized mice were bled retro-orbitally and serum was isolated as described earlier¹⁹. The presence of anti-Sap2 antibodies was confirmed using Sap2-specific ELISA as described previously¹⁹.

1. Perform heat-inactivation of the serum (source of polyclonal antibodies) at 56 °C for 30 min before use to rule out the role of complement in the inhibitory activity. Heat-inactivate the serum before making serum dilution.
   NOTE: Use serum from sham-immunized mice, preimmune mice, and Sap2-specific antibody-depleted serum as additional controls¹⁹. Antibody-depleted serum was prepared as per a previous study¹⁹. Among the serial dilutions (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, and 1:12,800) for serum tested in this protocol, inhibition of biofilm maturation was observed at 1:25, 1:50, and 1:100; hence, 1:50 was selected to strike a balance between inhibition and serum consumption.
2. Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium (1:50). Use a common serum dilution (1:50) for all the serum samples to be tested for inhibition of biofilm maturation³⁰.
3. Add 100 µL of the selected serum dilution to each well of the 96-well microtiter plate. For each sample, add serum dilutions in duplicate, as per the layout attached (Figure 2B).
   1. In column 10, do not add serum dilution; add only RPMI 1640 MOPS medium for the fungus-only positive control.
      NOTE: Column 10, rows G1-G8 and H1-H8 initially had fungal cells in RPMI-MOPS. However, while RPMI-MOPS was added to column 10 even after 24 h, rows G1-G8 served as PBS control and rows H1-H8 as no-serum control after 24 h.
   2. In column 11, add a 1:50 dilution of serum to all wells to serve as the no fungus plus serum negative control.
   3. In column 12, do not add serum dilution to any well; keep this as the no fungus no serum negative control.
4. Cover the plate with a lid and aluminum foil. Incubate the plate for 24 h at 37 °C.

4. Biofilm metabolic activity estimation

1. The next day, aspirate the serum carefully using a multichannel pipette (without touching or disrupting the biofilms). Tap the plate gently in an inverted position on blotting sheets to remove any residual serum.
2. Wash the plate with 200 µL of 1x PBS (per well) using a multichannel pipette, adding the PBS along the side walls of the well to avoid disrupting the biofilms. Aspirate the PBS carefully using a multichannel pipette and repeat the PBS wash 2x (a total of three washes). Air-dry the plate (without the lid) for 30 min at room temperature, inside a biological safety cabinet to dry any excess PBS.
3. Preparation of XTT/menadione:
   1. Prepare XTT in sterile Ringers Lactate as a 0.5 g/L solution. Dissolve 25 mg of XTT in 50 mL of filter-sterilized Ringers Lactate. Aliquot 10 mL in separate tubes covered with aluminum foil and store at -80 °C.
   2. Prepare menadione as a 10 mM stock. Dissolve 8.6 mg of menadione in 5 mL of acetone and distribute 50 µL in 100 separate microtubes. Store the aliquots at -80 °C.
   3. Prepare XTT/menadione solution just before use by taking 10 mL of XTT and adding 1 µL of menadione to obtain a 1 µM working solution.
4. Add 100 µL of the XTT/menadione solution per well of the 96-well microtiter plate. Cover the plate with a lid and aluminum foil. Incubate the plate for 2 h at 37 °C in the dark.
5. Transfer 80 µL of the colored supernatant from each well into a fresh 96-well plate. Read the plate at 490 nm.
6. Calculate the mean of the absorbance values of the wells in column 10 (fungus-only positive control), which will serve as a reference value for calculating the percentage biofilm inhibition by each serum sample using equation (1).
   % Biofilm inhibition = 100 - × 100    (1)

Representative Results

Candida tropicalis biofilms were grown in 96-well microtiter plates and imaged at 40x using an inverted microscope (Figure 1A). The biofilm was further stained using crystal violet and observed at 40x using an inverted microscope (Figure 1B). Scanning electron microscopy shows a representative image of C. tropicalis biofilm (Figure 1C). For performing the biofilm inhibition assay, 10⁵ cells of Candida were added to the wells of the 96-well microtiter plate at time 0, as per the layout (Figure 2A). After 24 h, the plate was washed, and serum samples were added to the preformed biofilms. A common serum dilution of 1:50 was selected to compare different serum samples obtained from different groups of Sap2-immunized and sham-immunized mice as per a pilot study and a previously published report³⁰.

