This work was supported by the Ramalingaswami grant DBT-843-BIO (Department of Biotechnology, Government of India) and Early Career Research Award SER-1058-BIO (Science and Engineering Research Board, Government of India) to S.R. The authors acknowledge an ICMR-JRF grant to P.C and DBT-JRF grant to P.S. The authors thank Dr. Ravikant Ranjan for suggestions on the manuscript and technical assistance by Mr. Pradeep Singh Thakur during SEM.

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 &#176;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...

Fungal infections&#160;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 ...

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 candidiasis9,10.
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,&#160;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 &#34;persister cells&#34; 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&#160;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&#180;-[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 reproducibility7,22.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>-</td>
			<td>Prepared in lab</td>
			<td>NaCl : 4 g 
			KCl : 0.1 g 
			Na2HPO4:&#160; 0.72 g 
			KH2PO4 : 0.12 g 
			Water 500 mL. Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtiter plates</td>
			<td>Nunc</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M5625</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter Plate Reader</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette and tips</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Tarson</td>
			<td>460090</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers Lactate</td>
			<td>-</td>
			<td>Prepared in lab</td>
			<td>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&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 MOPS</td>
			<td>Himedia</td>
			<td>AT180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud dextrose Agar</td>
			<td>SRL</td>
			<td>24613</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud dextrose Broth</td>
			<td>SRL</td>
			<td>24835</td>
			<td></td>
		</tr>
		<tr>
			<td>XTT&#160;</td>
			<td>Invitrogen</td>
			<td>X6493</td>
			<td></td>
		</tr>
	</tbody>
</table>

a soluble tetrazolium-based reduction assay to evaluate the effect of antibodies on candida tropicalis biofilms

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.

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 Candid...

Watch this Scientific Journal Video about A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms at JoVE.com

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

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.

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

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

This in vitro, 96-well-plate-based protocol can be used to test the inhibitory effect of antibodies on novel antifungal agents on the metabolic activity of fungal cells in Candida biofilms. Compared to other methods, this is a highly sensitive, accurate, high-throughput, reproducible, user-friendly, and cost-effective technique for evaluating the effect of antibodies or antifungals on the development of Candida biofilms. This assay can be used for assessing the efficacy of novel drug-based or antibody-based therapeutics, as well as vaccine candidates against Candida biofilms, which are associated with severity of invasive candidiasis.This method can be applied for testing the effect of novel antifungal agents or antibodies during biofilm formation or maturation in different settings using different fungal strains or antibody sources. Demonstrating this procedure will be Mr.Pankaj Chandley, a PhD research scholar working in my laboratory. To begin, add 100 microliters of Candida tropicalis culture to a 96-well microtiter plate.Using a multi-channel pipette, fill the last two columns with 100 microliters of RPMI 1640 MOPS medium alone. Cover the microtiter plate with a lid and aluminum foil and incubate the plate for 24 hours at 37 degrees Celsius under stationary conditions. The next day, aspirate the medium carefully using a multi-channel pipette.Invert the plate gently on blotting sheets and tap to remove any residual medium. Wash the plate with 200 microliters of 1X PBS using a multi-channel pipette. Aspirate the PBS carefully using a multi-channel pipette, and wash twice with PBS.To remove excess PBS, air dry the plate for 30 minutes at room temperature inside a biological safety cabinet. Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI 1640 MOPS medium. Add 100 microliters of the selected serum dilution to each well of the 96-well microtiter plate.In column 10, add only RPMI 1640 MOPS medium for the fungus-only positive control. Add a one to 50 dilution of serum to all wells in column 11 to serve as the no-fungus plus serum negative control. After covering the plate with a lid and aluminum foil, incubate the plate for 24 hours at 37 degrees Celsius.The next day, after aspirating the serum carefully using a multi-channel pipette, tap the inverted plate gently to remove any residual serum. Repeat the PBS wash three times, and air dry the plate for 30 minutes at room temperature inside a biological safety cabinet to dry any excess PBS. Then, dissolve 25 milligrams of XTT in 50 milliliters of filter-sterilized Ringer's lactate.Aliquot 10 milliliters in separate tubes. Cover it with aluminum foil and store it at minus 80 degrees Celsius. Next, dissolve 8.6 milligrams of menadione in five milliliters of acetone.And after distributing 50 microliters in 100 separate micro tubes, store the aliquots at minus 80 degrees Celsius. Take 10 milliliters of XTT and add one microliter of menadione to obtain a one micromolar working solution. Add 100 microliters of the XTT menadione solution per well of the 96-well microtiter plate.Next, after covering the plate with a lid and aluminum foil, incubate the plate for two hours at 37 degrees Celsius in the dark. Finally, transfer 80 microliters of the colored supernatant from each well into a fresh 96-well plate, and read the plate at 490 nanometers. Serum from Sap2-parapsilosis-immunized mice could prevent the maturation of preformed Candida tropicalis biofilms by 65%as compared to 45%in serum from Sap2-albicans, and 55%in Sap2-tropicalis-immunized mice.In general, biofilm inhibition by Sap2 immune serum was significantly higher than that by sham immune serum, which is 16%and 13%in pre-immune serum. Biofilm inhibition was close to negligible on using PBS and no serum control. Utmost care should be taken while performing the washing steps to prevent disruption of biofilms.The XTT solution should be prepared just before use, and added to the plate immediately. Following this procedure, users can evaluate the effect of novel antifungal agents, vaccine candidates, or antibodies during Candida biofilm formation and maturation in different research settings using different fungal strains.

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

Abstract