Different serum samples were added to the 96-well microtiter plate as per layout (Figure 2B) and assessed in duplicate. Sera obtained at day 30 post immunization from three mice per group (Sap2-albicans immunized, Sap2-tropicalis immunized, Sap2-parapsilosis immunized, sham-immunized group, preimmune mice, and Sap2-depleted control sample) were analyzed at a 1:50 dilution. The plate was read at 490 nm wavelength using an ELISA reader for obtaining XTT-colorimetric readings (OD₄₉₀ values) for C. tropicalis biofilms formed in the presence or absence of serum (Table 1). Negative blank was calculated using the average of columns 11 and 12 (0.04). Positive control was calculated by calculating an average of fungus-only wells (column 10; 0.8165). Before calculations, the mean absorbance value of the control wells in columns 11 and 12 (0.04) was subtracted from the absorbance measurements of the experimental wells. Thus, the reference value of the biofilm positive control (column 10) was set to 0.7765 (= 0.8165 − 0.04).

The values (average minus blank) obtained for the experimental wells were then divided by this positive control (0.7765) and the percentage was obtained by multiplying by 100. Percentage biofilm inhibition was calculated by further subtracting the value obtained from 100. A bar graph shows all the values plotted (Figure 3). Ordinary one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons was used to calculate p values for assessing statistical differences between different serum groups. A p value of <0.05 was considered statistically significant. Serum from Sap2-parapsilosis immunized mice could prevent the maturation of preformed C. tropicalis biofilms by 65%, as compared to serum from Sap2-albicans (45%) and Sap2-tropicalis (55%) immunized mice. In general, biofilm inhibition by Sap2-immune serum was significantly higher than that by sham-immune serum (16%) and preimmune serum (13%), respectively. On depleting Sap2-specific antibodies from serum as described elsewhere¹⁹, the biofilm inhibition ability was reduced to 10%. Biofilm inhibition was close to negligible on using PBS (5%) and no-serum control (5%).

Figure 1: Imaging Candida tropicalis biofilm. (A) Visualization of Candida tropicalis biofilm formed on the bottom of a 96-well microtiter plate after removal of RPMI medium, using an inverted microscope. The image was captured using brightfield microscopy (no stain was used). (B) Visualization of C. tropicalis biofilm formed on the bottom of a 96-well microtiter plate after crystal violet staining. (C) Visualization of C. tropicalis biofilm formed on glass slides using scanning electron microscopy. Scale bars = 100 µm (A,B), 10 µm (C). Please click here to view a larger version of this figure.

Figure 2: Layout of the 96-well plate format. Addition of (A) fungal cells to the wells and (B) serum dilutions from different mice groups (Sap2-albicans immunized, Sap2-tropicalis immunized, Sap2-parapsilosis immunized, and sham-immunized; n = 3) assessed in duplicate at a 1:50 dilution. Additional controls included Sap2-depleted serum, preimmune serum, PBS, and no-serum control. Column 10 had no serum added (fungal cells present, positive control). Column 11 had no fungal cells added (serum present). Column 12 had no fungal cells added (serum absent). Abbreviations: PBS = phosphate-buffered saline; Sap2 = secreted aspartyl proteinase 2. The terms m1, m2, and m3 refer to different mice in each group (n = 3). Please click here to view a larger version of this figure.

Figure 3: Graph showing the effectiveness of Sap2-specific polyclonal antibodies against preformed C. tropicalis biofilms. The source of immune serum is shown on the x-axis, while the percentage of inhibition of C. tropicalis biofilm maturation is shown on the y-axis. Bars represent mean ± SEM (n = 3). Ordinary one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons was used to calculate p values. Bars and symbols represent the differences between Sap2-immunized mice groups with sham-immunized mice. ****, p < 0.0001. Abbreviations: PBS = phosphate-buffered saline; Sap2 = secreted aspartyl proteinase 2. Please click here to view a larger version of this figure.

<>	1	2	3	4	5	6	7	8	9	10	11	12
A	0.475	0.4141	0.0441	0.4637	0.485	0.0484	0.5179	0.4068	0.0442	0.8826	0.0452	0.0404
B	0.3848	0.3773	0.0428	0.369	0.3781	0.0465	0.4105	0.3773	0.0447	0.8428	0.0426	0.041
C	0.2754	0.2728	0.0427	0.305	0.3083	0.0415	0.3531	0.3213	0.0432	0.9661	0.0444	0.0453
D	0.6965	0.7185	0.043	0.6681	0.6711	0.0403	0.6346	0.728	0.0433	0.8643	0.0384	0.0358
E	0.7031	0.7117	0.0481	0.7806	0.688	0.0521	0.7095	0.6998	0.0525	0.7883	0.0491	0.0416
F	0.7588	0.7441	0.0456	0.7173	0.7233	0.0437	0.7733	0.6808	0.0462	0.7256	0.0468	0.0354
G	0.7681	0.7708	0.0435	0.7949	0.7496	0.0445	0.7695	0.7898	0.0451	0.7335	0.0429	0.0371
H	0.7652	0.7855	0.0469	0.7818	0.7766	0.0428	0.7592	0.7765	0.0445	0.7295	0.044	0.0341

Table 1: Absorbance readings at 490 nm for the 96-well microtiter plate. A standard plate reader (Tecan) was used to obtain the absorbance readings and the readings were matched to the layout described in Figure 2. Subsequent calculations were performed using these data.

Discussion

Fungal infections caused by Candida species are associated with high morbidity and mortality rates worldwide. The growing threat of invasive fungal infection requires the early management of such life-threatening diseases. Most Candida infections involve the formation of biofilms, which adhere to a variety of medical devices and are responsible for the persistence and recurrence of fungal infections in hospital settings³¹. Biofilms are composed of yeast or hyphal cells, and they exhibit considerable resistance to a majority of conventional antifungal drugs³². Antifungal resistance by Candida biofilms has been attributed to several mechanisms, including decreased antifungal penetration, presence of extracellular matrix, overexpression of drug-efflux pumps, altered cell membrane sterol composition, slow growth and spatial heterogeneity, activation of various signaling pathways, and the presence of drug-tolerant persister cells^33,34. Inhibition of Candida adhesion and biofilm formation are the most important strategies to prevent Candida infection.

Various in vitro assays, such as cell viability assays, microtiter plate assays, and dry weight measurement assays, have been utilized to study Candida biofilm formation, which are based on the specific time point assessment of biofilms²³. More advanced assays such as microfluidic device-based assays have been developed, which can be utilized to assess real-time biofilm formation³⁵. So far, biofilm formation has been studied using in vitro assays, but there is a need to understand the dynamic process of biofilm formation under in vivo conditions as well^36,37. Currently, most of the biofilm inhibition studies apply to C. albicans and few studies are available for the eradication of non-albicans Candida biofilms. The spectrum of Candida infections has changed gradually over the past few decades and emerging non-albicans Candida species pose a high burden of morbidity and mortality worldwide. Therefore, there is an emergent need for the development of novel strategies to control biofilm formation and the development of biofilm inhibition assays focusing on non-albicans Candida species. Immunotherapy, particularly antibodies, has a great potential to inhibit biofilm formation and can be used to treat systemic Candida infection³⁸. Several reports have demonstrated the role of antibodies in biofilm inhibition at earlier stages of biofilm formation. It was reported that polyclonal antibodies generated in response to complement receptor 3-related protein (CR3-RP, having a potential role in fungal adherence) immunization in rabbits reduced the adherence and biofilm formation of C. albicans on buccal epithelial cells³⁹. Further, Candida strains including catheter isolates were preincubated with polyclonal anti-CR3-RP antibody and monoclonal antibody, OKM1, which reduced the adherence and biofilm formation. Cell viability of C. albicans was evaluated using the XTT assay during the adherence phase and the biofilm formation phase⁴⁰. Few studies have evaluated the role of antibodies against non-albicans Candida species biofilm formation. Chupacova et al. evaluated the effectiveness of polyclonal anti-CR3-RP antibodies against C. albicans along with C. dubliniensis using the XTT assay. Polyclonal anti-CR3-RP antibodies inhibited adherence and biofilm formation of both C. albicans and C. dubliniensis⁴¹.

In this study, a simple, rapid, and user-friendly protocol is described for assessing the effect of antibodies on biofilm maturation and development. This 96-well microtiter plate-based XTT reduction assay measures the metabolic activity of viable cells in biofilms and has been adapted from earlier studies^7,8. The XTT assay described herein is extensively used to estimate viable biofilm growth. It has become a commonly used cell viability test because it is rapid, convenient, and can be used in high-throughput format such as a 96-well plate. Further, it can be used to quantify both yeast and hyphal forms of Candida biofilms. It can also be used for the measurement of Candida biofilms on a variety of substrates such as medical devices (e.g., catheters) and contact lenses.

Some of the critical steps of this protocol include vigorously vortexing the cell suspensions before pipetting in all steps, to avoid fungal aggregation⁷. Poor biofilms are likely to develop when cell densities are either too high or too low. Therefore, users should follow ideal fungal cell density in the initial inoculum⁷. The washing steps are very critical and excessive cell washing should be avoided to prevent disrupting the biofilm²². The bottom layer of the biofilm should not be disturbed during the washing process. The assay should always be performed in dark conditions, and it is necessary to include multiple replicates²². A fresh solution of XTT should be prepared every time just before use. As the time difference between two reactions may skew results, users should work fast to ensure a minimum time difference when adding the XTT solution to the first and last well of the 96-well plate⁴². The use of foil or parafilm is recommended for preventing evaporation. Several troubleshooting steps can be carried out if required. In case the biofilm growth is absent or unsatisfactory, appropriate seeding inoculum dilution and calculations should be used. For better biofilm formation, subculturing conditions can be changed, and biofilm growth can be tried using different surface materials. To significantly improve the XTT assay's sensitivity, one could decrease the time needed for biofilm to form, increase the antifungal agent's concentration, or shorten the assay's incubation period⁷. To avoid disrupting the biofilm, excessive cell washing should be avoided²². One can either avoid the outer wells to keep the assay agents from being exposed to light or add liquid to the outer wells to keep the agents from evaporating from the inner wells. Gentle washes should be given if the biofilm is becoming disrupted during washing²². While this protocol tests the effect of serum on 24 h preformed biofilms for assessing the inhibition of biofilm maturation, users can also modify this protocol to add serum at time 0 for assessing inhibition of biofilm formation, as per an earlier report³⁰.

Compared to other alternative methods for quantifying biofilm growth, the XTT method is the most sensitive, reproducible, accurate, cost-effective, and specific method. Although the XTT assay is a commonly used rapid and easy technique for evaluating biofilms, it does have certain limitations. While it is useful for determining the dosage or effects of various agents in a biofilm from a single species, it should be used with caution to compare multiple isolates simultaneously due to metabolic variability between isolates²¹. For interstrain biofilm comparisons, detailed technique standardization should be carried out²¹. Furthermore, while the XTT formazan product readily dissolves in solution, some strains may retain some amount within the cell²¹. Since there is a lack of clarity involving variables, such as the choice of media, time of biofilm development, and specifics of serum, it is advisable to perform confirmatory bioassays in addition to the XTT assay, for example, the measurement of biofilm thickness/biomass (using crystal violet) or cell viability (using the CFU method)⁴³. Of note, the results of this XTT assay correlated well with the crystal violet assay and the CFU method (data not shown). Notwithstanding the limitations mentioned above, this method can be extrapolated to evaluate different groups of serum samples (source of polyclonal antibodies), monoclonal antibodies, or adjunctive immunotherapies. Although the XTT reduction protocol presented here only shows the effect of antibodies on C. tropicalis biofilms, it has been used by researchers to study the effect of antibodies on C. auris biofilms as well¹⁸. Additionally, this protocol derives from previous studies performed with other Candida species such as C. albicans, C. parapsilosis, C. glabrata, C. dubliniensis, C. tropicalis, and C. auris, which can merit the extension of this protocol to the genus Candida^{16,41,44,45,46,47}.

Optimization, standardization, and regular improvement in biofilm quantification assays can enhance the accuracy, throughput, and reproducibility. The XTT reduction assay on which this protocol is based has been widely used for the assessment of metabolic activity and viability of biofilms. Since Candida biofilm is often resistant to antifungal medications, the exploration of new drugs and new combinations is an urgent necessity³³. To combat the issue of biofilm-associated infections, more antibiofilm compounds must be explored using biofilm screening assays⁴⁸. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms. Future research based on standardization and improvement of XTT reduction assay involving antibody-based immunotherapies may aid in the development of improved therapeutics for effective disease management and control.

Biofilm inhibition by Sap2-specific antibodies has been reported as an antibody-mediated protection mechanism in Sap2-vaccine mediated protection during murine systemic candidiasis caused by C. tropicalis¹⁹. An earlier study also showed that CAGTA potentially recognizing Sap antigens reduce the growth and biofilm formation of C. albicans in vitro¹⁶. In another study, Sap2-deleted mutants exhibited a biofilm growth defect in the presence of pepstatin A⁴⁹. Additionally, during biofilm formation, the Sap2 antigen played a role in proteolytic processing of the mucin Msb2 mediated through Cek1 mitogen-activated protein kinase signalling pathway⁵⁰. Therefore, it can be speculated that Msb2 processing is inhibited by antibody-mediated Sap2 neutralization, which affects biofilm integrity. In summary, this article provides a detailed protocol for assessing the role of serum antibodies in C. tropicalis biofilm maturation and development, which can be applied for the determination of biofilm metabolic activity in different settings, using different fungal strains or antibody sources.

Materials

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tubes	BD Falcon	546021
1x PBS	-	Prepared in lab	NaCl : 4 g KCl : 0.1 g Na2HPO4:  0.72 g KH2PO4 : 0.12 g Water 500 mL. Adjust pH to 7.4
50 mL conical centrifuge tubes	BD Falcon	546041
96-well microtiter plates	Nunc	442404
Incubator	Generic
Menadione	Sigma	M5625
Microtiter Plate Reader	Generic
Multichannel pipette and tips	Generic
Petri dishes	Tarson	460090
Ringers Lactate	-	Prepared in lab	sodium chloride 0.6 g sodium lactate 0.312 g potassium chloride 0.035 g calcium chloride 0.027 g Water 100 mL. Adjust to pH 7.0
RPMI 1640 MOPS	Himedia	AT180
Sabouraud dextrose Agar	SRL	24613
Sabouraud dextrose Broth	SRL	24835
XTT	Invitrogen	X6493